Muscarinic Receptors: From Clinic to Bench to Clinic

Abstract

Efforts to target muscarinic acetylcholine receptors in the brain have been hampered by dose-limiting side effects. In a tour de force of team science, Brown and colleagues have designed a muscarinic agonist that has been optimized to possess properties that could position it to succeed where other agonists have failed.

Alzheimer’s disease is characterized by the death of cholinergic neurons and this neurodegeneration is thought to play a key role in the cognitive deficits observed. Currently used therapeutics for treating Alzheimer’s disease act by promoting cholinergic signaling in brain areas that are both hypocholinergic in patients and critical to cognitive processing. However, these therapies also promote cholinergic signaling in peripheral tissue which can lead to dose-limiting side effects. A seminal study using the muscarinic acetylcholine receptor agonist xanomeline demonstrated that targeting muscarinic receptors could provide robust, dose-dependent efficacy in ameliorating cognitive deficits in Alzheimer’s disease patients [1]. However, gastrointestinal side-effects ultimately prevented this compound from continuing in the clinic. The task of designing a therapeutic that retains the efficacy of xanomeline but with reduced side effect liability has been tackled by numerous groups that have employed different strategies. One of these strategies has been to target specific subtypes of acetylcholine receptors that are best associated with pro-cognitive effects. The muscarinic receptor family contains 5 subtypes (M₁-M₅) and a wealth of previous work has identified the M₁ receptor as a major player in regulating cognitive function [for review see 2]. Accordingly, the focus of numerous drug discovery efforts has been to find compounds with improved selectivity for receptors that mediate pro-cognitive effects (M₁), without activating receptors that mediate negative side effects (M₂, M₃). Building upon previous advances in our understanding of the structures of muscarinic receptors [3], Brown and colleagues determined the structures of the M₁ receptor bound to several different agonists and performed molecular dynamics simulations to help determine ligand binding sites and interactions [4]. These atomic-level insights were ultimately leveraged to help design HTL9936, a compound with agonist activity at the M₁ receptor and no activity at the M₂ or M₃ receptor subtypes.

In addition to receptor subtype-selectivity, it has also become clear that the mode of interaction between drug and receptor can play a key role in determining the therapeutic window between efficacy and unwanted side effects. Compounds with agonist activity can activate receptors directly upon binding whereas positive allosteric modulators (PAMs) can alter the affinity or efficacy through which an agonist can induce receptor activation. Agonists can be further classified based on the degree to which they induce receptor activity with full agonists inducing maximal receptor activation and partial agonists producing less than maximal activation. Some compounds that are highly selective for the M₁ receptor subtype and possess both allosteric agonist activity and PAM activity have been observed to induce side effects that are similar to those seen with non-selective compounds in pre-clinical settings [5]. However, PAMs that have little to no agonist activity have been shown to mediate pro-cognitive efficacy in rodent models with little to no side effects [6,7]. These findings have led to a working hypothesis that higher levels of agonist activity may be associated with greater side effect liability. Here, Brown and coworkers report that HTL9936 is a partial agonist in that it can activate the M₁ receptor but with reduced efficacy compared to other synthetic agonists or the native neurotransmitter acetylcholine [4]. Furthermore, HTL9936 showed little to no bias across the signaling pathways examined, another attribute that could be favorable towards avoiding cholinergic side effects [8]. The pharmacological profile of a non-biased partial agonist activity may represent a sweet spot in the pharmacological landscape that could both i.) diminish the adverse effect profiles seen with full agonists or compounds with certain forms of signal bias and ii.) maintain utility in advanced Alzheimer’s disease patients in which extensive cholinergic neurodegeneration may diminish the potential efficacy of pure allosteric modulators. This hypothesis is supported by the behavioral efficacy observed with HTL9936 in several pre-clinical models across numerous species [4]. The behavioral efficacy observed in rodents and dogs was observed at doses well below those where adverse effects were seen. Classic cholinergic side effects were observed in dogs at high doses but not in either rodents or non-human primates at equivalent doses. This could suggest that the side effect profiles of M₁ agonists may be species-dependent and highlights the importance of utilizing multiple models/species. Ultimately, Brown et al. report that HTL9936 was well tolerated in elderly volunteers and produced changes in brain activity that are associated with cognition enhancement [4]. While further studies in patient populations will be required to determine the clinical utility of M₁ agonists such as HTL9936, the work by Brown and colleagues is an excellent example of how insights from previous clinical trials can inform drug discovery efforts (Figure 1).

Figure 1.

Flowchart of the discovery and validation of HTL9936, a drug with great potential for treating cognitive disturbances in Alzheimer’s disease. Insights from previous clinical studies guided efforts employing structural biology to obtain drugs with enhanced selectivity for the M₁ muscarinic receptor. Compounds were then optimized to contain partial agonist activity and a pharmacological profile that demonstrated pro-cognitive efficacy in multiple pre-clinical models with little to no adverse side effects. The lead compound HTL9936 was then moved into clinical trials where it was well tolerated and produced changes in brain activity that are associated with cognition enhancement.

In addition to Alzheimer’s disease, muscarinic receptors are being targeted for the treatment of schizophrenia and xanomeline has been shown to have efficacy across multiple symptom domains in schizophrenia patients [9,10]. One working hypothesis is that the majority of cognition-enhancing effects seen with xanomeline may be mediated primarily via the M₁ receptor while the antipsychotic-like effects may be more M₄-dependent [2]. Interestingly, despite possessing M₄ agonist activity in vitro, HTL9936 administration did not produce antipsychotic-like effects in vivo [4]. Determining if HTL9936 is a full or partial agonist or possesses any signal bias at the M₄ receptor could shed light on this discrepancy. In addition, because xanomeline possesses agonist activity at M₂, M₃, M₅ (while HTL9936 does not), there is also the possibility that the efficacy of xanomeline is dependent on the engagement of numerous muscarinic receptor subtypes in the brain. Further studies will be needed to determine the disconnect between HTL9936 and xanomeline in terms of antipsychotic-like efficacy.

Over the past few decades, there has been tremendous progress made in terms of identifying and understanding pharmacological properties that could provide roadmaps for developing therapeutics targeting muscarinic receptors. One recent approach that has shown great promise has been the co-administration of xanomeline with a peripherally restricted antagonist [9]. Other approaches have included developing PAMs that are highly selective for either the M₁ receptor [7, ClinicalTrials.gov/show/NCT03220295] or the M₄ receptor (ClinicalTrials.gov/ct2/show/NCT04136873). As highlighted above Brown et al. have provided evidence that the partial agonism of the M₁ receptor is a very compelling path towards harnessing the clinical potential of muscarinic receptors. While these efforts each have different rationale and goals they all strive to achieve some degree of the clinical efficacy observed with xanomeline with reduced side effect liability. With so many diverse approaches heading to the clinic, this is an exciting time in the development of muscarinic receptor-based therapeutics which have the potential to improve the treatment of patients suffering from multiple disorders.