By Jana Hennelová
G protein-coupled receptors (GPCRs), which mediate the transduction of various extracellular signals to the cell’s interior, belong to one of the largest protein superfamilies in the mammalian genome. As many of these receptors are involved in various physiological processes, they are often targeted in development of therapeutic drugs (Odoemelam et al., 2020). Namely, the calcitonin-gene related peptide receptor has been implicated in migraine pathology and therefore is a target in migraine prophylaxis. Recently, monoclonal antibodies targeting this receptor (anti-CGRP mAbs) have been shown to be effective for this purpose (Bhakta et al., 2021). Nevertheless, more tests examining their efficacy and safety are needed. Firstly, this review article summarises the structure and function of GPCRs, outlining structural characterisation and mode of activation of CGRPRs. Then, it compares the differences in mechanisms of action, efficacy, and safety of different therapeutic drugs for migraine treatment, including anti-CGRP mAbs.
2.1 GPCRs overview – structure and mode of action
GPCRs are a family of cell surface receptors consisting of seven transmembrane domains that are coupled to different classes of trimeric G proteins. Ligand binding to GPCRs leads to the activation of both the receptor and the associated G protein (Figure 1). This induces various signal transduction pathways that modulate effector molecules (Odoemelam et al., 2020). To understand the regulation and processing of these pathways and how they lead to a specific physiological response of cells or tissues, it is essential to understand the molecular mechanisms and structural changes of GPCRs and its corresponding G proteins. For a long time, a ternary complex model has been widely accepted as a mechanism of GPCR-mediated signal transduction (Samama et al., 1993). This model proposes that GPCR exists only in two conformational states: as an inactive form in the absence of a ligand, and in an active form that is triggered by ligand binding. This leads to the formation of a ternary complex which is composed of a ligand, an active GPCR and a corresponding G protein (Samama et al., 1993). However, current studies show that there are numerous intermediate conformations of GPCRs (Gurevich & Gurevich, 2017). However, these structures are difficult to determine by conventional structural biology techniques, such as X-ray crystallography, which rely mainly on inhibitors bound to inactive forms of GPCRs (Josephs et al., 2021). With new advances, such as cryo-EM, it became possible to determine the active forms of GPCR in ternary complexes. However, to determine the key intermediate states of these receptors (e.g. apo and agonist-bound), it is essential to isolate non-modified GPCRs. This can be achieved by using protein engineering techniques, such as the removal of mobile domains or increasing receptor thermostability (Josephs et al., 2021).
Figure 1. Activation of a G protein upon ligand binding to GPCRs.
In the absence of a ligand, GPCR is in an inactive state and the alpha subunit of its coupled G protein binds GDP (a). Upon ligand binding to either extracellular N-terminus/loops or transmembrane helices of a GPCR (b), a conformational change allows the receptor to function as a guanine nucleotide exchange factor (GEF). GEF mediates the exchange of GDP for GTP on the alpha G protein subunit (c, d). The activated G protein then dissociates into GTP-bound alpha subunit and beta-gamma dimer (e), which can stimulate downstream signalling pathways by the activation of effector molecules. The hydrolysis of GDP to GTP by G protein alpha subunit, triggered by GTP-ase activating proteins, leads to G protein re-association and a start of a new cycle in the presence of a ligand. If the ligand is absent, GPCR exists in its inactive form and is no longer able to activate the corresponding G protein, and signal transduction is thus terminated (f, a) (Luo et al., 2019).
2.2 CGRPR overview
Josephs and colleagues (2021) focused on the molecular dynamics of a particular class of GPCRs, the calcitonin-gene related peptide receptor (CGRPR), to understand its implications in migraine pathology. CGRPR is a heterodimer composed of calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein 1 (RAMP1) (Liang et al., 2018). CGRPR is activated by binding of its ligand, calcitonin-gene related peptide (CGRP), which activates downstream signalling pathways that regulate various physiological and metabolic functions (Josephs et al., 2021). It has been shown that CGRP release is triggered by neurogenic inflammation, resulting in acute and chronic migraine pathology (Charles & Pozo-Rosich, 2019). Therefore, it is important to understand the structural changes that occur upon ligand binding to the receptor, as such data would have important implications for developing and assessing the efficacy and safety of various migraine-specific drugs targeting CGRPR.
2.3 Migraine prophylaxis targeting CGRP pathway
Migraine has been listed as the second main cause of disability in society (Stovner et al., 2018). Despite such a high prevalence of this condition, until recently, there have been very few migraine-specific therapies available, as migraine treatment has been mainly relying on drug repurposing. Nevertheless, with the discovery of CGRPR involvement in migraine pathology, various novel approaches for migraine treatment have been developed (Charles & Pozo-Rosich, 2019). Clinically approved monoclonal antibody (mAb) treatments include antagonists of neuropeptide CGRP, such as fremanezumab, galcanezumab, and eptinezumab, and the CGRPR antagonist erenumab. These antibodies are used as a means of preventative migraine treatment. Furthermore, small molecule drugs such as ubrogepant and rimegepant, act as CGRPR antagonists and are used to treat acute migraine. All these drugs demonstrate variable efficacy, safety, and tolerability when used for therapeutic purposes (Bhakta et al., 2021). To assess their benefit-to-risk ratios in the prevention of episodic and chronic migraine, Drellia and colleagues (2021) conducted a likelihood-to-help versus harm analysis of anti-CGRP therapeutics and compared the results with conventional treatments for this condition.
3. CGRPR structural characterisation study
Josephs and colleagues (2021) conducted a study to determine the intermediate stages of an active CGRPR, building upon their previous work on structural characterisation of CGRP in a complex with a G protein (Liang et al., 2018). Using single-particle cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange mass spectrometry (HDX-MS), the research group determined the structure of CGRPR in the absence (apo form) or the presence (peptide-bound form) of its ligand (Figure 2). Importantly, by using an unmodified receptor, it was possible to observe the molecular dynamics of the receptor upon ligand binding. This provided essential information about the conformational changes of CGRPR during its activation process which are described in Figure 3.
Figure 2. Cryo-EM density map of the apo (A) and peptide-bound (B) CGRPRs.
The colour-coding of the density maps corresponds to the local resolution in Angstroms, ranging from the highest resolution (dark blue) to the lowest resolution (red). The structures of the apo and peptide-bound form of CGRPRs, mainly their intracellular domains, exhibit a high degree of similarity.
Adapted from: (Josephs et al., 2021).
Figure 3. Overview of the peptide binding and activation process for the CGRPR.
In the CGRPR unbound state, the TM bundle exhibits a relatively open conformation (1). Ligand binds to the dynamic ECD which stabilises the RAMP1-ECD interaction. Subsequently, RAMP1 interacts with ECL2 of the receptor while the N-peptide terminus stays dynamic (2). To achieve a fully-active CGRPR state, the dynamic intracellular loops change their conformation to bind G protein. This results in the shift of TM5 and TM6 domains which has two consequences. Firstly, this conformational change enhances the engagement of G protein with the receptor. Secondly, the interaction of the peptide N-terminus is stabilised through the additional conformational changes of TM1, TM7 and ECL2 domains (3).
Adapted from: (Josephs et al., 2021).
4. Anti-CGRP mAbs mode of action study
Bhakta and colleagues (2021) conducted a study to provide the missing data about the potential differences in the mechanism of action of migraine therapeutics. Specifically, the research group focused on three agents, i.e. fremanezumab (anti-CGRP mAb), erenumab (anti-CGRPRmAb), and telcagepant (small molecule CGRPR antagonist). Several assays, comparing the mode of action of these therapeutics, showed substantial differences between them (Bhakta et al., 2021).
Flow cytometry-based binding assay showed that erenumab bound to CGRPR with high efficiency (i.e. it bound to 98 percent cells expressing CGRPR), and only in the presence of RAMP1. This is in line with previous studies showing that both erenumab and telcagepant binding epitopes are present at the CLR/RAMP1 interface of CGRPR (Liang et al., 2018). Moreover, receptor occupancy in the presence of erenumab and an endogenous ligand (alpha-CGRP) was lower than with erenumab alone, suggesting the competitive nature of erenumab binding to the CGRPR. Alternatively, the observed reduction in receptor occupancy might have been a consequence of ligand-induced receptor downregulation. Neither a control mAb nor fremanezumab bound to the receptor (Bhakta et al., 2021).
An inhibition assay was conducted to assess the ability of the three drugs to inhibit CGRPR-mediated signalling in the presence of its agonists alpha-CGRP, adrenomedullin, and intermedin. All of these agonists act by increasing the intracellular concentration of cAMP. Results from this experiment showed that all three drugs are able to antagonise the action of an agonist alpha-CGRP. However, only CGRPR antagonists (erenumab and telcagepant) inhibited cAMP accumulation in the presence of either adrenomedullin or intermedin, while the CGRP antagonist (fremanezumab) showed no antagonism in the presence of these two agonists. Moreover, it was discovered that while erenumab binding to the receptor triggered its internalisation via receptor-mediated endocytosis, fremanezumab administration did not change the levels of cell surface CGRPR. This suggests that no internalisation of the receptors occurred in the presence of fremanezumab (Bhakta et al., 2021).
Further experiments showed that erenumab can bind not only CGRPR, but also another, closely related receptor, called amylin receptor (AMY1). This is due to the structural similarities between AMY1 and CGRPR, as AMY1 is also formed by RAMP1 dimerisation, except with calcitonin receptor (CTR) rather than CLR (Liang et al., 2018). Since erenumab binds to CGRPR at the interface of CLR/RAMP1, it is also able to bind to AMY1 at a similar epitope on the CTR/RAMP1 interface. This cross-reactivity of erenumab suggests potential adverse effects as a migraine therapeutic treatment. In comparison, no cross-reactivity was observed in case of fremanezumab, making it a better candidate for anti-migraine treatment.
5. Anti-CGRP mAbs efficacy, safety, and benefit-risk ratios study
The mode of action research led to further studies testing the efficacy of such therapeutics against migraine. Drellia and colleagues (2021) analysed data from phase 3 randomised clinical trials (RCTs) to compare the efficacy and safety of mAbs to conventional migraine therapeutics, which mainly include repurposed drugs (e.g. antiepileptics, beta-blockers, or anti-depressants). For episodic migraine, these are topiramate and propranolol; whereas topiramate and onabotulinumtoxinA are used to treat chronic migraine (Drellia et al., 2021).
Likelihood to help vs harm (LHH) ratios showed that all tested drugs were efficient in migraine prophylaxis. The only exception to this was topiramate whose efficacy was comparable to the placebo group, and various adverse effects were observed during treatment. However, anti-CGRP mAbs showed higher efficacy than conventional drugs for both episodic and chronic migraine. Moreover, their LHH ratios were also higher, suggesting greater safety and fewer adverse effects. This might be a solution for the low treatment adherence often observed during migraine treatments, which is also the main cause of failure in migraine treatment (Hepp et al., 2017).
Despite differences in the mode of action of anti-CGRP and anti-CGRPRmAbs described previously (Bhakta et al., 2021), the efficacy and safety ratios did not differ significantly between the tested agents. This is a promising result, showing a great potential of migraine-specific therapeutic agents. Nevertheless, it has been predicted that the preference of these novel drugs over conventional migraine therapeutics will most likely be a consequence of patients’ intolerance to the conventional treatments, rather than the greater efficacy of mAbs. This is due to the limited timescale of clinical trials evaluating mAbs in chronic migraine, during which not all adverse effects may have been observed and some may arise later. Therefore, the preference of using mAbs over conventional migraine treatments might not be as profound due to the lack of head-to-head comparison studies, and will most likely depend on various factors, including patients’ comorbidities.
Structural studies of GPCRs, showing their different conformational stages in the presence and absence of a ligand, have different applications in the development of therapeutic agents to treat various diseases. Namely, studies focusing on the structure and dynamics of CGRPR have proved to be useful in understanding and comparing the mode of action of conventional and antibody-based migraine prophylaxis. From those, mAbs targeting CGRPR and its ligand showed higher efficacy, specificity and safety compared to conventional drugs. Nevertheless, the data about safety and adverse effects is incomplete due to the limited timescale of clinical trials. Therefore, further studies to evaluate benefit-risk ratios for these migraine-specific treatments are needed to confirm the preferential use of these agents for episodic and chronic migraine treatment.
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