Drug Discovery: Decoding the mechanisms of allostery
G protein-coupled receptors are transmembrane proteins that help to regulate a wide array of biological processes, which makes them important drug targets. However, different receptors often share a high similarity in their sequences, especially at their binding sites, which often results in challenges to develop drugs that target a specific receptor (Vuckovic et al., 2019; Singh and Karnik, 2021).
For example, the five members of a muscarinic acetylcholine receptor subfamily (M1-M5 mAChR) have essential roles in various physiological processes (Wess et al., 2007). In particular, M4 mAChR is of major therapeutic interest due to its involvement in regulating dopaminergic neurons involved in cognition, psychosis and addiction, while others, such as M1 mAChR, can be targeted to treat cognitive decline in Alzheimer’s disease (Wess et al., 2007). However, these receptors share highly similar binding sites, and drugs that target a particular mAChR receptor often inadvertently activate other receptors in the subfamily, thereby causing adverse side effects (Felder et al., 2018).
As an alternative to targeting the primary binding site on the receptor (also known as the orthosteric site) with a drug, it is sometimes possible to regulate a receptor by targeting a remote (or allosteric) site. Since there is much less similarity in the sequences of allosteric sites, this approach makes it possible to design highly selective drugs with reduced side effects.
Now, in eLife, David Thal, Arthur Christopoulus and Celine Valant (all at Monash University), Yinglong Miao (University of Kansas) and colleagues – including Ziva Vuckovic, Vi Pham and Jesse Mobbs (all at Monash) and Jinan Wang (Kansas) as joint first authors, along with colleagues in Japan, the United Kingdom and the United States – report on the molecular mechanisms that govern allostery in human M4 AchR (Vuckovic et al., 2023). The researchers used two ligands that targeted the orthosteric site (acetylcholine and iperoxo), and two positive modulators that targeted the allosteric site (VU154 and LY298). Both modulators have shown antipsychotic efficacy in preclinical rodent models, but these results have failed to translate into human studies (Suratman et al., 2011; Dupuis et al., 2010). Nevertheless, they remain useful tools for investigating allostery in G protein-coupled receptors (Bubser et al., 2014).
Vuckovic et al. used two types of biochemical assays to determine the pharmacological characteristics of the allosteric modulators. This revealed that both LY298 and VU154 display a phenomenon called ‘probe dependence’, meaning that they had a stronger effect when the orthosteric ligand was acetylcholine rather than iperoxo. They also showed that these effects were caused by an increase in the binding affinities of the orthosteric ligands, rather than by any modulation of signaling through the receptor (Figure 1A). Moreover, LY298 was the more potent modulator as it caused a 400-fold increase in binding affinity, compared with a modest 40-fold increase for VU154.
To uncover the molecular basis for these results, Vuckovic et al. used cryogenic electron microscopy (cryoEM) to obtain four structures of M4 AChR bound to its cognate G protein and in complex with various ligands. The structural analyses – combined with molecular dynamics simulations – enabled the authors to uncover the underlying dynamics and conformational changes that are otherwise missed through static snapshots of cryoEM structures.
The experiments revealed that the allosteric sites for both VU154 and LY298 were, as expected, located in a region of the receptor called the extracellular vestibule. The orthosteric sites overlapped with those in other members of the mAChR subfamily and were located inside a central ‘pocket’ in the receptor; However, it was noticed that this pocket was contracted around iperoxo but not around acetylcholine. The smaller binding pocket, along with the rotation of a specific tyrosine residue, resulted in more stable interactions for iperoxo within the orthosteric site. On the other hand, the binding of acetylcholine was seen to be more dynamic with fewer stable interactions (Figure 1B).
Surprisingly, even though iperoxo bound to the receptor more tightly than acetylcholine, its ability to promote signaling through the receptor was lower. Vuckovic et al. suggest that since the acetylcholine-bound M4 AChR is more dynamic, it can sample a large range of conformations, including those that couple to and activate G protein. This allows the receptor to efficiently activate the G protein and increase the signaling response.
The structures and molecular dynamics simulations also helped uncover the molecular basis for the probe dependence of the allosteric modulators. It is possible that both had stronger effects on the acetylcholine-bound receptor due to the stabilization of an inherently dynamic structure. Conversely, since the iperoxo-bound receptor was already very stable, the modulatory effects were negligible. This result provides a key future consideration for the development of allosteric drugs that target G protein-coupled receptors.
Using mutational studies, Vuckovic et al. also identified a network of amino acids that were important to the conformational dynamics of the protein, some of which showed maximum variability between structures and modulated the signaling efficacy of both orthosteric and allosteric ligands.
Lastly, the researchers investigated why VU154 is potent in some species but not in others. Based on their initial findings, VU154 was a weaker positive allosteric modulator than LY298 in humans because it poorly stabilized the active receptor conformation. However, its effects in mice were stronger, and were comparable to the effects of LY298 in humans. Using mutational studies, Vuckovic et al. identified three important residues on the human receptor that confer species-selectivity. Mutating these to the equivalent residues in the mouse sequence resulted in improved allostery by VU154 in functional studies and stable binding in simulations.
In conclusion, Vuckovic et al. have described the complex interplay between structure, conformational dynamics and pharmacology that defines allostery at G protein-coupled receptors. Their work provides a detailed framework to guide future drug discovery efforts focused on the muscarinic receptor subfamily.
References
-
Current trends in GPCR allosteryThe Journal of Membrane Biology 254:293–300.https://doi.org/10.1007/s00232-020-00167-6
-
DataStructure of the M5 muscarinic acetylcholine receptor (M5-T4L)Worldwide Protein Data Bank.https://doi.org/10.2210/pdb6ol9/pdb
-
Muscarinic acetylcholine receptors: mutant mice provide new insights for drug developmentNature Reviews. Drug Discovery 6:721–733.https://doi.org/10.1038/nrd2379
Article and author information
Author details
Publication history
Copyright
© 2023, Khan and Gati
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 862
- views
-
- 99
- downloads
-
- 2
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.
-
- Biochemistry and Chemical Biology
- Cell Biology
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.