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Use for Diagnosis and Treatment

Sermorelin, a functional peptide fragment of GHRH1–29, has been used in the diagnosis and treatment of children with idiopathic growth hormone deficiency [13]. Tesamorelin, a stabilized synthetic peptide analog of GHRH1–44, received US Food and Drug Administration approval in 2010 for the treatment of lipodystrophy in HIV patients under highly active antiretroviral therapy, and was investigated for effects on certain cognitive functions in adults with cognitive impairment and healthy older adults [4].

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Chapter

Name of the clinical form Sermorelin acetate Related names Source: EMTREE Sermorelin; Geref (trade); geref; ghrh[1-29] amide; groliberin; groliverin; growth hormone releasing factor[1-29] amide; somatotropin releasing hormone[1-29] amide Chemical names CAS number 86168-78-7

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4.1.1 Active pharmaceutical ingredient

Most of the encountered falsified peptides described in case-reports relate to doping purposes or image enhancers. Particularly popular are the growth hormone secretagogues ipamorelin, GHRP-2 and GHRP-6, the anti-obesity peptide AOD-9604, hGH-RH analogues sermorelin and CJC-1295 and the tanning peptides melanotan I and II [96,97]. Melanotan II is mainly used for cosmetic reasons and to a lesser degree for weight loss, aphrodisiac and to counter skin conditions such as rosacea [98,99]. The GHRPs and hGH-RH analogues are used to secrete endogenous hGH. The main advantage of these peptides is the pulsatile release of hGH, which results in less side effects, compared to the steady increase when hGH is exogenously administered. In addition, their popularity might also be explained due to their lower cost compared to full length hGH.

Multiple reports in the literature also mention the seizure of samples containing lyophilized powder without any API present [19,23,25,28,100]. Moreover, multiple reports mention the presence of other API’s or API’s with a different sequence than the proclaimed API [25,32–34,84]. Additionally, in cases where the correct API was present, samples were predominantly found to be under-dosed [26,78]. Another remarkable observation is the presence of sometimes high numbers of samples without any labelling or indication of the respective content [25,28,100]. Moreover, most vials are shipped in envelopes or carton boxes without any medicinal leaflet or instructions on how to administrate these injectable drugs.

Next to the lack of medical guidance, almost all of these peptides are unauthorized drugs. Only GHRP-2 is approved in Japan as a diagnostic agent to assess the functionality of the hypothalamus-hypophysis axis [101], while AOD-9604 has been granted the generally regarded as safe or GRAS-status in the USA [102]. The GRAS-status however doesn’t apply for parenteral administrations. The only authorized peptide, sermorelin, was approved in 1997 but is already withdrawn by the manufacturer from the market due to commercial reasons [103]. Other peptides such as epitalon, melanotan II and CJC-1295 have never successfully completed clinical trials.

Although it is not straightforward to link adverse health or even toxic effects with the administration of these illegal injectables, multiple case-reports have reported sometimes serious side-effects associated with the use of these drugs [104,105]. In the case of the α-melanocyte-stimulating hormone analogues (melanotan I, melanotan II), Habbema et al. [98] have summarized in their review the putative dermatological and non-dermatological effect of these molecules. These reported non-dermatological effects included the development of sympathomimetic toxidrome and reversible encephalopathy syndrome. Moreover, four case-reports have described the formation of melanomas from existing moles during or shortly after the administration of these analogues. Although the growth hormone secretagogues (GHRP-2, GHRP-6 and ipamorelin) have been thoroughly assessed for their activity [96,97], the abuse of these anabolic peptides can lead adverse clinical effects related to the abuse of hGH such as arrhythmia due to the high fatty acid levels, while reported chronic effects are the development of neurological and cardiovascular disorders and acromegaly [106,107]. In addition, since some of these peptides have not completed clinical trials (e.g., epitalon, CJC-1295) their potential health effects are unclear.

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4.1 Falsified synthetic peptide drugs

4.1.1 Active pharmaceutical ingredient

4.1.2 Chemical impurities or contaminants

Potential chemical impurities that might be present are modifications of the peptide itself (API-related impurities) and chemicals related to the synthesis process or contaminations with chemical substances due to low quality reagents and improper handling or storage [37].

The first category comprises all impurities that are related to the original API. Possible modifications are oxidations, reductions, insertions or deletions of amino acids, incorporation of the wrong amino acid(s), truncations, racemization’s and the formation of aggregates [108]. Although impurities such as the insertion or deletion of amino acids and the incomplete removal of protection groups are impurities, which find their origin in the synthesis of these peptides, other impurities such as aggregates, oxidations, deamidations and succimide formations can also be formed due to improper handling and storage conditions. D’Hondt et al. [108] have made a comprehensive summarization of the possible related impurities in peptide medicines. Furthermore, for multiple peptide medicines (e.g., oxytocin, leuprorelin) several impurities have been identified in their respective monographs in the European Pharmacopeia [37,109].

With regard to falsified synthetic peptide drugs, only two studies described the presence of API-related impurities [26,110]. The first study, presented by Breindahl et al. [26] screened multiple samples of melanotan II, acquired via three different suspected illegal internet pharmacies, for API-related impurities via mass spectrometry and UV-detection. For the first two outlets, API-related impurities were found to range between 4.1 and 5.9% while for the third outlet no impurities could be quantified via UV.

The second study, performed by Janvier et al. [110] presented a similar set-up. Briefly, ten peptides were acquired from three different illicit internet shops and analysed for the API and API-related impurities. The study corroborated the findings of Breindahl et al. [26] for melanotan II, but also identified and semi-quantified multiple impurities for the nine other peptides, thereby demonstrating that most API-related impurities varied far more between peptides than between vendors. Particularly for peptides containing the amino acid cysteine (oxytocin and AOD-9604), multiple related impurities such as reductions of the cystine bridge and formation of aggregates via cystine bridges were detected. For some preparations of AOD-9604 the total amount of impurities amounted to 95.5%. Other numerously detected impurities were also related to sites known to be prone to modifications under specific conditions such as aspartimide formation, deamidations and oxidations of Trp and Met. With the exception of one impurity for GHRP-2 (extra Ala insertion) no other clear synthesis artefacts or unremoved protective groups were found [110].

There are multiple examples in the literature were changes in activity and stability were observed when the configuration of a peptide or protein was altered, although it is generally acknowledged that most modifications in peptide drugs do not necessary affect the functionality or stability. Morever, even the change of one amino from the L to D-conformation can change the potency of the respective peptide when coupled to a receptor, while insertions or deletions of amino acids may even transform agonist /-antagonistic functionality. In even worse cases, certain API-related impurities such as aggregates (dimerization) may induce toxic responses or immunological reactions [111,112]. For instance, in the case of the GHRPs, ligand-receptor assays showed that incorporation of an amino acid at position 1 increased the respective equilibrium constant (K)i. Moreover, for some GHRP’s without a C-terminal Lys no Ki could be established (loss of almost all affinity for the receptor). In addition, high Ki-values were measured for GHRP’s with a free carboxylic acid group at the C-terminus instead of the amidated C-term. Thus, an amidated C-term, a 0.9 Da modification, appeared to be relevant for the biological function of a molecule with a molecular weight of more than 800 Da [113]. Another remarkable observation is the (reversible) loss in biological functionality of oxytocin when it is present as a dimer [114]. Reports show that dimers of oxytocin have only 1/500 of the potency of their monomeric counterpart. In addition, these modifications can be induced due to improper handling and storage.

Other peptides such as epitalon, for which almost no biological or toxicological data are available, are another risk regarding the usage of falsified synthetic peptide drugs. No data is for instance available on the biological effect of the major related peptide impurity of epitalon observed by Janvier et al. [110] (succimide formation). In addition, it can be envisaged that impurities of a non-selective receptor agonist such as melanotan II also pose a potential health risks. Reports in literature, namely described melanotan II as peptide with affinities for multiple melanocortin receptors (MC1, MC3, MC4 and MC5) which (partially) regulate different physiological systems such as energy homeostasis, the immune system and the cardiovascular system next to the pigmentary system [115]. Therefore, the effect of related impurities in falsified peptide preparations cannot be neglected in the risk assessment regarding the illicit use of falsified synthetic peptides.

With regard to the second category, the process-related impurities or chemical contaminants information in the literature is quite scarce. No case-reports describe the presence of residual solvents, residual reagents, by-products and scavengers used during the chemical synthesis of peptides in falsified preparations. Only the presence of the polyol mannitol, detected via either LC–MS, GC–MS and/or infrared spectroscopy, was described by multiple reports [24,100,110]. This polyol is presumably used for its cryoprotective properties during freeze-drying or its usage as filler substance or cutting agent [116]. Nonetheless mannitol is generally well tolerated, clinical case reports have documented the occurrence of type I (anaphylaxis) and type IV immune reactions related to the use of mannitol [117,118].

One comprehensive study, described the screening of multiple falsified samples for residual solvents and small molecule contaminants (e.g., other APIs, residual reagents, scavengers) by means of respectively a GC–MS coupled to a headspace autosampler and a GC–MS and LC–MS in parallel. In addition, a screening via ICP-MS was performed for elemental impurities. Only for elemental impurities, multiple samples were found to contain arsenic and lead at concentrations exceding the respective safety thresholds for parenteral injections. Both are class one elemental impurities, human carcinogens, with limited or no functionality in drug products [119]. More specifically lead, a heavy metal, has limited acute toxicity, but accumulates in soft tissues (±10%; brain, spleen, kidneys, liver and bone marrow) and hard tissues (90%; bones and teeth). The chronic exposure to lead can cause adverse effects on the central nervous system, hematopoietic system, the immune system, the renal system and the reproductive system. Moreover, if untreated, lead poisoning has been known to develop into encephalopathy, lethargy, delirium, convulsions, coma and even death [120]. Arsenic, reported to be carcinogenic but not mutagenic, is known through history as a criminal toxin. Arsenic poisoning may result in adverse effects on the skin, respiratory tract, central nervous system, cardiovascular system, liver and immune system (humoral and cellular response). Subsequent speciation performed via HPLC-ICP-MS revealed all As to be inorganic As, which is generally deemed to be the most toxic form [110], although it’s toxicity is also primarily dependent on the manner of administration, the rate of absorption and excretion, metabolism (phase I and phase II biotransformation reactions) and the affected tissue(s) [121,122].

4.1.3 Biological impurities or contaminants

Hence most of these illegal preparations are purposed for parenteral injection (a minority is also available for nasal administration), it stands to reason that the sterility of these preparations is mandatory. Although contaminations with fungi and/or bacteria are less likely in chemically synthesized peptides than in biologicals, due to the presence of solvents (e.g., Triton-X) and the continuous use of non-physiological pH (during coupling and deprotecting functional groups), contaminations after the actual synthesis may present a critical point. Indeed, one study identified a haemolytic strain of Bacillus cereus in falsified synthetic peptide samples [123]. The study was based on the methodology described by the European pharmacopeia and employed MALDI-TOF biotyper technology as a screening tool and polymerase chain reactions (PCR) to corroborate the presence of the virulent B. cereus strains. Although B. cereus is generally known for its role as a food pathogen, their involvement in morbidity and mortality in intravenous drugs users and patients with invasive treatments has been described in the literature [124]. The documented spectrum of adverse health effects ranges from fulminant bacteraemia, infections with involvement of the central nervous system, pneumonia to necrotizing cutaneous infections resembling gas-gangrene.

Although no clinical cases have been reported regarding falsified synthetic peptides, severe cases of bacteraemia have already been reported for classical injectable drug users (e.g., narcotics) due to contaminated injection syringes [124]. Accordingly, falsified synthetic peptide drugs are sometimes delivered with injection material and bacteriostatic water which may or may not be bacteriostatic and/or devoid of microorganisms.

Additionally, also related virulent substances such as endotoxins and exotoxins may be present in these samples. Although there are no reports in the literature regarding contaminations of falsified synthetic peptides with toxins, it stands to reason that also pyrogenic substances (e.g., lipopolysaccharides and lipoteichoic acid) could be present in these preparations due to sterility issues. Pharmaceutical companies are obliged to incorporate quality controls which demonstrate the absence of these pyrogenic substances due to the possible adverse health effects such as fever, hypotension, multiple organ failure and, in severe cases, septic shock and death [125]. Accredited analytical techniques used to assess the putative pyrogenic potential of parenteral preparations are the rabbit pyrogen test (RPT), the Limulus amebocyte lysate assay (LAL) and its derivatives and the monocyte activation test (MAT) [125].

Other possible biological contaminations such as DNA, host cell proteins (HCP) and viral contaminations are unlikely to occur, but cannot be excluded seen the remarkable contaminants found in falsified samples in the past.

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Indications

Empty Cell Value Units Prep. and route of admin. Reference Comments Diagnosis of growth hormone deficiency Dosage 1 mgkg-1 Sermorelin acetate injection subcutaneously Sermorelin is administered in the morning after an overnight fast. Growth hormone therapy should be discontinued 1–2 weeks before test. A plasma GH response of 28+ 15 ngml-1 within 30 min demonstrates that the pituitary somatotrophs are intact. GH deficiency may still be the result of hypothalamic dysfunction in the presence of an intact somatotroph (http://www.bnf.org/). Treatment of growth hormone deficiency Dosage 30 mgkg-1 Sermorelin acetate injection subcutaneously Thorner et al. (1996) Sermorelin is administered at bedtime in the treatment of growth hormone deficiency.

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3.2.1.1 Peptide agonists

The first described agonist for MRGPRX2 was cortistatin-14 (CST-14), a neuropeptide consisting of 14 amino acids, which is expressed in DRG (Robas et al., 2003; Volante et al., 2008; Zhang et al., 2005). CST-14 showed an EC50 value of 25 nM in calcium assays performed in HEK cells recombinantly expressing MRGPRX2 (X2-1, Table 8). CST-17, a peptide that is three amino acids longer than CST-14, also activated MRGPRX2 showing an EC50 value of 99 nM in the same test system (X2-2, Table 8). Another closely related neuropeptide, somatostatin-14 (SS-14), displayed an EC50 value of 780 nM (X2-3, Table 8) (Robas et al., 2003). Somatostatin-derived peptides were reported to activate MRGPRX2 with potencies ranging from 200 nM to 10 μM (X2-4 to X2-8, Table 8) (Robas et al., 2003) In addition, CST-14 was found to induce MRGPRX2-dependent MC activation, degranulation, and cytokine release with potencies in the lower micromolar range (X2-1, Table 8) (John et al., 2021; Lafleur et al., 2020). This peptide also binds to all somatostatin receptors without subtype selectivity (in subnanomolar concentrations) (Siehler, Nunn, Hannon, Feuerbach, & Hoyer, 2008; Siehler, Seuwen, & Hoyer, 1999). However, it may have its own receptor since CST-14 induced some physiological functions that somatostatin-14 did not (Spier & de Lecea, 2000). Another well-studied agonist of MRGPRX2 is substance P (SP), a neuropeptide that acts as a basic secretagogue (Robas et al., 2003). Its MRGPRX2-activating activity was observed in calcium assays, β-arrestin assays, and CD34+ stem cell-derived mature human MC degranulation assays showing EC50 values that ranged from 100 nM to 20 μM (X2-9, Table 8) (Lafleur et al., 2020; Lansu et al., 2017; McNeil et al., 2015; Robas et al., 2003; Tatemoto et al., 2006). SP binds with higher potency to the neurokinin-1 receptor. However, SP-induced MC activation is specifically mediated by MRGPRX2 (Lu, Kulka, & Unsworth, 2017). Further neuropeptides were reported to activate MRGPRX2 in calcium assays and induce MC degranulation, e.g. vasoactive intestinal peptide (X2-10, Table 8) and its homolog pituitary adenylate cyclase-activating peptide (X2-11 and X2-12, Table 8), dynorphins A and B (X2-13 to X2-16, Table 8) and BAM peptides (X2-17 to X2-19, Table 8) (Lafleur et al., 2020; Lansu et al., 2017; Robas et al., 2003; Tatemoto et al., 2006). In addition, neuropeptide FF (X2-20, Table 8), hemokinin-1 (X2-21, Table 8), oxytocin (X2-22, Table 8), and vasopressin (X2-23, Table 8) were reported to activate MRGPRX2 in calcium assays (Robas et al., 2003). All the aforementioned neuropeptides, except CST-14, showed potencies at MRGPRX2 in the range of 100 nM to 40 μM in calcium and β-arrestin assays, respectively. These are relatively high effective concentrations for endogenous peptide ligands of GPCRs (Foster et al., 2019). Recently, CST-14 was shown to induce human skin MC degranulation in vivo and ex vivo (Kolkhir et al., 2022). Besides neuropeptides, another endogenous peptide, proadrenomedullin N-terminal peptide-12 (PAMP-12) activated MRGPRX2 in calcium, cyclic adenosine monophosphate (cAMP) inhibition, and β-arrestin assays with EC50 values ranging from 40 nM to 3 μM (X2-24, Table 8) (Kamohara et al., 2005; Lansu et al., 2017). PAMP-20, a PAMP 12-derivative containing nine additional amino acids on the C-terminus, showed lower potency than PAMP-12 in both cAMP and β-arrestin assays using CHO cells recombinantly expressing MRGPRX2 (X2-25, Table 8) (Kamohara et al., 2005). Kallidin, a bradykinin homolog cleaved from kininogen by kallikreins, caused a concentration-dependent calcium signal in HEK293T cells recombinantly expressing MRGPRX2 with an EC50 value of 11.5 μM (X2-26, Table 8) (Lansu et al., 2017) Endogenous secretagogues that bind to MRGPRX2 are typically less than 4000 Da in size and contain cationic amino acid residues like arginine (R) and lysine (K), and hydrophobic, aromatic amino acids such as tryptophan (W), tyrosine (Y) and phenylalanine (F). However, they otherwise only display moderate structural similarity among each other (see Table 8 for peptide sequences) (Kamohara et al., 2005; Robas et al., 2003; Tatemoto et al., 2006). MRGPRX2 is also activated by proteins and protein fragments as detected in calcium assays, e.g. by platelet factor 4, albumin(407-423), chaperonin-10(1-20), 2,3-cyclic nucleotide 3-phosphodiesterase(1-17), and tropomyosin(1-12), although with moderate potency (X2-27 to X2-33, Table 8) (Karhu et al., 2017b; Tatemoto et al., 2018). Eosinophil granule proteins such as eosinophil peroxidase and major basic proteins were previously known to cause MC degranulation by a mechanism that was unknown at that time (Patella et al., 1996). The discovery of MRGPRX2 has now provided a mechanism by which these peptides induce MC degranulation (Ogasawara, Furuno, Edamura, & Noguchi, 2020). Eosinophil peroxidase and major basic proteins caused significant histamine release from skin-derived cultured MCs, at 0.39 μM and 0.72 μM, respectively (X2-34 and X2-35 , Table 8). MRGPRX2 was reported to be the MC receptor that mediates the effect of host defense peptides (HDPs), including MC degranulation and cytokine production (Subramanian, Gupta, et al., 2011). HDPs, or antimicrobial peptides, are cationic short peptides, such as the human cathelicidin LL-37 and β-defensins, that play a role in innate immunity, in addition to their antimicrobial activity (Mookherjee, Anderson, Haagsman, & Davidson, 2020). The human cathelicidin LL-37 (LL-37) was found to induce MRGPRX2-dependent MC degranulation (at 1 μM concentration in LAD2 cells), chemokine generation and chemotaxis (at 3 μM concentration in MCs recombinantly expressing MRGPRX2), and calcium signaling (at 10 μM concentration in LAD2 cells) (X2-36, Table 8) (Subramanian, Gupta, et al., 2011). LL-37 displayed an EC50 value of 65.3 μM in calcium assays in HEK293 cells expressing MRGPRX2, and catestatin, magainin 2 and indolicidin showed EC50 values of 28.3 μM, 28.1 μM and 2.94 μM, respectively (X2-37 to X2-39, Table 8) (Tatemoto et al., 2006). Protegrin-1 (PG-1), also belonging to the cathelicidin family, activated MRGPRX2 and caused degranulation of MC, namely rat basophilic leukemia MC (RBL-2H3) recombinantly expressing MRGPRX2, when used at a concentration of 1.39 μM (X2-40, Table 8) (Gupta, Kotian, Subramanian, Daniell, & Ali, 2015). Peptide 17 (P17) is a short HDP that was screened at 314 GPCRs (in the tango-presto β-arrestin assay) showing specific activation of MRGPRX2 at a concentration of 100 nM (X2-41, Table 8) (Duraisamy et al., 2022). P17 was further tested at MRGPRX2 in different assay systems, e.g. calcium assays and MC degranulation assays, exhibiting potency in the lower micromolar concentration range. Homology modeling based on the κ-opioid receptor (PDB ID: 6B73) as a template was applied to locate the pharmacophore region of peptide P17 (sequence LFKEILEKIKAKL). Docking studies predicted that the binding site of MRGPRX2 is shallow and P17 interacts with the amino acid residues Y89, F172, S173, D174, G175, W250, K251, and S253 of MRGPRX2 (see Fig. 3A ). In particular, the K8 moiety of P17 may specifically form cation-π interactions with the F172 residue of MRGPRX2. The latter interaction was confirmed by exchanging K8 of P17 for alanine which abolished P17 activity as shown in β-arrestin and MC degranulation assays. P17 activity was also abolished at an MRGPRX2 mutant in which phenylalanine-172 was replaced by alanine (F172A) (Duraisamy et al., 2022).

Fig. 3. MRGPRX2 snake plots: A. amino acids of MRGPRX2 predicted to interact with P17 (sequence LFKEILEKIKAKL) highlighted in red. B. Molecular docking using the human C5a anaphylatoxin chemotactic receptor 1 (PDBID 5O9H) as a template revealed two binding pockets of MRGPRX2. One postulated binding site of MRGPRX2 comprised of F239, Y113, T187, S268, N271, G236, G116, F232, L191, and I192, was proposed to interact with the agonist C48/80 (amino acids shown in blue). A second binding pocket proposed to bind genistein is formed by C258, P262, W179, N85, V88, C95, Y89, and T106 (labeled in green) (Harding et al., 2022; Kooistra et al., 2021; Kumar et al., 2020; Munk, Harpsøe, Hauser, Isberg, & Gloriam, 2016; Pándy-Szekeres et al., 2022).

The human β-defensins (hBD) are cationic peptides that are mainly produced by epithelial cells and platelets. Concentrations of 0.3 - 3 μM of hBD2 and hBD3 caused MC degranulation in LAD2, CD34+ MC and RBL cells (expressing MRGPRX2) (X2-42 and X2-43, Table 8) (Subramanian et al., 2013). Retrocyclin-100, is a synthetic humanized θ-defensin with antimicrobial and antiviral effects, in particular anti-HIV activity. This peptide caused RBL-2H3 cell (expressing human MRGPRX2) degranulation at a concentration of 1.54 μM (Gupta, Subramanian, & Ali, 2016). MRGPRX2 is activated by other cationic host defense, antimicrobial peptides such as the ones derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) (X2-45, Table 8), and modified peptide-30/5C (AG-30/5C) (X2-46, Table 8) (Niyonsaba et al., 2020; Roy, Ganguly, Haque, & Ali, 2019). Interestingly, AG-30/5C was reported to act as a G protein-biased agonist on MRGPRX2 since it did not induce β-arrestin recruitment (X2-46, Table 8) (Lansu et al., 2017; Roy et al., 2019). MRGPRX2 is activated by several peptides that interact with immune targets. For example, E7, a C-terminal synthetic analog of the complement factor C3a that activates the complement C3a receptor 1, induced MC degranulation via MRGPRX2 activation at 1 μM concentration (X2-47, Table 8) (Kashem et al., 2011). The synthetic peptide PMX-53, a potent complement C5a receptor 1 antagonist, caused degranulation of LAD2 and CD34+ cell-derived MCs when used at concentrations higher than 30 nM (X2-48, Table 8) (Subramanian et al., 2011). Several cationic peptidic drugs display an off-target activation of MRGPRX2 and therefore cause MC degranulation. For example, angiopeptin, a synthetic cyclic octapeptide analog of somatostatin that inhibits the production of growth hormone (used for acromegaly treatment), was found to cause MC degranulation (EC50 value of 7 μM) and induce calcium signals via MRGPRX2 activation (X2-49, Table 8) (Lafleur et al., 2020; Tatemoto et al., 2006). Angiopeptin is a potent inhibitor of growth hormone release (in the lower nanomolar range), however, it is administered as an intramuscular injection that leads to sufficient concentrations at the site of injection to activate MRGPRX2 followed by subsequent MC degranulation. Octreotide, another somatostatin analog that has the same indications as angiopeptin, was also reported to activate MRGPRX2 and cause MC degranulation (EC50 value of 35 μM) (X2-50, Table 8) (Lafleur et al., 2020; McNeil et al., 2015). Since subcutaneous injection is the usual route of octreotide administration, MRGPRX2 activation may explain the reported side effects, namely, pain and irritation at the site of injection (Battershill & Clissold, 1989). Interestingly, therapeutic peptides that cause the opposite therapeutic effect than angiopeptin and octreotide were also reported to activate MRGPRX2. In particular, sermorelin (a synthetic growth hormone) and hexarelin (a synthetic growth hormone-releasing peptide) were reported to activate MRPGRX2 with potencies in the lower micromolar range (X2-51 and X2-52, Table 8) (Lafleur et al., 2020; Lansu et al., 2017; McNeil et al., 2015). Sermorelin and hexarelin are administered subcutaneously, and skin irritation at the site of injection, possibly mediated by MRGPRX2-induced MC degranulation, was reported as a side effect (Prakash & Goa, 1999). The gonadotropin-releasing hormone antagonist cetrorelix, and the agonist leuprolide were both found to activate MRGPRX2 (Lafleur et al., 2020; Lansu et al., 2017; McNeil et al., 2015). These two peptide drugs were tested thoroughly on MRGPRX2 using calcium assays in both HEK and CHO cells, in β-arrestin assays, and in MC degranulation assays; they showed moderate potencies in the 100 nM to 10 μM range (X2-53 and X2-54, Table 8). Icatibant, a selective antagonist of bradykinin B2 receptors, also displayed an off-target activation of MRGPRX2. Interestingly, icatibant was reported to be a G protein-biased MRGPRX2 agonist since it failed to induce β-arrestin recruitment (X2-55, Table 8) (Alkanfari, Gupta, Jahan, & Ali, 2018; Lansu et al., 2017; McNeil et al., 2015). Exogenous toxins from bacteria (Staphylococcus δ-toxin, X2-56, Table 8) (Azimi, Reddy, & Lerner, 2017), insects (mastoparan, MC degranulating peptide, and melittin X2-57 to X2-59, Table 8), and even frogs (granuliberin R, X2-60, Table 8) (Tatemoto et al., 2006) were found to activate MRGPRX2 thereby inducing MC degranulation. Finally, endogenous proteases such as cathepsin S (X2-61, Table 8) (Reddy et al., 2015), and exogenous enzymes, e.g. mucunain, were found to activate MRGPRX2 (X2-62, Table 8) (Reddy et al., 2018). A protease from Dermatophagoides pteronyssinus, designated Der p 1, was reported to induce calcium signals in HeLa cells recombinantly expressing MRGPRX1 when used at a concentration of 2 μM (see Table 4) (Reddy & Lerner, 2017). Der p 1 did not activate MRGPRX2 overexpressed in HeLa cells in calcium assays. Its predicted cleavage products from the N-terminus of MRGPRX1 were synthesized and tested at MRGPRX1 and MRGPRX2 in calcium assays. Interestingly, three fragments selectively activated MRGPRX2 in calcium assays at 2 μM concentration (X2-63 to X2-65, Table 8) (Reddy & Lerner, 2017). Overall, peptidic MRGPRX2 agonists are diverse but mostly activate other targets with higher potencies. They feature common motifs such as alternating basic and aromatic amino acids. Thus, any peptide with these amino acid residues might activate MRGPRX2.

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3 Modulators, pharmacology, and signaling

3.1 The human MAS-related G protein-coupled receptor X1

The human MRGPRX1 was the first receptor of the primate-specific subfamily for which an agonist, namely bovine adrenal medulla 8-22 peptide (BAM8-22), was identified (Lembo et al., 2002). Meanwhile, several natural and synthetic agonists, antagonists, and allosteric modulators have been described (Table 4-6). This receptor still remains an orphan receptor to date, since its proposed agonist, BAM8-22, has not been confirmed to be the actual physiological agonist of MRGPRX1 (Alexander et al., 2021).

Table 4. MRGPRX1-activating peptides.

Compound No. Name Peptide sequence EC50(μM) Cells stably expressing human MRGPRX1 (assay)a,b References Endogenous peptides X1-1 Bovine adrenal medulla 22 peptide (BAM-22) YGGFMRRVGRPEWWMDYQKRYG 0.016 HEK293 (Ca2+) (Lembo et al., 2002) 0.800 HEK293 (R-SAT) (Malik et al., 2009) X1-2 Bovine adrenal medulla 8-22 peptide (BAM8-22) VGRPEWWMDYQKRYG 0.014 HEK293 (Ca2+) (Lembo et al., 2002) 0.025 HEK293 (Ca2+) (Burstein et al., 2006) 0.04 HEK293 (Ca2+) (Tatemoto et al., 2006) 0.008-0.050 CHO (Ca2+) (Kunapuli et al., 2006) X1-3 Vasoactive intestinal peptide (VIP) HSDAVFTDNYTRLRQMAVKKYLNSILN 17.7 HEK293 (Ca2+) (Tatemoto et al., 2006) X1-4 Indolicidin ILPWKWPWWPWRR-NH2 7.59 HEK293 (Ca2+) (Tatemoto et al., 2006) X1-5 Cathelicidin (LL-37) LLGDFFRKSKEKIGKEFRIVQRIKDFLRNLVPRTES 96.4 HEK293 (Ca2+) (Tatemoto et al., 2006) X1-6 Catestatin SSMKLSFRARAYGFRGPGPQL 4.57 HEK293 (Ca2+) (Tatemoto et al., 2006) X1-7 Hemoglobin β-chain(33-42) LVV-hemorphin-7 LVVYPWTQRF 8.3 HEK293 (Ca2+) (Karhu et al., 2017a) X1-8 Hemoglobin β-chain(34-42) VV-hemorphin-7 VVYPWTQRF 4.5 HEK293 (Ca2+) (Karhu et al., 2017a) X1-9 Hemoglobin β-chain(36-42) PWTQRF >10 HEK293 (Ca2+) (Karhu et al., 2017a) X1-10 Hemoglobin β-chain(33-40) LVVYPWTQ >10 HEK293 (Ca2+) (Karhu et al., 2017a) X1-11 P60 (part of C5orf29) GIGCVWHWKHRVATRFTLPRFLQ 0.3 CHO-K1 (Gα16) (Ca2+) (Shemesh et al., 2008) Exogenous peptides and proteases X1-12 Conopeptide (CNF-Tx2) HSGILLAWSGPRNRFVRI 0.54 KNRK cells (Ca2+) (Espino et al., 2018) X1-13 Conopeptide CNF-Vc1 HSGFLLAWSGPRNRFVRF 1.8 KNRK cells (Ca2+) (Espino et al., 2018) X1-14 IPDef1 (IP-O) [C4 -C25] [C11-C33] [C15-C35] disulfide bond GFGCPFNQGACHRHCRSIGRRGGYCAGLFKQTCTCYSR 4.32 HEK293T (Ca2+) (Li et al., 2021) X1-15 Granuliberin R FGFLPIYRRPAS NH2 38.2 HEK293 (Ca2+) (Tatemoto et al., 2006) X1-16 Der p1 216-amino acid sequence (0.1)c HEK293 (Ca2+) (Reddy & Lerner, 2017) X1-17 Mucunain 332-amino acid sequence 0.50 HEK293 (Ca2+) (Reddy et al., 2018)

a Ca2+: Ca2+ assays b R-SAT: receptor selection and amplification technology, an assay that measures cell growth in response to the addition a compound over several days c Only a single concentration was tested

Table 5. Small molecule MRGPRX1 agonists and an allosteric modulator.

Compound No. Name Structure EC50(μM) Cells stably expressing human MRGPRX1a,b References X1-18 Compound 36 0.0063 HEK293 (Ca2+) (Wroblowski et al., 2009) X1-19 Compound 14 0.316 NIH3T3 (R-SAT) (Malik et al., 2009) X1-20 Compound 5e (best agonist in the series) 0.29 HEK293 (Ca2+) (Prchalová et al., 2019) X1-21 Compound 11g 0.10 HEK293 (Ca2+) (Prchalová et al., 2019) X1-22 Compound 16 (best agonist in the series) 0.05 HEK293 (Ca2+) (Prchalová et al., 2019) X1-23 Chloroquine 297 HEK293 (Ca2+) (Liu et al., 2009) X1-24 ML382 (PAM) 0.19c HEK293 (Ca2+) (Wen et al., 2015)

a Ca2+: Ca2+ assays b R-SAT: receptor selection and amplification technology, an assay that measures cell growth in response to the addition of a compound over several days c in the presence of 10 nM BAM8-22

Table 6. MRGPRX1 antagonists.

Compound No. Name Structure IC50(μM) Cells stably expressing human MRGPRX1 References X1-25 1 0.050b CHO (beta-lactamase assay) (Kunapuli et al., 2006) 0.103b CHO (Ca2+)a (Kunapuli et al., 2006) 0.050c CHO (radioligand whole-cell binding assay) (Kunapuli et al., 2006) 0.320b U2OS-β-arrestin-GFP stable cells (receptor tracking assay) (Kunapuli et al., 2006) X1-26 27k 0.014b HEK293 (Ca2+)a (Bayrakdarian et al., 2011)

a Ca2+: Ca2+ mobilization assays b In the presence of BAM8-22 (at its EC70 concentration) c Radioligand binding vs. [3H]BAM8-22 (10 nM)

3.1.1 MRGPRX1 agonists and positive allosteric modulators

MRGPRX1 agonists can be classified based on their structure and origin into three categories: endogenous peptides, exogenous peptides, and small molecules.

3.1.1.1 Endogenous peptides

Following its expression in DRG, MRGPRX1 was found to be activated by opioid-related peptides including γ1-melanocyte-stimulating hormone (MSH), γ2-MSH, and BAM-22 (Lembo et al., 2002). Further pharmacological characterization revealed that BAM-22 (X1-1, Table 4) was the most potent MRGPRX1 agonist, which, however, also activates the δ-opioid receptor. Its opioid receptor activity is conserved within the Met-enkephalin motif (YGGFM), which is located at the N-terminus of BAM-22. In contrast, its C-terminal fragment (BAM8-22) potently activated MRGPRX1, stably expressed in HEK293 cells, in a concentration-dependent manner, with an EC50 value of 14 nM (X1-2, Table 4) (Lembo et al., 2002). BAM8-22 was validated as an agonist for MRGPRX1 by several groups, consistently giving sub-micromolar potencies (X1-2, Table 4) (Lembo et al., 2002) (Burstein et al., 2006; Kunapuli et al., 2006; Solinski et al., 2014). Although the required machinery to process BAM8-22 is present in the brain, it remains unclear whether this peptide could be produced in the vicinity of DRG. Hence, MRGPRX1 remains an orphan receptor (Alexander et al., 2021; Solinski et al., 2014). Vasoactive intestinal peptide (VIP), a peptide hormone, activated MRGPRX1, expressed in HEK cells, in a concentration-dependent manner showing an EC50 value of 17.7 μM in calcium assays (X1-3, Table 4) (Tatemoto et al., 2006). The same group also demonstrated that the host defense peptides (HDPs) indolicidin, cathelicidin, and catestatin activate MRGPRX1 with potencies in the micromolar range, determined in the same assay system (X1-4 to X1-6, Table 4). Two other endogenous peptides, leucine-valine-valine-hemorphin-7 (LVV-H7), and valine-valine-hemorphin-7 (VV-H7), were reported to activate MRGPRX1 with potencies in the lower micromolar range (8.3 μM and 4.5 μM, respectively) (X1-7 and X1-8, Table 4) (Karhu et al., 2017a). Both peptides are natural proteolytic products of the hemoglobin β-chain that were described previously to activate opioid receptors (Yukhananov, Glämsta, & Nyberg, 1994). Despite the similar functions of hemorphins (LVV-H7, VV-H7) and BAM-22, they do not share any significant sequence similarity. Physiologically, hemorphins are produced by macrophage proteases in the spleen. They may also be produced from the hemoglobin β-chain in nervous tissues by proteases. In pathological conditions e.g. upon cerebrovascular bleeding, LVV-H7 was detected in the human cerebrospinal fluid (CSF) at concentrations of several hundred nM (Glämsta, Meyerson, Silberring, Terenius, & Nyberg, 1992). The EC50 value of the most potent hemorphins, VV-H7, was 4.5 μM determined in calcium assays in HEK293 cells recombinantly expressing MRGPRX1 (Karhu et al., 2017a). Any further truncation of this 9-amino acid peptide abolished activity (X1-9 and X1-10, Table 4). Although the reported peptides are fragments of the hemoglobin β-chain, intact hemoglobin did not activate MRGPRX1 (Karhu et al., 2017a). Previously, in a deorphanization campaign, ligand-like peptides from the human proteome were identified by machine learning algorithms (Shemesh et al., 2008). One of these peptides, P60, induced calcium mobilization in CHO-K1 cells transfected with MRGPRX1 and the promiscuous Gα15/16 protein in a concentration-dependent manner showing an EC50 value of 0.3 μM (X1-11, Table 4) (Shemesh et al., 2008). The same peptide also activated MRGPRX2 with an EC50 value of 1 μM. P60 is a potential fragment of a transmembrane growth factor receptor-bound protein 2-binding adapter protein; however, the 23-amino acid fragment P60 has so far not been detected naturally.

3.1.1.2 Exogenous peptides

Exogenous peptides derived from different organisms were found to activate MRGPRX1 as well. For example, the venoms from the snails Conus victoriae and Conus textile contain RF-amide conopeptides that activated either MRGPRX1 (CNF-Tx2), mouse MRGPRC11 (CNF-Sr1), or both (CNF-Vc1) (Espino et al., 2018). The RF-amide peptide family is characterized by possessing an arginine-phenylalanine-NH2 motif on the peptide’s C-terminus. The most potent conopeptide was CNF-Tx2 with an EC50 value of 0.54 μM determined in rat kidney (KNRK) cells transfected with MRGPRX1 using a calcium mobilization assay (X1-12 and X1-13, Table 4) (Espino et al., 2018). This activity translated into in vivo effects, namely scratching behavior after intradermal cheek injection of CNF-Tx2 (10 nM) and CNF-Vc1 (10 nM), as observed in MRGPRX1-humanized mice (hMRGPRX1; Mrgpr-cluster knockout) (Espino et al., 2018). In addition to peptides expressed by cone snails, (Ixodes persulcatus) was reported to activate MRGPRX1 namely Ixodes persulcatus defensin 1 (IPDef1), isolated from its saliva (Li et al., 2021). This peptide was resynthesized and tested at different MRGPRs using calcium imaging in transfected HEK293T cells (Li et al., 2021). IPDef1 in its folded oxidized form (IP-O), but not its reduced form, activated MRGPRX1 and mouse MRGPRC11 with potencies in the lower micromolar range (MRGPRX1: EC50 = 4.32 μM, MRGPRC11: EC50 = 4.63 μM) (X1-14, Table 4) (Li et al., 2021). Additionally, IP-O slightly activated MRGPRX2 and the mouse MRGPRA3 but not MRGPRX3 and MRGPRX4 (Li et al., 2021). Granuliberin R, an MC-degranulating peptide originally isolated from the skin of frogs, induced calcium signals when tested in HEK293 cells recombinantly expressing MRGPRX1 with an EC50 value of 38.2 μM (X1-15, Table 4) (Tatemoto et al., 2006). A major allergen from the house dust mite Dermatophagoides pteronyssinus known as Der peptidase1 (Der p1, a cysteine protease) induced significant calcium mobilization in HeLa cells expressing MRGPRX1, but not in cells expressing other MRGPRX receptors (X1-16, Table 4) (Reddy & Lerner, 2017). Furthermore, 100 nM of Der p1 was sufficient to induce the release of the pro-inflammatory cytokine interleukin 6 (IL-6) from the same cells (Reddy & Lerner, 2017). The protease Der p1 also activated mouse receptors, namely MRGPRC11 and MRGPRA3. However, the active cleavage product(s) of Der p1 action, assumed to be an endogenous peptide, is/are still unknown. The same was reported for other proteases, such as mucunain, which showed MRGPRX1 activation in calcium assays without identifying the active cleavage product that caused the effect (X1-17, Table 4) (Reddy, Azimi, Chu, & Lerner, 2018; Tatemoto et al., 2006).

3.1.1.3 Small molecule agonists and positive allosteric modulators

High-throughput screening (HTS) campaigns using cells expressing human MRGPRX1 led to the discovery of various agonistic small molecule scaffolds (Table 5). MRGPRX1 is at present the best investigated MRGPRX with regard to the number of discovered ligands and known structure-activity relationships (SARs). A series of pyridazinone derivatives was synthesized and optimized starting from a screening hit (Wroblowski et al., 2009). Initial SARs of this scaffold led to methylbenzylpyridazinone as a core structure bearing an amide-linked substituent (see, e.g., X1-18, Table 5). A model of the MRGPRX1 was built by homology to the bovine rhodopsin crystal structure (PDB code: 1f88), and docking studies were performed (Wroblowski et al., 2009). The following SARs were established: For an interaction with the acidic amino acid residues E157 and D177 of the receptor protein substitution at the 4-position of the pyridazinone ring with a positively charged moiety, such as piperazine, appeared to be essential. Due to key interactions formed with the aromatic residues Y99, Y106, F232, and F236, amide-substitution with a linear, extended, aromatic, and lipophilic residue is highly beneficial. This SAR-guided optimization process culminated in the development of a potent selective agonist, compound 36 (X1-18, Table 5), for which an EC50 of 6.3 nM was determined in calcium assays using HEK293 cells stably transfected with MRGPRX1 (Wroblowski et al., 2009). Another HTS campaign of 250,000 small molecules yielded tetracyclic benzimidazoles as agonists of both MRGPRX1 and MRGPRX2 (Malik et al., 2009). Further development investigating a focused tetracyclic benzimidazole library led to the discovery of an MRGPRX1-selective agonist with an EC50 of 316 nM (X1-19, Table 5) determined using the receptor selection and amplification technology (R-SAT), which measures an increase or decrease in cell growth (regardless of the native signaling pathway). MRGPRX1 selectivity versus MRGPRX2 was achieved by introduction of phenylpiperazine to the benzimidazole core structure (see Table 5) (Malik et al., 2009). Such a beneficial impact of a basic piperazine residue for MRGPRX1 interaction was also reported by Wroblowski et al. Two further lead structures were discovered in an HTS approach using calcium assays and HEK293 cells stably expressing human MRGPRX1, benzamidines and 1-aminoisoquinolines (Prchalová et al., 2019). SAR analysis and optimization yielded benzamidines 5e (X1-20, Table 5) and 11g (X1-21, Table 5), and aminoisoquinoline 16 (X1-22, Table 5) with EC50 values of 0.29, 0.10, and 0.05 μM, respectively (Table 5). All of the potent MRGPRX1 small molecule agonists feature peptide-like or peptidomimetic structures with amide or sulfonamide linkers, basic moieties (e.g. amidines), and extended linear structures. Besides the aforementioned drug-like molecules, the Food and Drug Administration(FDA)-approved drug chloroquine (X1-23, Table 5) was found to activate MRGPRX1 (Liu et al., 2009) in DRG neurons of humanized mice (hMRGPRX1; Mrgpr-cluster knockout), specifically generating action potentials in response to chloroquine treatment as measured by whole-cell patch-clamp recordings. However, the potency of chloroquine was very low when tested in calcium assays on HEK293 cells stably transfected with human MRGPRX1 (EC50 = 298 μM) (X1-23, Table 5) (Liu et al., 2009). Besides agonists, potent and selective positive allosteric modulators (PAMs) have been identified for MRGPRX1 possessing an arylsulfonamide structure (Wen et al., 2015). The best PAM, ML382 (X1-24, Table 5), displayed an EC50 value of 0.19 μM in calcium assays in the presence of the agonist BAM8-22, increasing its effect from 100% to 190% (Wen et al., 2015).

3.1.2 MRGPRX1 antagonists

Potent antagonists for MRGPRX1 have also been described. The first identified antagonists share a 2,3-disubstituted azabicyclooctane scaffold (Kunapuli et al., 2006). This compound series was identified by an ultra-HTS using a beta-lactamase gene reporter assay and a stable CHO-hMRGPRX1 cell line (see Table 6). All of the potent antagonists of this series feature a basic and lipophilic structure with a diphenylmethyl moiety at C2 of the bridged heterocyclic ring system, and a short linker attaching, for example, a substituted aromatic ring at C3. The linker can be diverse, including ethers, secondary amines, esters, ketones, carbamates, and ureas. The substitution at C3 strongly affects the compounds' antagonistic potency with various functional groups tolerated in this position. The best antagonist of this series had an IC50 value of 50 to 320 nM as determined in different assay systems (X1-25, Table 6). This antagonist had a 2-carbon linker (an α,β-unsaturated ketone or enone) attached to an ortho-methoxyphenyl residue (X1-25, Table 6) (Kunapuli et al., 2006). Later on, these type of compounds were found to block the rat MRGPRC as well (He et al., 2014). 2,4-Diaminopyrimidine derivatives were developed as MRGPRX1 antagonists starting from a quinazoline core structure, and evaluated in calcium assays using HEK293 cells recombinantly expressing MRGPRX1. Structural simplification of the bicyclic quinazoline resulting in pyrimidine analogs led to MRGPRX1 antagonists with improved solubility while maintaining potency. All potent 2,4-diaminopyrimidine derivatives share a peptidomimetic structure substituted with a basic moiety, in which position C2 of the pyrimidine ring is substituted with a 4-chlorophenylethylamino residue, while a broad range of functional groups are tolerated at C4. Whereas a significant decrease in activity was reported in case of methylation at C5 of X1-26 (see Table 6), substitution at C6 with a methyl group was tolerated. The most potent and selective antagonist among these derivatives inhibited the BAM8-22-induced effects with an IC50 value of 14 nM determined in calcium assays (X1-26, Table 6) (Bayrakdarian, Butterworth, Hu, Santhakumar, & Tomaszewski, 2011).

3.1.3 MRGPRX1 signaling

The signaling pathways of MRGPRX1 were investigated using in vitro assays involving heterologous expression in cell lines and rodent primary neuronal cell cultures, in addition to in vivo transgenic mouse models. Since the discovery of MRGPRX1, it was suggested to be coupled to Gq/11 proteins because calcium mobilization induced by the agonist BAM8-22 in recombinant HEK cells was insensitive to the Gi/o protein inhibitor pertussis toxin (PTx). In contrast, when the human receptor was overexpressed in rat neuronal cells, the BAM8-22-induced effect was PTx-sensitive indicating coupling to Gi/o proteins and potential Gβγ-mediated phospholipase C (PLC) activation. An electrophysiological study of neurons from MRGPRX1-humanized mouse DRG confirmed that BAM8-22 induced action potentials by PTx-sensitive and PLC-mediated pathways (sensitive to the PLC inhibitor U73122) in a Gβγ-dependent manner (gallein-sensitive) (see Fig. 2 for an overview of MRGPRX1 signaling pathways) (Tseng, Zheng, Li, & Dong, 2019). These differing observations may be due to the different cell systems employed, raising the question whether the suggested pathways are actually functional for MRGPRX1 natively expressed in human DRG. Recently, it was found that calcium signals induced by BAM8-22 were almost completely abolished by the specific Gαq/11 inhibitor YM-254890 in peripheral neuronal equivalents (PNEs) derived from human dental pulp stem cells natively expressing MRGPRX1 (McMillan et al., 2021). Using a chimeric G protein-based tumor growth factor α (TGFα) shedding assay, the MRGPRX1 activated by BAM-22 was reported to promiscuously couple to almost all Gα proteins including Gαs, Gαi/o Gαq/11 and Gα12/13 family members showing similar potencies, as determined in HEK293 cells recombinantly expressing MRGPRX1 (Inoue et al., 2019). Moreover, BAM8-22 was shown to induce β-arrestin-2 recruitment in β-arrestin assays (Southern et al., 2013). The downstream effectors of MRGPRX1 have been a source of controversy, especially when it comes to transient receptor potential channels (TRP), namely vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1). BAM8-22-stimulated MRGPRX1 activated TRPV1 via phosphatidylinositol bisphosphate (PIP2) and diacylglycerol (DAG) in F11 rat neuronal cells recombinantly expressing human MRGPRX1 (Solinski, Zierler, Gudermann, & Breit, 2012). Additionally, BAM8-22-induced-MRGPRX1 activation led to an increased TRPV1 sensitivity toward protons and heat (TRPV1 agonists) in the same cells (Solinski et al., 2012) (see Fig. 2 for an overview of MRGPRX1 signaling pathways). This effect was mediated by protein kinase C (PKC) as a downstream effector of MRGPRX1. These findings are in agreement with the almost exclusive expression of MRGPRX1 in TRPV1+ human DRG neurons (Klein et al., 2021). On the contrary, BAM8-22-induced action potentials in MRGPRX1-humanized mouse DRG were insensitive to HC030031 (a selective TRPA1 blocker) and ruthenium red (a broad-spectrum TRP channel blocker) (Tseng et al., 2019). Thus, the involvement of TRPA1 and TRPV1 upon MRGPRX1 activation appeared to differ in various cell systems. Interestingly, PNEs, which natively express MRGPRX1, showed a crosstalk with TRPA1 wherein MRGPRX1 activation sensitized TRPA1 to agonist stimulation via PKC (McMillan et al., 2021). In addition, MRGPRX1-induced membrane depolarization was blocked by the TRPA1 blocker HC030031 (McMillan et al., 2021). Although the involvement of TRPV1 was not investigated, this human PNE model paves the way to study MRGPRX1 downstream signaling pathways in an environment close to the native one. The use of this model also offers the advantage of avoiding interspecies and heterologous expression differences. Other potential downstream effectors of MRGPRX1, like tetrodotoxin-resistant sodium channels, M-type potassium channels, and chloride channels, were investigated in an electrophysiological recording study of DRG neurons isolated from humanized MRGPRX1 mice (Tseng et al., 2019). This study revealed that MRGPRX1 activation induced inward sodium currents and reduced the activation threshold of tetrodotoxin-resistant sodium channels (NaV 1.8 and NaV 1.9) through a PTx-sensitive and Gβγ-dependent pathway (Tseng et al., 2019). Further ion channels were reported as downstream effectors of MRGPRX1, namely high-voltage-activated (HVA) Ca2+ channels. The BAM8-22-induced activation of MRGPRX1, overexpressed in rat primary neurons, inhibited both high-voltage-activated (HVA) calcium currents and excitatory postsynaptic currents (Chen & Ikeda, 2004). The same effect was observed in MRGPRX1-humanized mouse sensory neurons and found to be mediated via Gαi/o and Gβγ proteins (Li et al., 2017). In conclusion, MRGPRX1 signaling appears to be complex, and it will require further investigation to understand how this receptor works in different cells and tissues. The aforementioned signaling pathways of MRGPRX1 are summarized in a signaling map (see Fig. 2 for an overview of MRGPRX1 signaling pathways).

Fig. 2. Overview of MRGPRX1 signaling pathways. MRGPRX1 couples to Gq/11 and Gi/o proteins, activating Gα and Gβγ pathways. The activation of the G proteins stimulates the downstream effectors, which leads to the opening or closing of transduction channels. The employed pharmacological tools for investigating the signaling pathways included BAM8-22 (MRGPRX1 agonist), YM-254890 (Gαq/11 protein inhibitor), pertussis toxin (PTx) (Gi/o protein inhibitor), gallein (Gβγ inhibitor), HC030031 (selective transient receptor potential ankyrin 1 (TRPA1) channel inhibitor), U73122 (phospholipase C inhibitor). The investigated ion channels include: high voltage-activated (HVA) channels, tetrodotoxin-resistant sodium channels (NaV 1.8 and NaV 1.9), TRPA1, transient receptor potential vanilloid 1 (TRPV1). β-Arrestin-2 recruitment was also reported to occur upon MRGPRX1 activation by BAM8-22 resulting in receptor internalization. Created with BioRender.com.

3.1.4 Functional orthologs of MRGPRX1

The expression of MRGPRX1 appeared to be exclusive for primates since no clear orthologs in rodents were identified based on sequence comparison. However, based on ligand binding, both mouse MRGPRA3, and MRGPRC11, the latter being currently identified in the NCBI database as the mouse MRGPRX1, were considered to be functional orthologs of MRGPRX1. These receptors share some agonists with MRGPRX1, but they have their distinctive pharmacological profiles. Furthermore, not all MRGPRX1 agonists activate MRGPRA3 or MRGPRC11 and vice versa. For example, chloroquine is a shared agonist between MRGPRX1 and MRGPRA3 (Liu et al., 2009). But its potency is 10-fold lower at the human receptor (Liu et al., 2009). Another example of a shared ligand is BAM-22, which activates both MRGPRX1 and MRGPRC11 (Lembo et al., 2002; Solinski et al., 2014). The rat MRGPRC is currently annotated as the rat MRGPRX1 ortholog according to the NCBI database. The MRGPRX1 of rhesus monkeys shares the highest degree of identity (87%) and similarity (91%) with the human MRGPRX1 (see Table 7). The suggested rodent orthologs share low sequence identities of 50-54% and similarities of 63-68% with the human MRGPRX1. Although these proposed functional orthologs have facilitated studying of the primate-specific MRGPRX1 receptor and the MRGPR family, significant differences related to their distinct expression pattern and pharmacology including differences in downstream signaling, have to be considered.

Table 7. Percentage of identity (and similarity) of putative MRGPRX1 orthologs.

Empty Cell MRGPRX1 (human) MRGPRX1 (rhesus monkey) 87% (91%) MRGPRC1=MRGPRX1a (rat) 53% (68%) MRGPRC11=MRGPRX1a (mouse) 54% (66%) MRGPRA3 (mouse) 50% (63%)

a according to the NCBI database

3.1.5 Conclusions

The MRGPRX1 is so far the best investigated MRGPRX subtype. Various potent MRGPRX1 agonists, antagonists, and PAMs have been developed as tool compounds to study this orphan receptor. It shows complex and promiscuous signaling, which requires further study.

3.2 The human MAS-related G protein-coupled receptor X2

The human MRGPRX2 has gained significant attention during the past years and has become an important target in MC research. It is still classified as an orphan receptor, despite having a plethora of agonists including small molecules, peptides, and proteins. This receptor breaches the dogma of receptor-ligand specificity since it is activated by a variety of structurally and functionally diverse ligands (Grimes et al., 2019; Kashem et al., 2011; Lansu et al., 2017; McNeil et al., 2015; Nothacker et al., 2005; Tatemoto et al., 2006; Zhang et al., 2005).

3.2.1 MRGPRX2 agonists

MRGPRX2 agonists can be divided into two main classes: peptides (Table 8) and non-peptidic agonists (Table 9). All of these agonists have been shown to activate MRGPRX2 in vitro and to induce MC degranulation. It is noteworthy that reported agonists are often more potent in heterologous systems than in nontransformed MCs. Some weakly potent MRGPRX2 antagonists have been reported in literature (Table 10).

Table 8. Peptide agonists of MRGPRX2.

Compound No. Name Sequence EC50(μM) Test system References Neuropeptides X2-1 Cortistatin-14 (CST-14) PCKNFFWKTFSSCK-OH 0.025 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) 0.048 CHO-MRGPRX2 cells (Ca2+ assay) (Kamohara et al., 2005) 5.13 HTLA-MRGPRX2 cells (β-arrestin assay) (Lansu et al., 2017) 0.126 CHO-MRGPRX2 β-arrestin cells (β-arrestin assay) (Southern et al., 2013) 0.063 CHO-MRGPRX2 cells (cAMP assay) (Kamohara et al., 2005) 0.501 NIH3T3-MRGPRX2 (receptor selection and amplification technology) (Malik et al., 2009) 2.20 CD34+ stem cell MC (β-hexosaminidase, degranulation assay) (Lafleur et al., 2020) X2-2 Cortistatin-17 (CST-17) DRMPCKNFFWKTFSSCK-OH 0.099 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-3 Somatostatin-14 (SST-14) [C3-C14] AGCKNFFWKTFTSC-OH 0.780 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) 0.350 CHO-MRGPRX2 (Ca2+ assay) (Akuzawa, Obinata, Izumi, & Takeda, 2007) X2-4 Somatostatin-28 (SST-28) [C17-C27] SANSNPAMAPRERKAGCKNFFWKTFTSC-OH 0.218 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) 4.30 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 9.50 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-5 Somatostatin 2-9 (SST-2-9) GCKNFFWK-OH 2.270 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-6 Somatostatin 3-10 (SST-3-10) CKNFFWKT-OH 0.540 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-7 Somatostatin 3-14 (SST-3-14) CKNFFWKTFTSC-OH 0.16 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-8 Somatostatin 7-14 (SST-7-14) FWKTFTSC-OH 1.81 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-9 Substance P (SP) RPKPQQFFGLM-NH2 8.02 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 0.152 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 0.700 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 0.145 HTLA-MRGPRX2 cells (β-arrestin assay) (Lansu et al., 2017) 20.1 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-10 Vasoactive intestinal peptide (VIP) HSDAVFTDNYTRLRQMAVKKYLNSILN 2.40 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 2.63 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 1.50 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 10.9 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-11 Pituitary adenylate cyclase-activating peptide(6-27) (PACAP6-27) FTDSYSRYRKQMAVKKYLAAVL-NH2 1.64 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) X2-12 Pituitary adenylate cyclase-activating peptide(1-38) (PACAP1-38) HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH2 0.60 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 0.60 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 0.30 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-13 Dynorphin A (1-9) YGGFLRRIR-OH 15.5 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) X2-14 Dynorphin A (1-13) YGGFLRRIRPKLK-OH 1.05 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) 30.2 HTLA-MRGPRX2 cells (β-arrestin assay)a (Lansu et al., 2017) X2-15 Dynorphin A (1-17) YGGFLRRXRPKL-NH2 1.85 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) 2.26 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) 4.00 HTLA-MRGPRX2 cells (β-arrestin assay)a (Lansu et al., 2017) 20.9 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 7.60 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-16 Dynorphin B (1-13) YGGFLRRQFKVVT-OH 6.46 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) 33.1 HTLA-MRGPRX2 cells (β-arrestin assay)a (Lansu et al., 2017) X2-17 BAM8-22 VGRPEWWMDYQKRYG 8.50 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) 8.91 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) 39.8 HEK293-MRGPRX2 (Ca2+ assay) (Burstein et al., 2006) 11.2 HTLA-MRGPRX2 cells (β-arrestin assay)a (Lansu et al., 2017) X2-18 BAM13-22 WWMDYQKRYG-OH 0.10 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-19 BAM-22 YGGFMRRVGRPEWWMDYQKRYG 0.42 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) >100 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 15.8 HEK293-MRGPRX2 (Ca2+ assay) (Burstein et al., 2006) X2-20 Neuropeptide FF (NPFF) FLFQPQRF-NH2 0.430 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-21 Hemokinin-1 (HK 1) TGKASQFFGLM-NH2 (3)b LAD2 (β-hexosaminidase MC degranulation) (Manorak et al., 2018) X2-22 Oxytocin [C1-C6] CYIQNCPLG 1.47 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-23 [Arg8]Vasopressin [C1-C6] CYFQNCPRG 2.02 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) Further endogenous peptides X2-24 PAMP-12, (PAMP[9-20]) FRKKWNKWALSR-NH2 0.041 CHO-MRGPRX2 (Ca2+ assay) (Kamohara et al., 2005) 0.166 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 3.16 HTLA-MRGPRX2 cells (β-arrestin assay)a (Lansu et al., 2017) 0.057 CHO-MRGPRX2 cells (cAMP) (Kamohara et al., 2005) X2-25 PAMP-20 ARLDVASEFRKKWNKWALSR-NH2 0.223 CHO-MRGPRX2 cells (Ca2+ assay) (Kamohara et al., 2005) 0.251 CHO-MRGPRX2 cells (cAMP) (Kamohara et al., 2005) X2-26 Kallidin KRPPGFSPFR-OH 11.5 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) Various proteins and protein fragments X2-27 Platelet factor 4 MSSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES 8.96 HEK293-MRGPRX2 (Ca2+ assay) (Karhu et al., 2017b; Tatemoto et al., 2006) X2-28 Albumin(407-423) LLVRYTKKVPQVSTPTL 49.5 X2-29 Albumin(408-423) LVRYTKKVPQVSTPTL 91.8 X2-30 Albumin(409-423) VRYTKKVPQVSTPTL 85.7 X2-31 Chaperonin-10(1-20) Acetyl-AGQAFRKFLPLFDRVLVERS 11.0 X2-32 2 ́,3 ́-Cyclic nucleotide 3 ́-phosphodiesterase (CNP)(1-17) Acetyl-NRGFSRKSQTFLPKIFF 9.00 X2-33 Tropomyosin(1-12) Acetyl-MDAIKKKMQMLK 12.0 X2-34 Eosinophil peroxidase - (0.39)b Skin-derived cultured MCs (histamine) (Fujisawa et al., 2014) X2-35 Major basic protein - (0.72)b Skin-derived cultured MCs (histamine) (Fujisawa et al., 2014) Antimicrobial peptides X2-36 Cathelicidin (LL-37) LLGDFFRKSKEKIGKEFRIVQRIKDFLRNLVPRTES (1)b LAD2 (β-hexosaminidase MC degranulation) (Subramanian, Gupta, et al., 2011; Tatemoto et al., 2006) 65.3 HEK293-MRGPRX2 (Ca2+ assay) (10)b LAD2 (Ca2+ assay) X2-37 Catestatin SSMKLSFRARAYGFRGPGPQL 28.3 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) X2-38 Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS 28.1 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) X2-39 Indolicidin ILPWKWPWWPWRR-NH2 1.30 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 2.94 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 0.70 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) X2-40 Protegrin-1 (PG-1) [C6-C15; C8-C13] RGGRLCYCRRRFCVCVGR-OH (1.39)b RBL-2H3-MRGPRX2 (β-hexosaminidase) (Gupta et al., 2015) X2-41 P17 LFKEILEKIKAKL-NH2 4.13 CHO-MRGPRX2 (β-arrestin assay) (Duraisamy et al., 2022) 3.5 CHO-MRGPRX2 cells (Ca2+ assay) 1.42 LAD2 (Ca2+ assay) X2-42 Human β-defensin 2 (hBD2) GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP (1)b LAD2 cellls (β-hexosaminidase) (Subramanian et al., 2013) (3)b RBL-2H3-MRGPRX2 (β-hexosaminidase) X2-43 Human β-defensin 3 (hBD3) GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK (0.1)b LAD2 cells (β-hexosaminidase) (0.33)b RBL-2H3-MRGPRX2 cells (β-hexosaminidase) (1)b CD34+ cell-derived MCs (β-hexosaminidase) (3)b RBL-2H3-MRGPRX2 (β-hexosaminidase) (Alkanfari et al., 2018) X2-44 Retrocyclin-100 (RC-100) [C3-C16; C5-C14; C7-C12] Cyclo(GICRCICGRGICRCICGR) (1.54)b RBL-2H3-MRGPRX2 (β-hexosaminidase) (Gupta et al., 2015) X2-45 AMP-IBP5 AVYLPNCDRKGFYKRKQCKP SR-NH2 (1.3)b LAD2 (β-hexosaminidase MC degranulation) (Niyonsaba et al., 2020) Synthetic host-defense drugs X2-46 Angiogenic peptide-30/5C (AG-30/5C) MLKLIFLHRLKRMRKRLKRKLRLWHRKRYK (0.01)b LAD2 (β-hexosaminidase MC degranulation) (Roy et al., 2019) X2-47 E7 C3a-receptor “superagonist“ WWGKKYRASKLGLAR (1)b LAD2 (β-hexosaminidase MC degranulation) (Kashem et al., 2011) Therapeutic peptides X2-48 PMX-53 Ac-Phe-Orn(1)-Pro-D-Cha-Trp-Arg-(1) (0.1)b LAD2 (β-hexosaminidase MC degranulation) (Subramanian, Gupta, et al., 2011) X2-49 Angiopeptin [C2-C7] β-Nal-Cys-Tyr-Trp-Lys-Val-Cys-Thr 2 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 4.39 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 0.80 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 7 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-50 Octreotide [C2-C7] D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol 6.48 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 7.90 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 25.9 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 35.2 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-51 Sermorelin Tyr-Ala-Asp-Ala-ξIle-Phe-ξ-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-ξIle-Met-Ser-Arg-NH2 1.34 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 7.10 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 3.60 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 31.1 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-52 Hexarelin His-2-methyl-D-Trp-Ala-L-Trp-D-phenyl-Ala-L-Lysin-NH2 9.5 HEK293-Gα15-MRGPRX2 (Ca2+ assay) (Robas et al., 2003) X2-53 Cetrorelix Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2 3.20 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 0.155 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 0.813 HTLA-MRGPRX2 cells (Lansu et al., 2017) 2.40 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 1.90 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-54 Leuprolide Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt 7.17 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) X2-55 Icatibant Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2 12.1 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 28.2 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+ assay) (Lansu et al., 2017) (19.2)b RBL-2H3-MRGPRX2 (β-hexosaminidase) (Alkanfari et al., 2018) Peptide toxins X2-56 Staphylococcus δ-toxin MAQDIISTIGDLVKWIIDTVNKFTKK (10)c LAD2 (β-hexosaminidase MC degranulation) (Azimi & Lerner, 2017) X2-57 Mastoparan INLKALAALAKKIL-NH2 3.90 HEK293-MRGPRX2 (Ca2+ assay) (McNeil et al., 2015) 8.50 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 10.4 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 10.9 CD34+ stem cell-derived mature human MCs (Lafleur et al., 2020) X2-58 Mast cell degranulating peptide (MCD peptide) [C3-C19; C5-C15] XKCNCKRXVXKPXXCRKXCGKN 0.30 CHO-MRGPRX2 cells (Ca2+ assay) (Lafleur et al., 2020) 1.25 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) 0.40 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 0.60 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-59 Melittin (1-26) GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 1.50 CHO-MRGPRX2 (β-arrestin assay) (Lafleur et al., 2020) 4.70 CD34+ stem cell MC (β-hexosaminidase, degranulation) (Lafleur et al., 2020) X2-60 Granuliberin R FGFLPIYRRPAS NH2 24.3 HEK293-MRGPRX2 (Ca2+ assay) (Tatemoto et al., 2006) Proteases (and their products) X2-61 Cathepsin S (-) (2)c HeLa-MRGPRX2 (Ca2+ assay) (Reddy et al., 2015), X2-62 Mucunain (-) 0.11 HEK293-MRGPRX2 (Ca2+ assay) (Reddy et al., 2018) X2-63 Der p1 (CYKQTL) (2)c HeLa-MRGPRX2 (Ca2+ assay) (Reddy & Lerner, 2017) X2-64 Der p1 (RSLIGK) (2)c HeLa-MRGPRX2 (Ca2+ assay) X2-65 Der p1 (SLIGKV) (2)c HeLa-MRGPRX2 (Ca2+ assay)

a HTLA cells (HEK-T cells stably expressing a β-arrestin2-TEV fusion protein and a tTa-dependent luciferase reporter) b Lowest concentration with significant degranulation c Tested at a single concentration

Table 9. Non-peptide MRGPRX2 agonists.

Compound No. Name Structure EC50 (μM) Cells (test system)a,b,c References Opiates and opioids X2-66 Dextrorphan 1.40 CHO-MRGPRX2 (Ca2+) (Akuzawa et al., 2007; Lansu et al., 2017) 6 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 4.3 HTLA-MRGPRX2 (β-arrestin) X2-67 Dextromethorphan 5.89 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) (Lansu et al., 2017) 3.24 HTLA-MRGPRX2 (β-arrestin) X2-68 Levorphanol 14 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 24 HTLA-MRGPRX2 (β-arrestin) X2-69 (+) Morphine 5.01 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 3.39 HTLA-MRGPRX2 (β-arrestin) X2-70 (-) Morphine 6.61 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 6.17 HTLA-MRGPRX2 (β-arrestin) X2-71 (+) Codeine 13.8 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+ ) 5.50 HTLA-MRGPRX2 (β-arrestin) X2-72 (-) Codeine 10.0 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 6.76 HTLA-MRGPRX2 (β-arrestin) X2-73 Thebaine 75.9 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 11.2 HTLA-MRGPRX2 (β-arrestin) X2-74 Dihydrocodeine 11.0 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+ ) 18.6 HTLA-MRGPRX2 (β-arrestin) X2-75 (+)-TAN-67 0.288 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 0.427 HTLA-MRGPRX2 (β-arrestin) X2-76 (-/+)-TAN-67 0.740 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) 1.82 HTLA-MRGPRX2 (β-arrestin) X2-77 Pethidine 27.4 HEK293-MRGPRX2 (Ca2+) (Liu, Wang, et al., 2018) (400)d LAD2 MCD (β-hexosaminidase) Neuromuscular blocking agents X2-78 Atracurium 30.8 HEK293-MRGPRX2 (Ca2+) (McNeil et al., 2015) X2-79 Cisatracurium 111 HEK293-MRGPRX2 (Ca2+) (Che, Wang, et al., 2018; Navinés-Ferrer et al., 2018) (53.8)d LAD2 MCD (β-hexosaminidase) X2-80 Mivacurium 37.9 HEK293-MRGPRX2 (Ca2+) (24.3)d LAD2 MCD (β-hexosaminidase) X2-81 Rocuronium 429 HEK293-MRGPRX2 (Ca2+) (Chompunud Na Ayudhya et al., 2021) (75)d LAD2 MCD (β-hexosaminidase) Fluoroquinolones X2-82 Ciprofloxacin 20.5 HEK293-MRGPRX2 (Ca2+) (Hamamura-Yasuno et al., 2020; Liu et al., 2019; McNeil et al., 2015) X2-83 Moxifloxacin 24.7 HEK293-MRGPRX2 (Ca2+) 142 X2-84 Levofloxacin 62.82 HEK293-MRGPRX2 (Ca2+) X2-85 Ofloxacin 75.0 HEK293-MRGPRX2 (Ca2+) X2-86 Norfloxacin 76.1 HEK293-MRGPRX2 (Ca2+) X2-87 Lomefloxacin 128 HEK293-MRGPRX2 (Ca2+) X2-88 Gatifloxacin 529 HEK293-MRGPRX2 (Ca2+) Nonribosomal cyclic lipopeptide antibiotics X2-89 polymyxin B 1.25 HEK293-MRGPRX2 (Ca2+) (Zhan et al., 2019) X2-90 polymyxin E 1.30 HEK293-MRGPRX2 (Ca2+) Antipsychotics X2-91 Chlorpromazine 25.0 HEK293-MRGPRX2 (Ca2+) (Hou et al., 2019) ~40 LAD2 MCD (β-hexosaminidase) X2-92 Thioridazine 214 HEK293-MRGPRX2 (Ca2+) X2-93 Trifluoperazine 296 HEK293-MRGPRX2 (Ca2+) Antidepressants X2-94 Desipramine 78.0 HEK293-MRGPRX2 (Ca2+) (Wolf et al., 2021) 21.6 β-arrCHO-MRGPRX2 (β-arrestin) 34 LAD2 MCD (β-hexosaminidase) X2-95 Clomipramine 15.0 HEK293-MRGPRX2 (Ca2+) 9 β -arrCHO-MRGPRX2 (β-arrestin) 16 LAD2 MCD (β-hexosaminidase) (15)d LAD2 MCD (β-hexosaminidase) X2-96 Paroxetine 34.0 HEK293-MRGPRX2 (Ca2+) 15.8 β -arrCHO-MRGPRX2 (β-arrestin) 18 LAD2 MCD (β-hexosaminidase) (75)d LAD2 MCD (β-hexosaminidase) Various synthetic scaffolds X2-97 (R)-ZINC-3573 1 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) (Lansu et al., 2017) 0.76 HTLA-MRGPRX2 (β-arrestin) ~1 LAD2 MCD (β-hexosaminidase) X2-98 (compound (+)-2a) (32)e HEK293-MRGPRX2 (NanoBit Go dissociation assays) (Iio et al., 2021) X2-99 C48/80 3.75 μg/ml HEK293-MRGPRX2 (Ca2+) (Tatemoto et al., 2006) 0.3 (0.57 μg/ml) CHO-MRGPRX2 (Ca2+) (Lafleur et al., 2020) 0.7 (1.34 μg/ml) β-arrCHO-MRGPRX2 (β-arrestin) (Lafleur et al., 2020) 1.84 (3.54 μg/ml) HTLA-MRGPRX2 (β-arrestin) (Lansu et al., 2017) 3.9 (7.5 μg/ml) CD34+ stem cell-derived mature human MCD (β-hexosaminidase) (Lafleur et al., 2020) (30 μg/ml)d LAD2 MCD (β-hexosaminidase) (Liu, Wang, et al., 2018) X2-100 Compound 13 0.158 NIH3T3 (R-SAT) (Malik et al., 2009) Small-molecule host-defense drugs X2-101 smHDP1 0.800 RBL-2H3-MRGPRX2 MCD (β-hexosaminidase) (Alkanfari et al., 2019) X2-102 smHDP2 3 X2-103 smHDP3 1.8 X2-104 smHDP4 0.4 X2-105 smHDP5 0.38 Natural products X2-106 Complanadine A 17.8 HTLA-MRGPRX2 (β-arrestin) (Lansu et al., 2017) 0.692 FLP-IN/T-REX HEK293T MRGPRX2 (Ca2+) X2-107 Salvianolic acid A 363 HEK293-MRGPRX2 (Ca2+) (Lin et al., 2018; Lin et al., 2019) X2-108 Salvianolic acid C 15.7 HEK293-MRGPRX2 (Ca2+) X2-109 Isosalvianolic acid C 38.3 HEK293-MRGPRX2 (Ca2+)

a Ca2+: Ca2+ assays b β-arrestin: β-arrestin assays c MCD (β-hexosaminidase): mast cell degranulation (using β-hexosaminidase) d Lowest concentration with significant degranulation; concentrations are in μM unless otherwise noted e Tested at a single concentration

Table 10. Non-peptide MRGPRX2 antagonists.

Compound No. Name Structure IC50 (μM) Tested vs. agonist (concentration in μM) Cell line (test system)a-c References X2-110 QWF (Boc-Gln-D-Trp(Formyl)-Phe benzyl ester trifluoro-acetate) (1)d SP (0.5) HEK293-MRGPRX2 (Ca2+) (Azimi et al., 2016) (100)d C48/80 (1.5 μM or 2.8 μg/ml) LAD2 (MCD (β-hexosaminidase)) (100)d atracurium (100) (100)d ciprofloxacin(100) X2-111 Saikosaponin A (0.5 mg/mL)d C48/80 (30 μg/ml) HEK293-MRGPRX2 (Ca2+) (Wang et al., 2018) (0.25 mg/mL)d C48/80 (30 μg/ml) LAD2 (Ca2+) (0.5 mg/mL)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (0.5 mg/mL)d C48/80 (30 μg/ml) (MCD histamine) X2-112 Isoliquiritigenin (50)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (Hou et al., 2018) (50)d C48/80 (30 μg/ml) (MCD histamine) (12.5)d C48/80 (30 μg/ml) LAD2 (Ca2+) X2-113 Resveratrol 77.0 C48/80 (30 μg/mL) LAD2 (MCD (β-hexosaminidase)) (Wang, Zhang, et al., 2021) X2-114 Osthole (72)d C48/80 (100 ng/ml) LAD2 (Ca2+) (Callahan et al., 2020) (72)d SP (0.3) LAD2 (Ca2+) (72)d LL-37 (3) LAD2 (Ca2+) (72)d C48/80 (100 ng/ml) LAD2 (MCD (β-hexosaminidase)) (36)d SP (0.3) LAD2 (MCD (β-hexosaminidase)) (72)d LL-37 (3) LAD2 (MCD (β-hexosaminidase)) X2-115 Shikonin 1.32 C48/80 (30 μg/ml LAD2 (MCD (β-hexosaminidase)) (Wang, Zhang, Li, et al., 2020) (1)d C48/80 (30 μg/ml) LAD2 (Ca2+) (1)d C48/80 (30 μg/ml) HEK293-MRGPRX2 (Ca2+) X2-116 Quercetin (50)d C48/80 (30 μg/ml) LAD2 (Ca2+) (Ding et al., 2019) (100)d SP (μg/ml) LAD2 (Ca2+) (100)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (50)d SP (4 μg/ml) LAD2 (MCD (β-hexosaminidase)) X2-117 Genistein 25 C48/80 (10 μM or 19.2 μg/ml) LAD2 (Ca2+) (Kumar et al., 2020) 38 C48/80 (10 μM or 19.2 μg/ml)) LAD2 (MCD (β-hexosaminidase)) 31 C48/80 (10 μM or 19.2 μg/ml)) HTLA- MRGPRX2 (β-arrestin) X2-118 Piperine (100)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (Qiao et al., 2020) (100)d LL-37 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (200)d ciprofloxacin(200) LAD2 (MCD (β-hexosaminidase)) X2-119 Imperatorin (50)d SP (4 μg/ml) LAD2 (MCD (β-hexosaminidase)) (Wang et al., 2021) (50)d SP (4 μg/ml) LAD2 (Ca2+)

X2-120 Paeoniflorin (500)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (Wang, Zhang, Wang, et al., 2020) (100)d C48/80 (30 μg/ml) LAD2 (Ca2+) X2-121 Hydroxysafflor yellow A (100)d C48/80 (30 μg/ml) LAD2 (Ca2+) (Liu, Zhao, et al., 2018) (50)d C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) X2-122 Roxithromycin 55 C48/80 (30 μg/ml) LAD2 (MCD (β-hexosaminidase)) (Zhang et al., 2020) (400)d C48/80 (30 μg/ml) LAD2 (Ca2+) X2-123 Compound 1 1.6 CST-14 (1) HEK293-MRGPRX2 (Ca2+) (Ogasawara et al., 2019) 0.3 Icatibant (30) Human cord blood-derived MCs (MCD (β-hexosaminidase)) X2-124 Compound 2 2.5 CST-14 (1) HEK293-MRGPRX2 (Ca2+) 1 Icatibant (30) Human cord blood-derived MCs (MCD (β-hexosaminidase)) X2-125 C9 0.043e (R)-ZINC-3573 (3) HEK293-MRGPRX2 (Ca2+) (Cao, Kang, et al., 2021) (100)d (R)-ZINC-3573 (2) LAD2 (MCD (β-hexosaminidase)) X2-126 C9-6 0.058e (R)-ZINC-3573 (3) HEK293-MRGPRX2 (Ca2+)

a Ca2+: Ca2+ assays b β-A: β-arrestin assays c MCD (β-hexosaminidase): mast cell degranulation (β-hexosaminidase) d Lowest concentration with significant degranulation; concentrations are in μM unless otherwise indicated e Ki value: inhibition constant

3.2.1.1 Peptide agonists

3.2.1.2 Non-peptide MRGPRX2 agonists

Many therapeutic drugs induce adverse reactions that appear to be allergic in nature. In recent years, MRGPRX2 emerged as a target that mediates pseudo-allergic events associated with a number of drugs. Its direct activation induces IgE-independent MC degranulation, designated pseudo-allergic reactions (Kumar, Duraisamy, & Chow, 2021). MRGPRX2 was found to be activated by opiates and opioids, including opioid peptides (see Table 9). Therefore, MRGPRX2 had been considered as a unique atypical opioid-like receptor (Akuzawa et al., 2007; Lansu et al., 2017). β-Arrestin recruitment assays (PRESTO-Tango) were utilized to screen 5,695 small molecules at MRGPRX2 in HTLA cells (HEK-T modified to stably expressing a β-arrestin-2 and a luciferase reporter) (Lansu et al., 2017). Subsequently, concentration-response curves were determined for the screening hits using β-arrestin recruitment as well as calcium mobilization assays. MRGPRX2, unlike the classical opioid receptors that are activated by levorotatory ligands, is activated by many dextrorotatory stereoisomers of opioids, including dextrorphan (X2-66, Table 9), dextromethorphan (X2-67, Table 9), and the dextrorotary morphinan sinomenine (X2-68, Table 9). For instance, dextrorphan (X2-66, Table 9), which displayed an EC50 value of 4.3 μM in β-arrestin assays and an EC50 value of 6 μM in calcium assays, was almost 10-fold more potent than levorphanol (X2-68, Table 9). Moreover, levallorphan was completely inactive at MRGPRX2. However, both (+)- and (−)-morphine enantiomers (X2-69 and X2-70, Table 9) and (+)- and (−)-codeine enantiomers (X2-71 and X2-72 , Table 9) activated MRGPRX2 with comparable potency in β-arrestin and calcium assays displaying EC50 values in the lower micromolar range, similarly as thebaine (X2-73, Table 9) and dihydrocodeine (X2-74, Table 9). N-methyl-substituted morphinan derivatives, e.g. codeine and morphine, and N-methyl-substituted benzomorphan, e.g. dextrorphan and dextromethorphan, resulted in MRGPRX2 activation, while the N-unsubstituted norcodeine was inactive as determined in calcium assays. Larger substituents than methyl were found to abolish MRGPRX2 activation. Opioids were also reported to induce LAD2 cell degranulation (Liu et al., 2017). TAN-67, a δ-opioid receptor agonist, was found to activate MRGPRX2 in β-arrestin and calcium assays. In particular, the purified dextro-enantiomer (+)-TAN-67 was more potent than the racemic (±)-TAN-67, showing an EC50 value of 288 nM versus 740 nM for the racemate (X2-75 and X2-76, Table 9). Despite its low sequence identity with the opioid receptors (approximately 26%), MRGPRX2 was considered as an atypical opioid receptor, showing, however, pivotal differences in ligand recognition patterns (Lansu et al., 2017). Pethidine, a weak δ-opioid receptor agonist, induced LAD2 cell degranulation at high concentration (400 μM) and showed an EC50 value of 27.4 μM in calcium assays in HEK293 recombinantly expressing MRGPRX2 (X2-77, Table 9) (Liu et al., 2017). Neuromuscular blocking agents, antagonists of the postsynaptic nicotinic acetylcholine receptors in skeletal muscle, e.g., atracurium, cisatracurium, and mivacurium, were also identified as MRGPRX2 agonists. These compounds share a tetrahydroisoquinoline (THIQ) core structure. They activated MRGPRX2 with low to moderate potencies as determined in calcium assays (30 to 111 μM) (X2-78 to 80, Table 9) (McNeil et al., 2015). Cisatracurium (53.8 μM) and mivacurium (24.3 μM) also induced LAD2 MC degranulation (Che et al., 2018; Che et al., 2018; Navinés-Ferrer et al., 2018). Another neuromuscular blocker, the steroidal rocuronium, lacking the THIQ ring system, was reported to activate MRGPRX2 in calcium assays and to induce MC degranulation with low potency (X2-81, Table 9) (Chompunud Na Ayudhya, Amponnawarat, Roy, Oskeritzian, & Ali, 2021; McNeil et al., 2015). By similarity search to the bicyclic THIQ motif, many fluoroquinolone antibiotics were identified as potential MRGPRX2 agonists. Indeed, fluoroquinolones activated MRGPRX2, with the following rank order of potency determined in calcium mobilization assays in HEK293 recombinantly expressing the human MRGPRX2: ciprofloxacin > moxifloxacin > levofloxacin > ofloxacin > norfloxacin > lomefloxacin > gatifloxacin (X2-82 to X2-88, Table 9). All of them feature a basic nitrogen atom that is protonated under physiological conditions (Liu et al., 2019; McNeil et al., 2015). Antibiotics such polymyxin B and polymyxin E activated MRGPRX2 recombinantly expressed in HEK293 cells in a concentration-dependent manner displaying EC50 values of 1.25 and 1.3 μM in calcium assays (X2-89 and X2-90, Table 9) (Zhan et al., 2019). The antipsychotic drugs chlorpromazine (X2-91, Table 9), thioridazine (X2-92, Table 9) and trifluoperazine (X2-93, Table 9) were reported to act as MRGPRX2 agonists in calcium assays (Hou et al., 2019). Among these phenothiazine derivatives, chlorpromazine was the most potent one with an EC50 value of 25 μM in calcium assays, and of around 40 μM in LAD2 MC degranulation assays (X2-91, Table 9). Utilizing a computer-based analysis of 3D molecular structures of these newly identified agonists, a pharmacophore model was generated. Structural features shared by the agonists and therefore assumed to be of major importance for receptor interaction include (1) a lipophilic, aromatic moiety consisting of two benzene rings, (2) an aliphatic, protonatable, sterically demanding (often tertiary) amino group, (3) a hydrophobic center connecting the aromatic ring system; and (4) a halogen substituent on one of the benzene moieties (see X2-91 and X2-95) (Wolf et al., 2021). The established SARs of a protonatable, sterically demanding amino group in combination with an aromatic ring system can be transferred to several other published MRGPRX2 agonists, such as opioids, neuromuscular blocking agents, fluoroquinolones, and phenothiazine derivatives (see Table 9). Based on these SARs, antidepressants, including desipramine (X2-94, Table 9), clomipramine (X2-95, Table 9), and paroxetine (X2-96, Table 9), were identified as MRGPRX2 agonists and validated in calcium, β-arrestin and MC degranulation assays showing potencies in the mid-micromolar range. Several further approved drugs including aminoglycoside antibiotics, sulfonamide antibiotics and anti-fungal drugs, in addition to the contrast agent iohexol (Yuan et al., 2021), were reported to activate MRGPRX2, however, they displayed low potencies in the high micromolar or even millimolar range (these compounds are not depicted in the table due to their low potencies) (Zhang et al., 2017).

Molecular modeling and subsequent virtual screening enabled the discovery of a highly selective MRGPRX2 agonist, (R)-ZINC-3573, showing moderate potency (1 μM in calcium assays and 0.79 μM in β-arrestin assays), that may be useful as a tool compound to study receptor activation in vitro (X2-97, Table 9) (Lansu et al., 2017). Recently, an unnatural morphinan (compound (+)-2a), which has no δ-opioid receptor activity but still activates MRGPRX2, was synthesized based on the structure of the MRGPRX2 agonist (+)-TAN-67 (Iio et al., 2021). This compound was reported to induce scratching behavior in mice at a low dose (100 μg/mouse) (X2-98, Table 9). Long before the breakthrough discovery of MRGPRX2 expression on MCs, compound 48/80 (C48/80) was used as an experimental drug in MC research (Paton, 1951). It is a synthetic oligomer obtained by condensation of N-methyl-p-methoxyphenylethylamine and formaldehyde. It is marketed as an oligomeric mixture or as a pure trimer, depending on the provider. Therefore, the results cannot be directly compared in cases where a mixture has been employed; here EC50 values can only be provided in μg/ml. This compound induces MRGPRX2-mediated degranulation of CD34+ stem cell-derived MCs and LAD2 cells (Lafleur et al., 2020; Liu et al., 2018). In calcium assays and β-arrestin assays, the potency of C48/80 at MRGPRX2 was reported to be in the lower micromolar or μg/ml range (X2-99, Table 9) (Kashem et al., 2011; Lafleur et al., 2020; Tatemoto et al., 2006).

A high-throughput screen of 250,000 small molecules yielded tetracyclic benzimidazoles as agonists of both MRGPRX1 and MRGPRX2. The most potent and selective compound for MRGPRX2 was compound 13 (X2-100, Table 9) which showed an EC50 value of 0.158 μM determined in R-SAT assays (Malik et al., 2009). Peptidomimetic drugs such as the small-molecule host-defense drugs (smHDP) were reported to activate MRGPRX2. Five synthetic smHDPs, smHDP1, smHDP2, smHDP3, smHDP4 and smHDP5, displayed EC50 values of 0.8 μM, 3 μM, 1.8 μM, 0.4 μM and 0.3 μM, respectively, determined in RBL-2H3 (expressing MRGPRX2) MC degranulation assays (X2-101 to X2-105, Table 9). Finally, the natural product “complanadine A” was described as a selective MRGPRX2 agonist after screening it at 165 G protein-coupled receptors; it showed an EC50 value of 5.5 μM in calcium assays (X2-106, Table 9) (Johnson & Siegel, 2014). Further natural products, including salvianolic acid A/C and isosalvianolic acid C, activated MRGPRX2 with moderate potencies as determined in calcium assays (X2-107 to X2-109, Table 9) (Han et al., 2018; Lansu et al., 2017; Lin et al., 2019).

3.2.2 MRGPRX2 antagonists

Up to now, only weakly potent MRGPRX2 antagonists have been described in literature (Table 10). For example, the tripeptide Gln-D-Trp-(Formyl-)Phe-benzylester (QWF), which is a neurokinin-1 receptor antagonist, was reported to reduce SP-induced calcium signaling in HEK293 cells stably expressing MRGPRX2; 1 μM of QWF inhibited the effect of SP at its EC50 value (500 nM) (Azimi et al., 2016). QWF (100 μM) further inhibited agonist-induced-LAD2 degranulation by SP (1 μM), C48/80 (1.5 μM or 2.8 μg/ml), atracurium (100 μM), and ciprofloxacin (100 μM). In addition, QWF (500 μM) blocked SP-induced itch (500 μM) in mice that did not express neurokinin-1 receptors (NK-1R–/–) (X2-110, Table 10) (Azimi et al., 2016).

Saikosaponin A, a triterpenoid glycoside extracted from the radix of Bupleurum falcatnum, was reported to reduce C48/80-induced pseudo-allergic shock in mice (the death rate was reduced by 0.25 mg/kg of saikosaponin A compared to 0.06 mg/kg of C48/80 alone). Moreover, saikosaponin A reduced C48/80-induced calcium release and degranulation in LAD2 cells (β-hexosaminidase release: C48/80 alone 70%, with 0.5 μM saikosaponin A 30%) (X2-111, Table 10) (Wang et al., 2018).

Isoliquiritigenin was identified as a result of molecular docking and virtual screening experiments showing that isoliquiritigenin binds to MRGPRX2 through hydrogen-bonding interactions. Indeed, Isoliquiritigenin, which is a chalcone derivative isolated from the roots and stems of Glycyrrhiza glabra, G. uralensis, and G. inflate, caused an inhibition of C48/80-induced LAD2 degranulation (β-hexosaminidase release: C48/80 alone, 35%; in the presence of 50 μM isoliquiritigenin, 15%) (X2-112, Table 10) (Hou et al., 2018). In calcium assays, 50 μM of isoliquiritigenin caused a 3-fold reduced potency of C48/80 in HEK293 cells (expressing MRGPRX2). Cytokine release was also reduced as a consequence of preincubation with isoliquiritigenin (50 μM). In vivo, C48/80-induced local anaphylaxis, measured by hind paw swelling, was reduced by isoliquiritigenin (0.6 mg/kg).

Resveratrol, a polyphenolic compound present in red wine, inhibited C48/80-induced calcium mobilization in MCs and inhibited their degranulation with an IC50 value of 77 μM, in an MRGPRX2-dependent manner (X2-113, Table 10) (Wang et al., 2021). In addition, resveratrol (20 mg/kg) suppressed C48/80-induced passive cutaneous anaphylaxis, which was measured by hind paw thickness, via a nuclear factor erythroid-derived 2-related factor 2(Nrf2)-dependent pathway.

Osthole, a natural coumarin present in the fruits of the Cnidium monnieri plant, inhibited MRGPRX2-induced MC degranulation. The effect of MRGPRX2 agonists, including SP (300 nM), C48-80 (100 ng/ml), and LL-37 (3 μM), was reduced by 72 μM of osthole in LAD2 cells as determined in calcium assays and MC degranulation assays (X2-114, Table 10) (Callahan et al., 2020). Osthole was additionally tested in vivo showing a protective effect (at 100 mg/kg) against C48/80-induced anaphylaxis.

Shikonin, a major component of the dried root of Lithospermum erythrorhizontha, is a commonly used herbal medicine in China and other countries (Chen, Yang, Oppenheim, & Howard, 2002). In LAD2 and HEK293 cells (expressing MRGPRX2), shikonin showed an inhibitory effect on C48/80-induced Ca2+ signaling. Consequently, shikonin induced LAD2 MC degranulation with an IC50 value of 1.32 μM (versus 30 μg/ml of C48/80). Shikonin (10 mg/kg) inhibited local C48/80 effects in vivo measuring hind paw thickness and systemic anaphylaxis (body temperature measurement) (X2-115, Table 10) (Wang et al., 2020).

Quercetin, an abundant flavonoid, showed an anti-pseudoallergic effect (at 4 mg/kg) protecting mice from C48/80(30 μg/ml)- or substance P(4 μg/ml)-induced hind paw swelling (X2-116, see Table 10; for results from in vitro assays) (Ding et al., 2019). A protective effect of the flavonoid genistein against C48/80-induced anaphylactoid shock was reported to be mediated via MRGPRX2 inhibition (Kumar et al., 2020). This non-steroidal polyphenol, genistein (100 μM), reduced the efficacy and the maximal response (Emax) of C48-80 in calcium, β-arrestin, and MC degranulation assays, but not its potency (X2-117, Table 10; for IC50 values). Therefore, genistein was suggested to bind to a different binding site, an allosteric site, on MRGPRX2 differing from that of C48/80. Molecular docking using the human C5a anaphylatoxin chemotactic receptor 1 (PDBID 5O9H) as a template revealed two binding pockets. One postulated binding site of MRGPRX2 comprised of F239, Y113, T187, S268, N271, G236, G116, F232, L191, and I192, was proposed to interact with C48/80. A second binding pocket proposed to bind genistein is formed by C258, P262, W179, N85, V88, C95, Y89, and T106 (see Fig. 3B). In vivo studies using a mouse model of anaphylactoid shock demonstrated a protective effect of genistein (20 mg/kg) (Kumar et al., 2020)

Piperine (20 mg/kg), an alkaloid extracted from pepper, showed anti-anaphylactoid effects when injected into the hind paw of C57 black 6 (C57BL/6) mice prior to C48/80 injection (X2-118, see Table 10; for results from in vitro assays) (Qiao et al., 2020).

Imperatorin is one of the active furocoumarins in Angelicae Dahuricae radix, which is a herb used in China as an anti-inflammatory medicine for the treatment of respiratory diseases (Lee, Shin, Ham, & Choi, 2015). Imperatorin decreased SP-induced MRGPRX2 activation in calcium assays in LAD2 cells (50 μM) and reduced degranulation (X2-119, Table 10) (Wang, et al., 2021). Moreover, imperatorin (6 mg/ml) reduced C48/80-induced local anaphylaxis in C57BL/6 mice. Additionally, imperatorin decreased asthma and lung inflammation in an ovalbumin-induced asthma mouse model, measured by examining lung sections and cytokine release.

Paeoniflorin (2.5 mg/kg), a monoterpene glycoside isolated from the Chinese medical herb Radix Paeoniae alba, was reported to reduce C48/80-induced hind paw swelling in mice (X2-120, Table 10; for in vitro assays) (Wang et al., 2020).

Hydroxysafflor yellow A, extracted from the Chinese medical herb Flos Carthami, has been demonstrated to suppress MC Ca2+ release (at 100 μM concentration vs. 30 μg/ml C48/80) and degranulation (at 50 μM concentration vs. 30 μg/ml C48/80) (X2-121, Table 10) (Liu et al., 2018). These effects resulted in a decreased C48/80-induced pseudo-allergic effect measured by decreased paw edema and Evans blue extravasation. Evans blue is a dye that binds albumin and can be visualized in paws (Hydroxysafflor yellow A: 10 mg/kg). Interestingly, most of the aforementioned natural products are used in Chinese traditional medicine for their potential anti-inflammatory and anti-allergic effects.

Roxithromycin, a macrolide antibiotic, was reported to decrease C48/80-induced passive cutaneous anaphylaxis in mice (at 80 mg/kg vs. 30 μg/ml C48/80) measured by decreased hind paw thickness. Hypothermia resulting from C48/80-induced active systemic allergy was reduced by roxithromycin (at 80 mg/kg roxithromycin vs. 300 μg/ml C48/80). In vitro, roxithromycin decreased C48/80-induced MC degranulation, Ca2+ mobilization, and cytokine release (X2-122, Table 10 for in vitro assays) (Zhang et al., 2020) A screening campaign of 12,000 commercially available compounds tested in calcium assays, using SP-activated HEK293 cells recombinantly expressing MRGPRX2, led to the discovery of two different heterocyclic scaffolds: compound 1 (1-(5H-10λ2-phenazin-5-yl)ethan-1-one) (X2-123, Table 10) and compound 2 (3-(pyridine-2-ylmethyl)-2-thioxo-2,3-dihydro-4H-1λ2-quinazolin4-one) (X2-124, Table 10) (Ogasawara, Furuno, Edamura, & Noguchi, 2019). Both compounds inhibited CST-14-induced MRGPRX2 activation measured in calcium assays, showing IC50 values of 1.6 μM (compound 1) and 2.5 μM (compound 2) (X2-123 and X2-124, Table 10). Based on the above scaffold (X2-124, Table 10), searching of the ZINC database (http://zinc15.docking.org) followed by two rounds of analog modeling of make-on-demand compound libraries led to the discovery of potent MRGPRX2 inverse agonists namely C9-6 and C9, for which Ki values of 58 nM and 43 nM vs. 3 μM of (R)-ZINC-3573 were determined in calcium assays in HEK293 cells expressing MRGPRX2 (X2-125 and X2-126, Table 10) (Cao et al., 2021).

3.2.3 MRGPRX2 signaling

The signaling of MRGPRX2 appears to be mainly mediated by activation of Gq/11 and Gi/o proteins (Burstein et al., 2006; Kamohara et al., 2005). The peptide CST-14, the first identified MRGPRX2 agonist, was reported to induce Ca2+ mobilization in HEK cells recombinantly expressing MRGPRX2 (Robas et al., 2003). Later on, PAMP-20-mediated MRGPRX2 activation was found to induce Ca2+ mobilization as well (Kamohara et al., 2005). In the same study, PAMP-20-mediated MRGPRX2 activation was reported to also decrease forskolin-induced cAMP accumulation in HEK cells recombinantly expressing MRGPRX2. MRGPRX2 agonists, e.g. the human host defense peptide LL-37 and hBDs, were shown to induce human MC degranulation by a PTx-sensitive G protein-dependent pathway (Subramanian et al., 2013; Subramanian, Gupta, et al., 2011). Similarly, PTx inhibited C48/80- and AG30/5C-induced rat MC degranulation (recombinantly expressing MRGPRX2) without affecting their Ca2+ mobilization response (Roy et al., 2019). Since PTx inhibited agonist-induced degranulation, but not calcium signaling, the Gi-protein pathway appeared to be mainly relevant for MC degranulation. In another study, PTx and YM-254890 (a Gαq protein inhibitor) both significantly and additively decreased SP-induced Ca2+ mobilization and rat MC (expressing MRGPRX2) degranulation (Chompunud Na Ayudhya, Roy, Alkanfari, Ganguly, & Ali, 2019). Similarly, both Gαq and Gαi1 were reported to be required for human skin MC degranulation, but Gαi was clearly more relevant for C48/80- and SP-induced ex vivo MC degranulation (Wang et al., 2022). In contrast to the predominant role of Gαi1 in MCs, Gαq was reported to be dominant after mastoparan-induced activation in MRGPRX2-transfected HEK293 cells as detected in reporter gene assays (Arifuzzaman et al., 2019). Thus, both Gαi and Gαq may be involved in MRGPRX2 signaling in MCs, although coupling might be agonist- as well as cell-specific. Using a chimeric G protein-based TGFα shedding assay, which measures direct G protein activation, the MRGPRX2 activated by CST-14 was reported to be coupled promiscuously to all G protein families, Gαs, Gαi/o, Gαq/11, and Gα12/13 like it had been observed for MRGPRX1 as well (Inoue et al., 2019). Accordingly, MRGPRX2 was found to couple to all Gα protein families using the TRUPATH activation platform, but showing robust coupling especially with Gαq and Gαi1 proteins (Cao, Kang, et al., 2021). MRGPRX2 was reported to be desensitized and internalized after incubation with CST-14 or morphine, receptor surface expression being quantified by enzyme-linked immunosorbent assays (Akuzawa et al., 2007). Later on, β-arrestin-1 and β-arrestin-2 assays were established providing useful tools for the pharmacological characterization of MRGPRX2 and its ligands (Babina et al., 2021; Lafleur et al., 2020; Lansu et al., 2017; Southern et al., 2013). The activation of MRGPRX2 leads to MC degranulation, which appears to be mediated through different downstream effectors. For instance, MRGPRX2-mediated degranulation was reported to be triggered by a rapid and transient rise in the intracellular Ca2+ concentration (Ali, 2017). Activation of phospholipase C-β (PLCβ) by Gαq/11 and Gβγ subunits leads to the hydrolysis of phosphatidylinositol bisphosphate (PIP2) yielding diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which binds to its receptor (IP3R) causing Ca2+ release from the endoplasmic reticulum (ER) (Cai, Stafford, Bryan, Mitchell, & Liu, 2005). In addition to the canonical activation of PLCβ by Gαq proteins, MRGPRX2 was reported to activate PLCγ, an enzyme that is typically regulated by receptor and non-receptor tyrosine kinases (Bunney & Katan, 2011; Liu et al., 2017). Using western blotting, the phosphorylation of PLCγ and IP3R was shown to be increased during MRGPRX2-mediated activation by Sinomenine in human MCs (Liu et al., 2017). The involvement of different PLC isoenzymes, PLCβ and PLCγ, might explain why, unlike FcεRI-mediated MC degranulation, MRGPRX2-induced MC degranulation is rapid and triggered by transient Ca2+ mobilization (see Fig. 4 for an overview of MRGPRX2-induced G protein activation and downstream effectors). Since PLCγ is activated by tyrosine kinases, the effect of MRGPRX2 agonists on different kinases such as Lck/Yes novel tyrosine kinase (Lyn), a Src family kinase, was investigated using western blotting (Ding et al., 2021; Zhang et al., 2022). C48/80 was reported to induce Lyn and PLCγ phosphorylation leading to increased Ca2+ mobilization in LAD2 cells (Ding et al., 2021). In a recent study, phosphorylated protein kinase B (AKT) and PLCγ expression were shown to be higher in LAD2 MCs following MRGPRX2 activation by C48/80 compared to untreated cells (Zhang et al., 2022). The inhibition of both phosphatidylinositol-3 kinase (PI3K) by LY294002 (a PI3K inhibitor) and PLCγ by U73122 (a PLC inhibitor) was shown to suppress the C48/80-induced Ca2+ signal and subsequent MC degranulation (see Fig. 4 for an overview of MRGPRX2-induced G protein activation and downstream effectors, and Fig. 5 for an overview of MRGPRX2-activated kinases).(Niyonsaba et al., 2020; Zhang et al., 2022). In animal studies, the allergic symptoms caused by C48/80 were reduced by inhibiting both PI3K and PLCγ phosphorylation (Zhang et al., 2022). Thus, PI3K/AKT and PLCγ pathways are likely to play a role in MC degranulation observed in pseudo-allergic reactions resulting in an increased intracellular Ca2+ level following MRGPRX2 activation. The link between MRGPRX2 and the activation of tyrosine kinases remains unclear. It might be explained by Gα protein-interacting vesicle-associated protein (GIV; a.k.a Girdin), an unusual protein that can bind both G proteins and receptor tyrosine kinases (RTK) (Garcia-Marcos, Ghosh, & Farquhar, 2015; Ghosh, 2015). GIV behaves like a signal transducer and a non-receptor guanosine triphosphate exchange factor for Gαi protein subunits (Garcia-Marcos, Ghosh, & Farquhar, 2009). However, the interaction between MRGPRX2 and RTK still awaits elucidation and clarification. 2-Aminoethoxydiphenyl borate, an inhibitor of IP3Rs, calcium release-activated channels (CRACs) and TRP channels, significantly blocked hBD3-induced Ca2+ signaling and MC degranulation in LAD2 cells (Subramanian et al., 2013). In the same study, bisindolylmaleimide (a PKC inhibitor) did not affect hBD3-induced Ca2+ mobilization via MRGPRX2, but significantly inhibited MC degranulation. Since the activation of MRGPRX2 induced a rapid and transient increase in Ca2+ concentrations, the role of CRACs namely ORAI-1 or CRAC1, on plasma membranes, and ORAI-2, on MC granules, was investigated (see Fig. 4 for an overview of MRGPRX2-induced G protein activation and downstream effectors) (Ikeya et al., 2014; Subramanian et al., 2013). An inhibitor of these channels, La3+, caused a decrease in MRGPRX2-induced Ca2+ mobilization and MC degranulation (LAD2 cells) (Subramanian et al., 2013). The IgE-dependent MC activation is mediated by the store-operated Ca2+ entry (SOCE) process via the stromal interaction molecule 1 (STIM1) sensor in the ER (Baba et al., 2008). This complex has a Ca2+-binding domain, which senses Ca2+ depletion in the ER and releases bound Ca2+. Subsequently, SOCE-STIM1 interacts with the membrane ORAI1, which leads to the opening of these CRAC channels and of transient receptor potential canonical (TRPC) channels (Baba et al., 2008). These channels will allow Ca2+ influx into the cytoplasm, which then induces cytoskeletal reorganization and subsequent degranulation (Baba et al., 2008). Non-IgE- versus IgE-dependent MC activation differ in the duration of the Ca2+ signal, which is slower and persistent in the IgE-dependent pathway (Gaudenzio et al., 2016). The content released from the granules is also different when degranulation is mediated via non-IgE- or IgE-dependent pathways. However, non-IgE-dependent MC activation via MRGPRX2 appears to share some elements with IgE-dependent pathways. For example, the activation of the human MC LAD2 via CST-14, SP, C48/80, LL-37 and (R)-ZINC-3573 was reported to be reduced by a CRAC channel antagonist (YM 58483) and by a SOCE inhibitor (SKF96365 HCl) (Occhiuto et al., 2019). In addition, synta66, a CRAC blocker, inhibited SP-induced calcium signaling and MC degranulation (Chaki et al., 2021). Hence, the SOCE-STIM1-ORAI pathway, which triggers extracellular Ca2+ influx into the cytoplasm, is suggested to be a downstream effector pathway of MRGPRX2. Conversely, sinomenine-activated LAD2 cells caused Ca2+ mobilization in a STIM1-independent manner (Liu et al., 2017). Moreover, sinomenine-induced intracellular Ca2+ influx was suggested to be caused by the release of Ca2+ from the ER (Liu et al., 2017). Nevertheless, this seems not to be the case for other MRGPRX2 agonists, thus the signaling pathway could vary depending on the applied agonist. Transient receptor potential vanilloid-type 4 (TRPV4) is a membrane ion channel that was suggested to be involved in MRGPRX2-mediated MC degranulation (Mascarenhas, Wang, Chang, & Di Nardo, 2017). LL-37 was reported to increase TRPV4 expression in human MCs (CD34+ derived) in a MRGPRX2-Gαi-dependent, PTx-sensitive manner. This ion channel was reported to be directly sensitized by the IP3R, which is a downstream effector of the MRGPRX2 pathway (see Fig. 4 for an overview of MRGPRX2-induced G protein activation and downstream effectors) (Garcia-Elias, Lorenzo, Vicente, & Valverde, 2008). The voltage-gated Ca2+ channel blocker nifedipine, and A425619, a TRPV1 selective antagonist, did not affect CST-14-induced degranulation or its calcium signal (Occhiuto et al., 2019). SP-induced human MC (LAD2) activation was reported to be mediated by Ca2+/calmodulin protein kinase II (CamKII), a kinase that is activated by Ca2+ released via IP3R (Wang, Wang, et al., 2021). Resveratrol, a suggested MRGPRX2 antagonist, inhibited MRGPRX2-mediated MC activation via the nuclear factor erythroid-derived 2-related factor 2- (Nrf2)-dependent pathway (Wang, Zhang, et al., 2021). Previous studies on β-defensin- and LL-37-induced MC degranulation, even before their link to MRGPRX2, suggested that their effect was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), MAPK p38, and c-jun N-terminal kinase (JNK) pathways, which increased the release of interleukin-31 (IL-31), prostaglandin E2 (PGE2), and leukotriene C4 (LTC4) (see Fig. 5 for an overview of MRGPRX2-actived kinases) (Chen et al., 2007; Niyonsaba et al., 2010). Recent studies revealed three functional kinase clusters that translate MRGPRX2 activation into degranulation and granule secretion (Lazki-Hagenbach, Ali, & Sagi-Eisenberg, 2021). The first cluster includes IkappaB kinase (IKK) and nuclear factor kappa B (NF-κB), mitogen-activated protein kinase kinase (MAPKK or MEK), MAPK, and ERK1/2, which might play a role in the granules’ exocytosis process (Franke, Wang, Zuberbier, & Babina, 2021; Lazki-Hagenbach et al., 2021). The mechanism by which these kinases are activated is unclear, however, PKC might take a role in activating a set of kinases (see Fig. 5 for an overview of MRGPRX2-actived kinases). The second functional cluster is PLC signaling (as described above) involving not only PLCβ, which is typically activated by GPCRs, but also PLCγ (Franke et al., 2021; Lazki-Hagenbach et al., 2021). The third cluster includes autophagy-related signaling such as PI3K and unc-51-like autophagy activating kinase 1 (ULK1). Autophagy was reported earlier to have an important role in MC degranulation (Ushio et al., 2011). In conclusion, MRGPRX2-mediated Ca2+ mobilization can be due to an interplay between various pathways including but not limited to Lyn, PLCγ, and IP3R phosphorylation cascades, which triggers Ca2+ influx into the cytoplasm via i) IP3Rs, ii) ORAI channels activated by the STIM1-SOCE complex, and iii) TRPV4 channel activation. Other downstream pathways like MAPK/p38/ERK/JNK, NF-κB, and CamKII induce the generation of cytokines and other complex processes like exocytosis of the granules, and autophagy, whereas the Nrf2 pathway inhibits MC degranulation upon MRGPRX2 blockade. A recent study addressed possible points of intersection between early and late events due to MRGPRX2 activation (Wang et al., 2022). Later events such as ERK phosphorylation were found to depend on multiple early upstream events such as Gαi and Gαq activation, while AKT (also known as protein kinase B) phosphorylation strictly depended on Gαi activation (see Fig. 5 for an overview of G proteins and MRGPRX2-activated kinases). The diversity and complexity of MRGPRX2 downstream signaling mirrors its ligand diversity, which makes the signal mapping of this receptor challenging and will certainly require further investigation.

Fig. 4. Overview of MRGPRX2 signaling pathways following G protein activation. MRGPRX2 couples to Gq/11 and Gi/o proteins. G protein activation stimulates the corresponding downstream effectors, which leads to an increased intracellular calcium concentration mediated by the opening of channels. The employed pharmacological tools for investigating the signaling pathways include: YM-254890: Gq protein inhibitor; pertussis toxin (PTx): Gi/o inhibitor; U73122: phospholipase C inhibitor; 2-aminoethoxydiphenyl borate (2-APB): inositol 1,4,5-trisphosphate receptor (IP3R) and TRP channels inhibitor; bisindolylmaleimide I: protein kinase C (PKC) inhibitor and store-operated Ca2+ entry (SOCE) inhibitor that blocks the stromal interaction molecule (STIM); YM 58483: calcium release-activated channel ORAI-1 (CRAC) blocker. Other involved downstream effectors: transient receptor potential vanilloid 4 (TRPV4). Dashed line indicates activation of transcription factors or upregulation. Created with BioRender.com.

Fig. 5. Overview of MRGPRX2 signaling pathways involving activation of kinases. As a consequence of MRGPRX2 activation, several kinases are phosphorylated as detected by western blotting, leading to a downstream cascade that results either in activation or deactivation of transcription factors. Abbreviations: Mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNKs), nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (p38), calcium/calmodulin dependent kinase II (CaMKII), pAKT (phosphorylated protein kinase B). MRGPRX2 activation also leads to β-arrestin recruitment. Solid lines indicate reported interactions between early and later events, e.g. Gαi/PI3K/AKT and Gαq-Gβγ/ PLCβ/PKC/ERK pathways (Wang et al., 2022). Dashed lines indicate direct or indicrect interactions. Created with BioRender.com.

3.2.4 Functional orthologs of MRGPRX2

The exclusive expression of MRGPRX2 in primates and the consequent lacking of animal models has hampered research efforts to characterize its (patho)physiological roles for quite some time, until a functional mouse ortholog of MRGPRX2, MRGPRB2, was identified (McNeil et al., 2015). Due to the low sequence similarities between MRGPRX2 and the 22 existing mouse MRGPRs, the mouse MRGPRB2 had to be determined as the functional mouse ortholog by analyzing and comparing expression patterns and pharmacology (McNeil et al., 2015). Shared ligands between MRGPRX2 and MRGPRB2 were identified using HEK293 cells recombinantly expressing MRGPRB2. Shared agonists include C48/80, neuropeptides, cationic peptidic drugs, fluoroquinolones, and neuromuscular blocking agents (McNeil et al., 2015). However, their potency at the mouse receptor was lower than at the human receptor, which suggests that crucial species differences exist (McNeil et al., 2015). The fact that studies reported MRGPRX2 expression in both DRG and MCs (Kamohara et al., 2005; McNeil et al., 2015; Robas et al., 2003; Tatemoto et al., 2006; Zhang et al., 2005) and an exclusive expression of MRGPRB2 in MCs, but not in DRG, led to the suggestion that MRGPRX2 may be represented in mice by two different orthologs, MRGPRB2 on MCs, and another, distinct MRGPR on mouse sensory neurons (Azimi et al., 2016). Previous studies already revealed the expression of MRGPRA1 in mouse DRG, but not on MCs (Dong et al., 2001; McNeil et al., 2015). Interestingly, MRGPRA1 was 10-fold more sensitive towards SP than MRGPRB2 (EC50 values, MRGPRX2: 0.1 μM; MRGPRA1: 5 μM; MRGPRB2: 50 μM) (Azimi et al., 2016). These findings might suggest that the mouse receptors MRGPRA1 and -B2 both act as functional orthologs of MRGPRX2, and this might also explain why SP evokes a similar number of scratching bouts in MC-deficient and wild-type mice (Andoh, Nagasawa, Satoh, & Kuraishi, 1998). Itch might be generated by the activation of MRGPRA1 on mouse sensory neurons by SP (Azimi et al., 2017). By bioinformatic approaches, expression analysis, and functional assays, the rat MRGPRB3 and other MRGPRs in rat, mouse, dog, minipig, and rhesus monkey were proposed as orthologs of MRGPRX2 (Grimes et al., 2019; Hamamura-Yasuno et al., 2020; Sahid, Liu, Mogi, & Maeyama, 2020; Subramanian, Gupta, & Ali, 2016; Tatemoto et al., 2006). The highest degree of sequence identity with the human MRGPRX2 is shared by the MRGPRX2 in rhesus monkey (91% identity) followed by the proposed orthologs in dog (62% identity) and minipig (59% identity) (see Table 11). Among all of the suggested rodent orthologs, MRGPRB1 (annotated as rat MRGPRX2) in rats shares the highest degree of identity (60%) and similarity (73%) with the human MRGPRX2. Other suggested rat orthologs, termed rat MRGPRX2-like (MRGPRB2) and rat MRGPRB3, displayed higher identities of 54-57% and similarities of 67-69% with the human MRGPRX2 than the mouse receptors, MRGPRB2 and MRGPRA1. Thus, rat may be a better model than mouse due to higher sequence similarities with the human MRGPRX2. Identifying functional orthologs is of great importance for preclinical and translational studies in drug development. This would enable the straightforward development of animal models for this primate-specific subfamily. Furthermore, mice expressing the human MRGPRX2 have recently been developed and will be useful for a number of studies (Mencarelli et al., 2020).

Table 11. Percentage of identity (and similarity) of proposed orthologs of the human MRGPRX2.

Empty Cell MRGPRX2 (human) MRGPRX2 (rhesus monkey) 91% (94%) MRGPRX2 (dog) 62% (73%) MRGPRX2 (minipig) 59% (72%) MRGPRX2= MRGPRB1 (rat) 60% (73%) MRGPRX2-Like=MRGPRB2 (rat) 54% (67%) MRGPRB3 (rat) 57% (69%) MRGPRX2=MRGPRB10 (mouse) 56% (67%) MRGPRA1 (mouse) 52% (63%) MRGPRB2 (mouse) 53% (65%)

3.2.5 Conclusion

MRGPRX2 plays a fundamental role in MC degranulation, and the development of potent and selective antagonists inhibiting MC degranulation and other MRGPRX2-related pathophysiological effects is of major importance. The receptor appears to be highly promiscuous especially with regard to agonists, being activated by a large variety of basic peptidic and non-peptidic compounds that bear a positive charge at physiologic pH value. There is still a lack of potent, selective antagonists that are suitable as tool compounds and for development as drugs. MRGPRX2 appears to have several binding sites that might be exploited for ligand development.

3.3 The human MAS-related G protein-coupled receptor X3

MRGPRX3 is the least investigated MRGPRX subtype, and information on this receptor is still extremely limited.

3.3.1 MRGPRX3 modulators

MRGPRX3 is the only receptor in the MRGPRX subfamily for which no agonist has been reported yet. A single study reported that RNA interference with MRGPRX3 reduced angiogenic peptide (AG)-30/5C-induced effects in keratinocytes, measured by cytokine/chemokine release (Kiatsurayanon et al., 2016). However, direct activation of MRGPRX3 by (AG)-30/5C leading to cytokine/chemokine release has not been demonstrated. Interestingly, transgenic rats overexpressing the human MRGPRX3 displayed abnormal skin phenotypes (Kaisho et al., 2005). These rats had transient epidermal desquamation associated with high MRGPRX3 expression. Thus, MRGPRX3 may play a role in the process of epidermal cell proliferation.

3.3.2 MRGPRX3 signaling

MRGPRX3 was suggested to mediate its effect via the Gq/11 protein family (Burstein et al., 2006). A cellular proliferation assay measuring constitutive activity of GPCRs showed that the effects induced by rhesus monkey MRGPRX3 were PTx-insensitive. (Burstein et al., 2006; Weissman, Ma, Essex, Gao, & Burstein, 2004) leading to the assumption that the receptor’s constitutive activity was mediated by Gq/11 proteins. However, this needs to be validated since no ligands for the receptor have been described so far.

3.3.3 Functional orthologs of MRGPRX3

An ortholog of MRGPRX3 in rhesus monkeys that shares 90% identity and 94% similarity was described (Burstein et al., 2006). Additionally, an Mrgprx3 gene in rat is listed in the NCBI database, that belongs to the MRGPRA subfamily (see Table 2). In rats, the MRGPR subtype MRGPRA, designated MRGPRX3 according to NCBI, is activated by the nucleobase adenine, but this is not the case for the human receptor (Bender et al., 2002; Knospe et al., 2013). The annotation remains purely hypothetical since the proposed orthologous receptor shares only a low degree of identity (54%) and similarity (64%), in addition to having different pharmacological properties.

3.4 The human MAS-related G protein-coupled receptor X4

MRGPRX4 is far less characterized than MRGPRX1 and MRGPRX2, although several compounds have been described to activate MRGPRX4 (see Table 12). These are valuable pharmacological tools for investigating the receptor and for elucidating its (patho)physiological roles. Like all other MRGPRX subtypes, MRGPRX4 is still an orphan receptor, and its actual endogenous ligand and function remain to be established.

Table 12. MRGPRX4 agonists (83L receptor variant).

Compound No. Name Structure EC50(μM) Cells stably expressing human MRGPRX4 (assays)a-e Reference X4-1 Nateglinide 4.7 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+) (Cao, Kang, et al., 2021) 9.1 HTLA cells (β-arrestin) (Kroeze et al., 2015) 2.10 HEK293T cells (PI hydrolysis) (Kroeze et al., 2015) X4-2 MS47134 0.149 FLP-IN/T-REX HEK-293T-MRGPRX2 cells (Ca2+) (Cao, Kang, et al., 2021) X4-3 AG-30/5C MLKLIFLHRLKRMRKRLKRKLRLWHRKRYK (10)f HTLA cells (Ca2+) (Roy et al., 2019) X4-4 Bilirubin 61.9 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Green, et al., 2019) X4-5 Deoxycholic acid (DCA) 2.6 HEK293 (Ca2+) (Yu et al., 2019) 6.19 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) 2.7 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-6 Lithocholic acid (LCA) 9.3 HEK293 (Ca2+) (Yu et al., 2019) 88.0 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-7 Chenodeo-deoxycholic acid (CDCA) 15.9 HEK293 (Ca2+) (Yu et al., 2019) 46.2 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) 15 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-8 Cholic acid (CA) 6.9 HEK293 (Ca2+) (Yu et al., 2019) 431 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) 15.2 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-9 Taurolitho-cholic acid (TLCA) >50 HEK293 (Ca2+) (Yu et al., 2019) 6.1 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-10 Taurodeoxy-cholic acid (TDCA) 14.1 HEK293 (Ca2+) (Yu et al., 2019) 18.9 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) 28.0 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-11 Taurocheno-deoxycholic acid (TCDCA) >100 HEK293 (Ca2+) (Yu et al., 2019) 30.1 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) >100 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-12 Taurocholic acid (TCA) >50 HEK293 (Ca2+) (Yu et al., 2019) 179 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019) X4-13 Glycodeoxycholic acid (GDCA) 14.4 HEK293 (Ca2+) (Yu et al., 2019) 28.3 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-14 Glycocheno-deoxycholic acid (GCDCA) > 50 HEK293 (Ca2+) (Yu et al., 2019) >100 HEK293T-based TGFα shedding reporter cells (shedding assay) (Yu et al., 2019) X4-15 Glycocholic acid (GCA) > 50 HEK293 (Ca2+) (Yu et al., 2019) X4-16 Ursodeoxy- cholic acid (UDCA) 4.93 HEK293-Gα15 cells (Ca2+) (Meixiong, Vasavda, Snyder, & Dong, 2019)

a Ca2+: Ca2+ assays b β-Arrestin: β-arrestin assays c PI hydrolysis: phosphatidylinositol hydrolysis assays d HTLA cells: HEK-T cells stably expressing a β-arrestin-2-TEV fusion protein and a tTa-dependent luciferase reporter e TGFα shedding reporter cells f Tested at a single concentration

3.4.1 MRGPRX4 modulators

The first described MRGPRX4 agonist, nateglinide, an antihyperglycemic drug, was found to activate both Gαq protein signaling and β-arrestin recruitment (X4-1, Table 12) (Kroeze et al., 2015). This compound had been tested at 91 different GPCRs in β-arrestin assays but was demonstrated to be selective for MRGPRX4, also versus the other MRGPRX subtypes, MRGPRX1, -2, and -3 (Kroeze et al., 2015). Interestingly, rash, itching, and urticaria have been reported as occasional side effects of nateglinide treatment (Twaites, Wilton, Layton, & Shakir, 2007). It was consequently suggested that pharmacological activation of MRGPRX4 might trigger these symptoms (Kroeze et al., 2015). The nateglinide analog MS47134 was developed as a more potent MRGPRX4 agonist, for which an EC50 value of 149 nM was determined using calcium assays (X4-2, Table 12) (Cao, Kang, et al., 2021).

The synthetic HDP, AG-30, its stable and improved modified version (AG-30/5C), and the antimicrobial peptide AMP-IBP5 were reported to promote proliferation and migration of keratinocytes (Chieosilapatham et al., 2017; Kiatsurayanon et al., 2016; Nakagami et al., 2012). Medium to high concentrations of these HDPs had to be used to observe statistically significant differences in cell migration and cell proliferation assays: 10 μM were used of AG-30, 0.15 to 2.5 μM of AG-30/5C, and 1.25 to 5 μM of AMP-IBP5. Furthermore, AG-30/5C and AMP-IBP5 were shown to increase the production of cytokines and chemokines (Chieosilapatham et al., 2017; Kiatsurayanon et al., 2016). Keratinocyte proliferation and migration, as well as the production of cytokines/chemokines were proposed to be primarily controlled by MRGPRX3 and MRGPRX4 since siRNA-mediated knockdown of MRGPRX3 and -X4 (but not of MRGPRX1 or -X2) suppressed HDP-induced responses (Chieosilapatham et al., 2017; Kiatsurayanon et al., 2016). Since these processes were not regulated by a single receptor subtype, it was assumed that both AG-30/5C and AMP-IBP5 act as agonists for MRGPRX3 and MRGPRX4. Later on, AG-30/5C was found to act as a G-protein-biased agonist for MRGPRX4 since AG-30/5C at 10 μM concentration caused Ca2+ mobilization in HTLA cells expressing MRGPRX4 but did not activate β-arrestin recruitment at the same concentration (X4-3, Table 12) (Roy et al., 2019).

MRGPRX4 was proposed to act as a bilirubin receptor that may contribute to jaundice and cholestatic pruritus (X4-4, Table 12) (Meixiong et al., 2019). Bilirubin is a degradation product of heme, which is constituent of hemoglobin in erythrocytes. The compound induced Ca2+ mobilization in a concentration-dependent manner displaying an EC50 value of 61.9 μM in HEK293 cells co-expressing MRGPRX4 and the promiscuous Gα15 protein.

In a transcriptome analysis study to identify a GPCR in human DRG neurons that mediates cholestatic itch in humans, MRGPRX4 and six other GPCRs had been identified. When expressed in HEK293T cells, only MRGPRX4-expressing cells showed a significant signal upon addition of bovine bile extract (10 μg/ml) as measured in TGFα shedding assays (Yu et al., 2019). Mass spectrometry and 1H-NMR spectroscopy identified DCA (X4-5, Table 12) and chenodeoxycholic acid (X4-7, Table 12) to be the key components in bovine bile extract that induced the detected signal. None of the bile acids showed any activity at MRGPRX1, -2 or -3. The bile acids induced Ca2+ mobilization in cultured rat DRG neurons only when these were transfected with MRGPRX4. Among all bile acids tested, DCA induced the most significant calcium response and activated the highest number of MRGPRX4-expressing rat DRG neurons. The percentage of DCA-responsive cells that were loaded with Fluo-8 AM and responded to DCA application was similar to the percentage of MRGPRX4-expressing cells measured by in situ hybridization (Yu et al., 2019). (Meixiong, Vasavda, Snyder, & Dong, 2019; Yu et al., 2019). In Gαq protein-dependent TGFα shedding assays as well as in calcium assays, DCA showed the highest potency with EC50 values in the lower micromolar range (X4-5, Table 12) (Yu et al., 2019). The following SARs were established: hydroxylation at position 7 and/or 12 and taurine/glycine conjugation at position 25 were shown to be essential for activation of MRGPRX4 (X4-6 to X4-16, Table 12)

Bilirubin (X4-4, Table 12) was found to potentiate the DCA-induced activation of MRGPRX4 in calcium assays acting as a positive allosteric modulator. The monocyclic terpene alcohol menthol, a natural product present in some medicinal and spice plants, which is also used as an additive in menthol cigarettes, was identified as a negative allosteric modulator of MRGPRX4. Menthol (100 μM) decreased the efficacy of the agonist nateglinide (also see Chapter 3.5.1) (Kozlitina et al., 2019).

It should be noted that different natural variants of MRGPRX4 exist. Two major variants differ in position 83, namely 83S, and 83L. Since the NCBI database denotes the 83L variant as the major one, most of the more recently published data refer to that variant. However, this is due to an error in the database. Looking at the underlying sequencing data it becomes clear that the main natural variant has a serine in position 83 (for discussion on variants see Chapter 3.5 and Table 13). Thus, published data utilizing the (minor) 83L variant may be misleading and not valid for the major variant, 83S.

Table 13. Polymorphisms and relevant genetic variants of MRGPRX subtypesa.

MRGPRX1 MAF (%) MRGPRX2 MAF (%) I36V 4 N16H 10 A46T 17 N62S|N62T 32 R55L 1 F273L 2 MRGPRX3 MAF (%) MRGPRX4 MAF (%) C82R 21 F8L 37 N169D 23 N25K 28 L198R 2 Y54C 38 T297P 2 S83L⁎ 3.4 W307R 4 V142M 3 S150F 1 A182V 14 N245S 2

(Harding et al., 2022; Kooistra et al., 2021; Munk et al., 2016; Pándy-Szekeres et al., 2022)

⁎ In the NCBI database this SNP is indicated as L83S meaning the wildtype amino acid L at position 83 is exchanged for the amino acid S at the same position. However, the MRGPRX4 83L receptor variant has only a frequency of 3.4% in the population. The main MRGPRX4 receptor variant (96.5%) has a serine at this position (MRGPRX4 83S). Therefore, the corrected designation is depicted. a SNPs are depicted in blue, genetic variants in yellow.

3.4.2 MRGPRX4 signaling

Already early on, MRGPRX4 was suggested to be Gq/11-coupled as determined in cell proliferation assays, in which the proliferative response was mediated by MRGPRX4 (Burstein et al., 2006). The effect was not blocked by PTx (Burstein et al., 2006). Later on, the coupling of MRGPRX4 was confirmed since the agonist nateglinide induced Gαq protein activation and subsequent Ca2+ mobilization (Kroeze et al., 2015). In addition, nateglinide-induced β-arrestin recruitment and subsequent MRGPRX4 internalization (Kroeze et al., 2015). Later on, bile acids were reported to activate MRGPRX4 inducing the Gq/11-dependent pathway (Meixiong, Vasavda, Snyder, & Dong, 2019). MRGPRX4 activation induced by deoxycholic acid and bilirubin was blocked by preincubation with either a PLC inhibitor (U73122) or a Gq protein inhibitor (YM254890) (Meixiong, Vasavda, Green, et al., 2019; Meixiong, Vasavda, Snyder, & Dong, 2019; Yu et al., 2019). The discovery of MRGPRX4 agonists and the recent elucidation of the MRGPRX4 structure in complex with Gq protein and the agonist MS47134 has now paved the way to further characterize this receptor and understand its downstream signaling pathways in physiological and pathological conditions (Cao, Kang, et al., 2021).

3.4.3 Functional orthologs

The suggested functional ortholog of MRGPRX4 in mouse is MRGPRA1 (Meixiong, Vasavda, Snyder, & Dong, 2019). The mouse MRGPRA1 shares only 50% identity and 63% similarity with MRGPRX4 in humans. MRGPRA1 was reported to be activated by bilirubin, which induced Ca2+ mobilization in HEK293 cells expressing the mouse MRGPRA1 (Meixiong, Vasavda, Green, et al., 2019; Meixiong, Vasavda, Snyder, & Dong, 2019). This effect was abolished by the Gαq-specific-inhibitor YM-254890 or the PLC inhibitor U73122. Although MRGPRX4 and MRGPRA1 share some ligands, both receptors have their distinct pharmacology. For example, MRGPRA1 was reported to be activated by SP, an MRGPRX2 agonist (Azimi et al., 2016). The existence of a mouse MRGPR that is activated by agonists of two different human MRGPRX increases the complexity of the interspecies relationships within the large MRGPR family. Like all other MRGPRX, MRGPRX4 has an ortholog in rhesus monkeys which shares lower sequence identity (81%) and similarity (87%) compared to identity/similarity of the other human MRGPRXs with their corresponding rhesus monkey orthologs (identities of 87-91% and similarities of 91-94%).

3.5 Single-nucleotide polymorphisms and structures of MRGPRX subtypes

3.5.1 Single-nucleotide polymorphisms of MRGPRX subtypes

Analysis of the MRGPR genes indicated that this family experienced a strong positive selection pressure, which may account for the frequent polymorphisms of its members (Choi & Lahn, 2003). In the MRGPRX subfamily, many genetic variants have been reported, however, only a few of them have more than 1% frequency and are thus considered as single nucleotide polymorphisms (SNPs) (see table 13 for all MRGPRX subtypes’ polymorphisms and variants). In general, SNPs are often cited due to their association to a certain disease, being causative or protective, in addition to their possible effect on drug effectiveness (pharmacogenetics). There is a limited number of studies that investigated the effects of SNPs (minor allele frequency (MAF) >1%) or naturally occurring genetic variants (MAF of >0.1% and <1%).

Genetic variants resulting in R131S (0.6%) and H133R (0.2%) exchange in MRGPRX1 altered the effect of its agonists BAM8-22 and γ2-MSH (Heller et al., 2016). The altered function was ligand-dependent as detected in luciferase gene reporter assays in HEK cells recombinantly expressing the receptor. The R131S variant caused a loss of function of both BAM8-22 and γ2-MSH analogs whereas the H133R variant resulted in a gain of function for γ2-MSH only. However, the clinical relevance of these variants remains unclear.

In MRGPRX2, SP-induced MC degranulation (using RBL-2H3 MC recombinantly expressing MRGPRX2) was decreased or blocked by the following natural variants (with less than 1 % frequency in the population): V123F, R138C, R141C, G165E, D184H, W243R, H259Y, and V282M; thus they might have a protective effect against pseudo-allergic reactions (Alkanfari et al., 2018; Chompunud Na Ayudhya et al., 2019). In contrast, other variants, namely S325L and L329Q, were found to cause the opposite effect increasing SP-induced MC activation (Chompunud Na Ayudhya et al., 2019).

A SNP in MRGPRX4, which results in the N245S MRGPRX4 variant (2%), shows an exclusive expression in Africans and was found to be associated with increased menthol cigarette preference. Menthol was identified as a negative modulator of MRGPRX4 (N245S) since it reduced the response of the agonist nateglinide in Ca2+ and β-arrestin assays in HEK293 cells expressing this receptor (Kozlitina et al., 2019). A SNP indicated in the NCBI and the IUPHAR/BPS databases regarding amino acid position 83 in MRGPRX4 was reported to have a frequency of 3.4% in the population. Careful checking of the nucleotide sequences revealed that the most frequent receptor variant is actually serine-83 (S83), not L83 (as indicated in the NCBI database). The codon TCG, encoding for the amino acid S, has a frequency of 96.5%, while the codon TTG (encoding for the amino acid leucine (L)) has only a frequency of 3.4% (evaluating 1000 genomes) (see Table 13 for an overview of the most common SNPs and their frequencies for all MRGPRX subtypes, in addition to the natural variants with less than 1% frequency).

3.5.2 Structures of MRGPRX subtypes

Solving a GPCR structure has great impact on medicinal chemistry and pharmacology, and 3D structures help to uncover how ligands are positioned within binding pockets, how receptors are activated, and how they may malfunction in disease. Moreover, resolved GPCR structures can greatly facilitate structure-based drug discovery (Stevens et al., 2013). In terms of protein sequence, class A GPCRs are activated through common pathways (Zhou et al., 2019). These pathways are typically triggered by conserved signaling and ligand recognition motifs in many GPCRs, including CWxP, PIF, a Na+ binding pocket, and DRY. Recently, high-resolution cryogenic electron microscopy (cryoEM) structures of MRGPRX2 and MRGPRX4 were solved (Cao, Kang, et al., 2021; Yang et al., 2021). Both receptors were determined as ternary complexes with their G proteins and ligands, MRGPRX4 with Gαq (determined at a resolution of 2.6 Å), and MRGPRX2 with both Gαq and Gαi1 (determined at resolutions ranging from 2.45 to 3.50 Å, see Table 14 for further information). These breakthroughs revealed important structural information about the receptors. The trigger motifs of class A GPCRs are absent or modified in the members of the MRGPRX subfamily. In particular, the essential glycine residue of the sodium binding motif DG (D2.50, G3.39) is replaced by S3.39, and the DRY3.49,50,51 motif (ionic lock for inactive conformation) is modified to ERC3.49,50,51. Likewise, the PIF motif (P5.50, I3.40, F6.44) is altered to MLF in MRGPRX2 and to VLF in the other MRGPRX members. Additionally, the tryptophan residue known as ‘toggle switch’ present in the CWxP motif (G6.47, W6.48, x6.49, P6.50) is exchanged for glycine. Interestingly, the MRGPRX subfamily lacks the disulfide bond between transmembrane helix 3 (TM3) and extracellular loop 2 (ECL2), which is conserved in many class A GPCRs (Wheatley et al., 2012). Instead, a disulfide bond between TM4 and TM5 (C1684.64 and C1805.34) was identified in the new structures (Cao, Kang, et al., 2021; Yang et al., 2021). MRGPRX2 is activated by many diverse ligands, which can now be explained by the absence of the canonical class A TM3– ECL2 disulfide bond. This results in a wide-open extracellular ligand-binding surface. Indeed, MRGPRX2 agonists are bound to a shallower binding pocket which is located closer to the surface of the receptor compared to conventional class A binding sites. The binding pocket is divided into a negatively charged site (sub-pocket 1) and an aromatic hydrophobic site (sub-pocket 2). Interestingly, CST-14 binds to both sub-pockets (see Fig. 6A), while ZINC-3573 only binds sub-pocket 1 (see Fig. 6A). Taking the acidic sub-pocket 1 into account, which contains pivotal acidic amino acid residues (D1845.38 and E1644.60), and regarding its position close to the extracellular domain, might explain why many basic peptides activate MRGPRX2 (see Fig. 6A). Moreover, sub-pocket 1 binds only one basic residue, which allows a single basic amino acid (K or R) of many peptides to activate the receptor (Cao, Kang, et al., 2021).

Table 14. The Protein Data Bank (PDB) ID codes of MRGPRX2 and MRGPRX4 cryo-EM structures.

Receptora Ligand PDB Resolution (Å) Reference Gq-coupled MRGPRX2 (R)-Zinc-3573 7S8N 2.90 (Cao, Kang, et al., 2021) Gq-coupled MRGPRX2 Cortistatin-14 7S8L 2.45 (Cao, Kang, et al., 2021) Gi-coupled MRGPRX2 (R)-Zinc-3573 7S8O 2.58 (Cao, Kang, et al., 2021) Gi-coupled MRGPRX2 Cortistatin-14 7S8M 2.54 (Cao, Kang, et al., 2021) Gi-coupled MRGPRX2 Cortistatin-14 7VDL 3.22 (Yang et al., 2021) Gi-coupled MRGPRX2, local Linear Cortistatin-14b 7VV4 2.97 (Yang et al., 2021) Gi-coupled MRGPRX2 Linear Cortistatin-14b 7VV3 2.97 (Yang et al., 2021) Gi-coupled MRGPRX2 Substance P 7VDM 2.89 (Yang et al., 2021) Gi-coupled MRGPRX2, local C48/80 7VV6 3.30 (Yang et al., 2021) Gi-coupled MRGPRX2, state1 C48/80 7VV5 2.76 (Yang et al., 2021) Gi-coupled MRGPRX2, state2 C48/80 7VDH 2.90 (Yang et al., 2021) Gi-coupled MRGPRX2, local PAMP-12 7VV0 3.50 (Yang et al., 2021) Gi-coupled MRGPRX2, state1 PAMP-12 7VUY 2.84 (Yang et al., 2021) Gi-coupled MRGPRX2, state2 PAMP-12 7VUZ 2.89 (Yang et al., 2021) Gq-coupled MRGPRX4 MS47134 7S8P 2.6 (Cao, Kang, et al., 2021)

a Local: The local resolution map was applied using an algorithm named ResMap; states 1 and 2 indicate different fittings of the secondary structure elements to EM maps b Linear cortistatin: cortistatin derivative, in which the disulfide bond is reduced

Fig 6. Cryo-EM structure of MRGPRX2. A. Cryo-EM structure of Gq protein-coupled MRGPRX2 (cartoon represented and color gray) with peptide agonist cortistatin-14 (stick representation, carbon atoms colored in green), PDB: 7S8L. B. Cryo-EM structure of the Gq protein-coupled MRGPRX2 (cartoon representation in gray) with small molecule agonist (R)-Zinc-3573 (stick representation, carbon atoms colored in magenta), PDB: 7S8N. Oxygen atoms are colored in red and nitrogen atoms in blue. (Cao, Kang, et al., 2021; Yang et al., 2021).

The only reported structure for MRGPRX4 is a ternary complex of MRGPRX4(83L variant)-MS47134-Gq determined at a resolution of 2.6 Å ( see Fig. 7 and Table 14 for further information) (Cao, Kang, et al., 2021). The MRGPRX4 binding pocket appears to be more compact than that of MRGPRX2. The agonist is likewise bound close to the extracellular space. The acidic residues D1775.38 and E1574.60, corresponding to the cationic agonist recognition site in MRGPRX2, are solvent-inaccessible, which might explain the insensitivity of MRGPRX4 towards positively charged ligands (see Fig. 7). MRGPRX4 in fact has a positive electrostatic surface potential, which makes it suitable for negatively charged ligands, in contrast to MRGPRX2. Residue R822.60 is mainly forming ionic interactions with the negatively charged carboxylate function of MS47134 (X4-2, Table 12), whereas R953.25 was found to interact through non-polar interactions with the phenyl group of MS47134 (see Fig. 7).

Fig 7. Cryo-EM structure of MRGPRX4: Cryo-EM structure of the Gq protein-coupled MRGPRX4 (cartoon representation colored in light brown) with small molecule agonist MS47134 (stick representation, carbon atoms colored in cyan) PDB: 7S8P. Oxygen atoms are colored in red and nitrogen atoms in blue (Cao, Kang, et al., 2021).

Unfortunately, only the cryo-EM structure of the MRGPRX4 variant 83L was determined, which is found in a minority (3.4%) of the population. The main variant (96.5%) harbors a 83S residue instead. This error occurred probably due to an error in the NCBI database which has not been corrected up to now. The potency of the agonist MS47134 was reported to be significantly lower in the most abundant MRGPRX4 83S variant as compared to the rare S83L variant, indicating that residue 83 is in fact of high relevance for ligand interaction.

In both receptors, MRGPRX2 and MRGPRX4, the binding pockets are shallow due to the exchange of the conserved ‘toggle switch’ residue W6.48 for G6.48. The recently obtained structural information on MRGPRX receptor subtypes will accelerate and facilitate structure-based drug design and thus the discovery of new therapeutic agents targeting MRGPRXs (see Table 14 for further information).

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Growth Hormone–Releasing Hormone.

GHRH is used for both diagnosis and treatment of GHD.50–52 GHRH cannot be used alone in the diagnosis of GHD as responses to GHRH are variable between individual subjects. GHD is credibly diagnosed by applying both GHRH and arginine. For this test, recombinant analogue GHRH (1-29) 1 μg/kg of body weight is injected intravenously at time 0 and arginine hydrochloride 0.5 g/kg of body weight is infused intravenously during the first 30 minutes, and GH levels are measured every 15 minutes for 90 minutes. Insufficient elevation of GH levels after stimulation is diagnostic of GHD.

Therapeutically, GHRH is used to treat GHD in children. Synthetic GHRH (Sermorelin) was approved in 1998 for GHD treatment, but the manufacturer (Serono) withdrew it from the U.S. market in 2002, so no U.S. Food and Drug Administration (FDA)–approved GHRH is available for routine use now. A synthetic GHRH analogue, CJC-1295, with a longer in vivo half-life has been tested in normal subjects and proven to increase GH and IGF-I.53

The GH Research Society (2000) and the Lawson Wilkins Pediatric Endocrinology Society (2003) have published guidelines on treatment of GHD in children.27,28 The American Association of Clinical Endocrinologists (AACE) has a 2003 guideline on rhGH use for GHD in adults and children.29 The Endocrine Society has a 2006 guideline on treatment of GHD in adults.30 Several authoritative reviews also give advice on GH use.31,33,41 We synthesize a summary here incorporating all agents. In children with GHD, rhGH should be started once the diagnosis is made and terminated when the target height is reached. rhGH should be restarted in the late teenage years or young adulthood if GHD proves to be permanent on repeat testing or with a low IGF-I level in the presence of a structural lesion. rhIGF-I can be used for children with GH resistance. It is not practical in most cases to use GHRH as it is not commercially available in the United States. Whichever agent is to be used, dosing should be individualized and clinical and laboratory responses monitored. rhGH therapy should be used in definitive adult GHD.

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3 Purification and application

Several impurities that must be removed after peptide production are produced along with the peptide [25]. This is because every impurity could potentially exert adverse biological effects on the human body. Therefore, pharmaceutical production has strict purity requirements. Different purification methods, each with its own advantages and disadvantages, have been developed. In turn, in light of the imposed requirements that vary depending on the particular application, the purification strategy must be evaluated for every case. A peptide produced through hydrolysis, for instance, can be separated from the enzyme employed during the process and from other protein residues with a higher molecular mass through ultrafiltration. In this case, the membranes of choice generally have a low molecular mass cutoff, and the size of the membrane pores depends on the molecular weight of the desired peptide. The main disadvantage of ultrafiltration is the poor selectivity of the membrane.

A technique particularly useful for separating charged peptides and proteins is isoelectric focusing (IEF). IEF is based on the same separation principle as electrophoresis. In IEF, the sample is injected into a chamber in which an electric field is applied in the presence of a pH gradient. Acidic species move toward the anode and basic species move toward the cathode. When a species reaches a zone with a pH identical to its isoelectric point, it stops migrating. Then, the species can be moved to a detection window for identification. Thus, IEF separates analytes depending on their isoelectric points. Several modes of IEF have been developed; some can be used on an analytical scale whereas others can be used on a preparative scale in which IEF is conducted in a solution or a cellulose-based separation medium.

Chromatography is the most suitable purification technique for valuable products such as pharmaceuticals, for which high resolution and selectivity are required. This technique allows the highly efficient separation of complex mixtures, in which components have similar chemical properties. This is a flexible and adaptable technique because it enables the choice of many stationary and mobile phases. Moreover, numerous well-established chromatographic methods have been developed at the industrial level and are available for practical biopharmaceutical applications. Different modes of chromatography will be considered in detail in the following sections.

3.1 Reverse-phase liquid chromatography

The mode of chromatography most frequently used to purify peptide and protein mixtures is reverse-phase liquid chromatography (RP-LC). This technique separates analytes based on their hydrophobic properties. C18 ligands are the most employed stationary phases in RP-LC, but occasionally C8 or even C4 ligands have better retentive characteristics, particularly in the case of extremely hydrophobic peptides. In other cases, monolithic, poly(styrene-divinylbenzene)-based columns have been used [26]. All of these stationary phases can distinguish diastereomers (peptide epimers for instance) but not enantiomers.

In RP-LC, the retention of macromolecules such as peptides decreases drastically with the organic modifier content. Therefore, the use of gradient elution is recommended for macromolecules. This is because gradient elution also improves separation of the target peptide from its product-related impurities, because since species with a similar structure can display different adsorption behaviors at a given mobile phase composition. Several peptide mixtures have been separated using gradient elution in RP-LC, such as insulin from its main degradation product A21-desamido insulin and octreotide from impurities. By using a shallow gradient (0.1% ACN [acetonitrile]/min), Harris and coworkers purified mixtures of different synthetic polypeptides 23–51 amino acids long by eluting impurities [27]. Samples varying from 145 to 900 mg of peptide mixtures could be purified through this one-step chromatographic method, including peptides modified with nonproteinogenic substituents (e.g., biotin, carboxyfluorescein). The purity achieved with this technique was almost always above 95%.

Other more eco-friendly solvents have been tested and compared with ACN for elution. Ethanol (EtOH), for instance, exhibited an elution strength and separation characteristics similar to those of ACN during the separation of three peptides (bradykinin, angiotensin II, and angiotensin I). It is a promising alternative for ACN in some cases.

Acidic ion-pairing agents (TFA or formic acid) are often included in the mobile phase to pair with basic amino acids, which are positively charged at acidic pH, improving the peak shape. A study reported that TFA concentration affects peptide or protein recovery. At low TFA concentrations (below 0.05%), strong ion exchange interactions are established between analytes and hydrolyzed silanols, causing peak broadening.

A remarkable advantage of RP-LC over other chromatographic modes is that because of the solvents employed, it can easily be coupled with MS, a detection technique widely used to characterize macromolecules such as peptides and proteins. For instance, MS has enables the separation of bioactive peptides and tryptic digests of different proteins in RP-LC under both acidic and alkaline conditions by using TFA or a buffer composed of triethylamine and acetic acid, respectively, and applying a gradient of ACN. The detectability of peptides under conditions of full-scan negative-ion electrospray ionization MS after the separation at a high pH was two to three times lower than that under conditions of full-scan positive-ion electrospray ionization MS after separation at a low pH.

3.2 Ion-exchange chromatography

In ion-exchange chromatography (IEX), the separation mechanism is based on electrostatic interactions between opposite charges of analytes and the stationary phase. Separation in IEX is modulated by the amount of competitive ions present in the mobile phase. This chromatographic technique is particularly suitable for peptide purification because most peptides have a net charge that can vary depending on the pH of the mobile phase. At acidic pH, carboxylate groups and basic residues (Arg, Hys, and Lys) are mainly protonated. Thus, IEX is often used to characterize charge variants of peptides and proteins, although it has been increasingly replaced by RP-LC because of its incompatibility with MS detection.

To identify peptide modifications such as deamidation or acetylation, which are difficult to detect through RP-LC, IEX is a good choice. Moreover, this technique can distinguish analytes with similar hydrophobicities. For example, using a strong cation-exchange column (Luna SCX containing a phenyl sulfonic acid exchanger) with a salt gradient, bradykinin variants differing only slightly in hydrophobicity or only by one charge could be separated through IEX.

3.3 Hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) is a variant of normal-phase chromatography. HILIC likely involves a partitioning mechanism between the more hydrophobic mobile phase and a layer of solvent rich in water adsorbed on the stationary phase. At a low ACN percentage, the separation mechanism is mainly based on ion exchange, whereas retention increases dramatically at high ACN levels (e.g., greater than 50%) owing to hydrophilic interactions. Moreover, electrostatic effects diminish in importance. The order of elution is opposite that in RP-LC, so retention increases from the least to the most hydrophilic component.

HILIC has been employed for different purposes. Nine peptides present in an illicit drug mixture, including oxytocin, leuprorelin, sermorelin, epitalon, and melanotan II, were separated within 35 min using a gradient elution method by employing LC-DAD-MS (liquid chromatography/photodiode array detection/mass spectrometry). The mobile phases used for the gradient were MP-A: acetonitrile, deionized water, and 100 mM ammonium formate pH 3.0 (80:10:10, v/v/v), and MP-B: acetonitrile/deionized water/100 mM ammonium formate pH 3.0 (40:50:10, v/v/v). For this purpose, a zwitterionic hydrophilic interaction chromatography (ZIC HILIC) column composed of bare silica modified with sulfobetaine was used. In addition, other stationary phases were tested, including CORTECS (made of bare silica) and the BEH HILIC column (composed of unbound porous particles hybridized via ethylene cross-linking). The three HILIC columns (XBridge amide from Waters, bare silica Kinetex HILIC from Phenomenex, and silica with quaternary ammonium and sulfonic acid Nucleodur HILIC from Macherey Nagel) were compared in the context of quantification of proteotypic tryptic peptides, in which chromatography was coupled with ESI (electrosprayionization)-MS.

Higher sensitivity was observed using the amide column without salt buffer in the mobile phases. The HILIC technique is a good solution for separating immunoglobulin-deriving peptides containing modified residues (deamidated asparagine and oxidized methionine) from their native forms. This technique enables the identification and quantification of critical impurities. The method involved a Penta-HILIC column and a gradient elution program in which the percentage of the strong solvent (water containing 0.1% formic acid and 50 mM ammonium formate) increased from 5% to 70% within 90 min, whereas the ACN content decreased.

In addition to several benefits including high productivity, chromatography has drawbacks such as high operating costs and large solvent volumes. A single chromatographic step is typically insufficient to achieve the desired product purity. Therefore, downstream processing usually represents the bottleneck of the entire production workflow in terms of time. This problem is the most concerning aspect related to the greenness of chromatography, because large volumes of non–eco-friendly solvents are required, with potential risks for the environment, human health, and safety.

In the following sections, trends toward the increasing sustainability of peptide processing are discussed [28,29].

A: Greener materials. The first approach that can be used to increase the sustainability of purification processes is to replace toxic materials with more eco-friendly materials. Polar biomolecules, such as some peptides, are usually purified under ion-pair RP-LC conditions by employing acetonitrile/water mixtures as mobile phases to which an ion-pairing reagent is added. This agent masks the charge of polar biomolecules, allowing their retention on hydrophobic adsorbents. Another option is to use EtOH as a solvent. This has several advantages over MeOH, including lower toxicity, generation from biomasses, faster biodegradation, and a slightly lower cost. EtOH has also a higher elution strength than methanol (MeOH), so less EtOH is required for the elution of the target at comparable retention times. The use of EtOH is disadvantageous because of its higher viscosity; most important, it has a lower vapor pressure than MeOH (and ACN). This last condition is particularly problematic in preparative chromatography because the solvent contained in the collected samples has to be evaporated, but removing EtOH is particularly difficult.

B: Green ion-pairing reagents. Ion-pairing reagents are mostly used for the chromatographic separation of peptides. TFA is the most widely used mobile phase additive. It is employed for purification as well as for peptide cleavage after synthesis. Therefore, cationic peptides are obtained as trifluoroacetate salts. Because the most used counterions for peptide drugs are chlorides and acetates, an ion exchange step (IEX or RP-LC) must be used before lyophilization. However, TFA represents a possible breakdown product of more than one million chemicals, including pharmaceuticals, which are potentially hazardous and toxic to the environment. TFA salts are stable and tend to accumulate in water basins such as sea coasts, salt lakes, and oceans. Moreover, TFA may interfere with physicochemical characteristics of the peptide as well as in biological experiments. Thus, TFA is believed to have a negative impact in terms of toxicity. For these reasons, replacing TFA with other ion-pairing reagents is a growing trend.

C: From extraction to catch and release methods for pre–reverse-phase-HPLC(high permeation liquid chromatography)treatments. One or more extraction steps are required for peptide sample pretreatment. Extraction processes principally aim to maximize the yield of the target compound without modifying its physiochemical properties while minimizing the extraction of undesirable compounds. The most common approach to extracting peptides from a solvent-based sample is liquid–liquid extraction. However, this requires large volumes of solvent per sample, usually volatile organic compounds. This is obviously unacceptable from the viewpoint of process sustainability, and requires a reduction in solvent consumption. The ideal extraction method uses minimal amounts of solvent. Water should be the ideal choice; however, for many systems its properties are inappropriate.

D: Greener technologies. In addition to replacing non–eco-friendly chemicals with greener ones, another choice for improving the sustainability of downstream processing is to use greener purification techniques.

In batch liquid chromatography, the injected sample is completely eluted before administering the next injection, and between two consecutive injections, the column is cleaned and regenerated. The amount of sample to be injected is selected to overload the column until a compromise is achieved among purity, yield, and productivity. Excessive overloading leads to worsening from the viewpoint of the resolution between the target peak and close-eluting species. Therefore, this behavior has a direct repercussion on the product's purity. In fact, in preparative single-column chromatography, running into a purity–yield trade-off is common. Some impurities are chemically similar to the target product, and therefore, their chromatographic behavior is analogous. Their resolution becomes insufficient and the baseline of their peaks is not separated. This effect worsens with increasing loadings. Thus, the front and/or the tail of the main peak overlaps with the peaks of the close-eluting impurities. They are typically not collected in the collecting pool, which must fulfill strict purity requirements. With the exclusion of overlapping windows, recovery unavoidably decreases. On the other hand, widening of the collecting window would be beneficial for recovery but detrimental to purity. In this situation, purity and yield cannot both be high at the same time. This represents a limit intrinsic to single-column chromatography, often referred to as the purity–yield trade-off [30]. This trade-off may be alleviated by decreasing the sample volume loaded or by using less steep gradients. However, this would result in lower productivity and higher solvent consumption. Therefore, downstream processing represents a manufacturing bottleneck for many biopharmaceuticals. Continuous chromatographic techniques can overcome this issue because of the countercurrent movement of the stationary phase with respect to the mobile phase.

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PITUITARY HORMONE DEFICIENCY

Hypopituitarism refers to decreased secretion of one or more hormones from the anterior pituitary.21,22 Hypopituitarism can be either due to diseases that damage the pituicytes or due to hypothalamic diseases that result in decreased secretion of hypothalamic releasing hormones. Hypopituitarism can be either acquired or congenital. Tumors, trauma, infiltrative diseases, and vascular accidents are among the most common causes of acquired hypopituitarism, while mutations of genes critical for pituitary development and birth trauma are the most common causes of congenital hypopituitarism.23 Therapeutic goals for hypopituitarism are to optimally replace the normal hormonal milieu.

Prolactin is not replaced, and lactation may proceed without prolactin.24 Hormonal treatment of GH deficiency is discussed here. Although hormonal replacement is a great achievement in medicine and reaches satisfactory clinical and biochemical end points in most patients, the current replacement therapy still does not completely mimic normal physiologic hormone secretion.25

Growth Hormone Deficiency

Pathophysiology

Growth hormone deficiency (GHD) is a syndrome of absolute or relative inadequate GH secretion. Regulation of GH action is summarized in Figure 40-3. During normal physiologic states, the principal regulators of the GH-secreting somatotrophic cells in the anterior pituitary are the stimulatory growth hormone–releasing hormone (GHRH) and inhibitory somatostatin secreted from the hypothalamus. GH acts on GH receptor in the liver to stimulate production of insulin-like growth factor-I (IGF-I) and IGF binding protein 3 (IGFBP3). IGF-I and GH then activate their respective receptors on bone and muscle to promote growth in children or preserve muscle mass and bone mass in adults. Abnormal functioning of any steps in this chain of action results in GHD. Inadequate GHRH secretion can be idiopathic, as in idiopathic isolated GHD in children, or secondary to tumors involving the hypothalamus, as in craniopharyngioma. Neonatal injury to the pituitary stalk cuts off GHRH stimulation and invariably causes GHD. Mutations in homeobox genes such as PROP1 and Pit1 disrupt pituitary organogenesis and cause a decrease in somatotroph numbers, resulting in GHD and other pituitary hormone deficiencies. Pituitary tumors and surgical and radiation therapy for pituitary tumors or other intracranial neoplasms can damage the somatotrophs as well. Mutations in the genes for GH receptor, postreceptor signaling molecules, and IGF-I can all contribute to resistance to GH (Laron syndrome).26 Rarely, IGF-I resistance occurs in patients with abnormal IGF binding proteins. Clinically, GHD is mostly idiopathic in children, but due to acquired pituitary or hypothalamic diseases in adults.

Goals of Therapy

GHD in children needs to be treated. The goals of treatment should be to restore normal growth as much as possible while avoiding GH excess and a long delay in puberty for several years.27–29 Although treatment of adult GHD offers some measurable benefits, the goals of treatment are less clearly defined.29,30 As its pathophysiology indicates, GHRH, GH, and IGF-I are each used in GHD treatment; among them, GH is by far the most important agent.

Growth Hormone.

The rationale of using GH to treat GHD in children is straightforward as GH is the hormone that is lacking in most patients with GHD. Since recombinant human GH (rhGH) made in genetically modified Escherichia coli strains was approved for treatment of childhood GHD in 1985, it has become the mainstay for GHD treatment. Several brands are available in the U.S. market.29 rhGH is a 22-kDa, single-chain polypeptide with 191 amino acid residues, similar to the native human pituitary GH.31 Numerous clinical trials and extensive clinical experience have convincingly demonstrated that rhGH is an effective and safe treatment for most forms of GHD.27–31 Data from National Cooperative Growth Study on more than 12,000 U.S. children demonstrated that rhGH therapy started at an average (±SD) age of 9.2 ± 4.1 years increased growth velocity from 4.4 ± 2.8 cm/yr before treatment to 10.0 ± 3.1 cm/yr 1 year after treatment, and improved the low height standard deviation scores from −2.6 ± 1.1 to −0.5 ± 1.1 after 7 years of treatment.32

After one subcutaneous injection of rhGH, plasma rhGH levels peak in 2 to 6 hours, and IGF-I levels peak in about 20 hours after injection.27–31 The plasma half-life of rhGH is 20 to 30 minutes, and it is metabolized by the liver, kidney, and other organs. For GHD in children, rhGH is administered subcutaneously every day in the evening for 6 to 7 days a week. For younger children, 1 day without injection per week constitutes a “rest” and may improve compliance. The starting dose is 0.3 mg/kg of body weight per week divided evenly into 6 or 7 daily injections. rhGH therapy should continue until final height or epiphyseal closure is reached. Dose titration is based on the growth curve and serum IGF-I and IGFBP3 levels and targeted to achieve sufficient catch-up linear growth and normalize IGF-I and IGFBP3 in order to avoid overdosing. Higher doses can be used if GHD is diagnosed in late childhood to maximize growth within the therapeutic window.

During the transition period from late adolescence to adulthood, GH function should be re-evaluated in those patients with childhood GHD.33,34 Patients with idiopathic or isolated childhood GHD tend to develop normal GH secretion, while those with GHD and multiple pituitary hormone deficiencies, genetic abnormalities, and structural damage to the hypothalamus or pituitary usually remain GH deficient. All those with clear GHD during the transition and afterward would benefit from GH therapy. Patients with irreversible causes of GHD should continue GH therapy, but rhGH doses should be continuously titrated down to avoid overdosing and to maintain normal GH status. When restarting GH therapy, it is suggested to begin with 0.4 to 0.8 mg/day and to increase the dose by 0.2 to 0.4 mg/day every 4 to 6 weeks until a maintenance dose of 1.2 to 2.0 mg/day is achieved.29 The maintenance dose may need to be titrated down periodically to avoid adverse effects.

Adverse effects of rhGH are related to overdosing.27–31 Children may develop coarse facial features. Rare allergic reactions may occur. Due to the concern that rhGH may promote tumor growth, rhGH treatment in tumor survivors should be coordinated with a pediatric oncologist or neurosurgeon in order to monitor for tumor relapse. Current data do not indicate that rhGH causes such a relapse, although the risk of secondary neoplasms appears to increase.35

rhGH therapy is also approved for treating other childhood diseases characterized by short stature or relative GHD. These diseases are Turner's syndrome, chronic renal failure, small for gestational age, Prader-Willi syndrome, and severe idiopathic short stature. rhGH treatment in these diseases varies, and readers should refer to published guidelines.29

The decision to treat GHD in adults should be made on an individual basis, and rhGH treatment probably most benefits those with clear structural pituitary diseases, with demonstrated laboratory abnormalities in GH secretion following provocative testing, and low IGF-I levels.29–31,36 In these patients, rhGH therapy increases lean body mass and decreases fat mass, increases bone mineral density, lowers LDL and total cholesterol levels, and improves the quality of life. rhGH is injected subcutaneously daily, and dosing should be titrated up from a conservative starting dose, which is usually 0.3 mg/day. A body weight–based regimen commonly causes overdosing and should not be used in adults. Dosing is increased by 0.1 to 0.2 mg/day every 1 to 2 months, targeting the clinical response (such as improvements in energy levels and muscle mass) and keeping IGF-I levels in the upper half of normal range. The optimal duration of rhGH treatment is not clear, but treatment perhaps can continue indefinitely if rhGH produces expected benefits. Although there is only weak and controversial evidence suggesting that rhGH treatment for adult GHD may facilitate tumor growth, current guidelines suggest that rhGH treatment is contraindicated in patients with concurrent malignancies; clinicians should make a balanced decision regarding rhGH usage with concurrent malignancy.

Several studies show that administration of exogenous GH to healthy elderly without GHD modestly increases lean body mass and decreases fat mass.37–40 The increase in muscle mass, however, does not translate into increase in muscle strength or functioning levels. If the healthy elderly undergo muscle strength training, GH does not give any additional benefits. In addition, some individuals treated with GH develop carpal tunnel syndrome, gynecomastia, or hyperglycemia. Malignancy associated with GH therapy is also a theoretical concern. Due to these issues, GH should not be used as an antiaging agent, and prescribing GH for “antiaging” purposes is unwise (and illegal) in the United States. The limited benefits extra GH can confer to a healthy elderly adult can be easily achieved with exercise.41,42 rhGH is also used for treating lipodystrophy in adult and pediatric patients infected with human immunodeficiency virus.43,44

Insulin-like Growth Factor-I.

As IGF-I is the major hormonal mediator of GH actions, IGF-I therapy is a rational treatment of patients with GH resistance.45–48 Recombinant human IGF-I (rhIGF-I) and recombinant human IGF-I/IGFBP3 complex (rhIGF-I/rhIGFBP3) were both approved for treatment of primary IGF-I deficiency. rhIGF-I is produced in genetically modified E. coli strains and is a 7.6-kDa single-chain polypeptide of 70 amino acid residues. The amino acid sequence of rhIGF-I is identical to that of natural human IGF-I. rhIGF-I binds to IGF-I receptor and stimulates skeletal growth. Five clinical trials including a total of 71 children (chronological age 6.7 years at time of starting treatment) with primary IGF-I deficiency treated with rhIGF-I for 1 to 8 years demonstrated that rhIGF-I accelerates height velocity from 2.8 cm/yr before treatment to 8.0 cm/year in the first year of treatment. In the 13 patients who completed 8 years of rhIGF-I treatment, standard deviation score went from −6.7 to −5.2. Bioavailability of rhIGF-I after one subcutaneous injection is close to 100% in normal volunteers. rhIGF-I binds to endogenous IGFBP3 in the plasma. Plasma rhIGF-I concentration peaks in 2 hours after injection, and half-time for elimination is 5.8 hours. rhIGF-I is metabolized by the liver and kidney. rhIGF-I dosing should be individualized. The starting dose is 40 to 80 μg/kg of body weight twice a day injected subcutaneously. The dose can be increased by 40 μg/kg per dose, to a maximum of 120 μg/kg given twice a day. Mild to moderate hypoglycemia occurs in 42% of patients, consistent with the structural homology between rhIGF-I and insulin. Other adverse events include lipohypertrophy at injection sites and rarely benign intracranial hypertension.

The adverse effects of rhIGF-I may be related to high free IGF-I levels. To avoid these adverse effects, rhIGF-I has been mixed at a 1 : 1 molar ratio with rhIGFBP3.49 The rhIGF-I/rhIGFBP3 complex has a longer plasma half-life than free IGF-I, which is more rapidly eliminated from the circulation. Produced also in E. coli, rhIGFBP3 is a 29-kDa single-chain polypeptide of 264 amino acid residues. The amino acid sequence of rhIGFBP3 is identical to that of natural human IGFBP3. The efficacy of rhIGF-I/IGFBP3 has been established in a prospective, open-label clinical trial of 36 children (chronological age 8.7 years at time of starting treatment) with IGF-I deficiency. rhIGF-I/IGFBP3 accelerates height velocity from 2 to 3 cm/yr before treatment to 7 to 9 cm/year in the first 6 months of treatment. Standard deviation score improved by 0.4. Plasma rhIGF-I and IGFBP3 concentrations peak in 11 to 19 hours after injection, and half-time for elimination is 13.4 hours for IGF-I and 54 hours for IGFBP3. rhIGF-I dosing should also be individualized. The starting dose is 0.5 mg/kg of body weight subcutaneously once a day. The dose can be increased to a maximal of 1 to 2 mg/kg daily. Adverse effects are fewer and less severe than those caused by rhIGF-I alone.

There has been limited experience with rhIGF-I and rhIGF-I/rhIGFBP3 in the treatment of primary IGF-I deficiency. Generally their efficacy is less impressive compared with that of rhGH for common GHD, suggesting that GH has IGF-I–independent growth benefits. These agents, however, are valuable as rhGH does not work well in the treatment of primary IGF-I deficiency.45–48

Growth Hormone–Releasing Hormone.

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URL: https://www.sciencedirect.com/science/article/pii/B9781416032915500445

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