Application of cryo-EM to understanding of GPCR structure, function and drug discovery

GPCRs are among the most important therapeutic drug target classes. The existing technical advances in GPCR cryo-EM provide a robust template for the generation of novel structures that could be used to facilitate new drug design and development. Moreover, increasing numbers of cryo-EM GPCR structures are being solved, both by our laboratories and others that enable innovative questions to be asked about how different drug chemotypes bind and how different ligands influence the conformational dynamics of the structures. Within this major Theme, using existing technologies and advances developed under Major Theme (i), structures will be determined for GPCRs that have validated and/or potential therapeutic interest. This will include new, first in class structures, and novel drug:GPCR complexes.

  

This work will be completed with the Monash University Node.

Project Areas

MIPS researchers are studying GPCRs from 3 of the major subclasses; Class A, Class B (particularly B1) and Class C. Receptor targets will be chosen based on alignment between potential industry partners and the host academic laboratories.

Novel GPCR structures

Although GPCRs account for ~30% of marketed drug targets, these account for only a small portion of potentially therapeutically important receptors. For many receptors, no structures current exist or the available structures are confined to a single receptor state (most also with only a single ligand). Within this project area, novel receptor structures will be determined, along with analysis of conformational dynamics of individual ligand receptor complexes. These novel structures can provide a template for future structure-directed drug discovery and development.

 

Efficacy and complex stability

The ability to generate stable complexes of GPCRs with agonists and transducer proteins is influenced by ligand affinity and ligand efficacy, with the latter likely the critical factor for success. Despite this, there is only limited information on efficacy thresholds that need to be met and how these might vary with different technologies for complex stabilisation and solubilisation. However, early chemical hits and leads often have low potency. Understanding the relationship between efficacy and complex stability for an individual receptor is critical for integration of cryo-EM in drug discovery and development workflows. Moreover, in the context of the ability to form complexes with different transducers, projects could also potentially address the ability of structures to assist in understanding of biased agonists.

This project area will address this important knowledge gap for individual receptors.

 

Ligand affinity and potential for fragment screening

As we move towards new technical advances that will enable the determination of apo and inhibitor-bound GPCR structures by cryo-EM, research that addresses the capability of cryo-EM for understanding of binding of low affinity ligands (including fragments) will be increasingly important. This project area overlaps research on technical development of cryo-EM and will explore, for individual GPCRs of interest, the balance between affinity, solubility and the ability to achieve structural resolutions that could support drug discovery and development programs.

Development of approaches for inactive, antagonist bound GPCR structures determination

A/Prof Denise Wootten, Dr Rachel Johnson, Dr Yan Jiang and Prof Patrick Sexton

Industry partner: Pfizer, USA

 

The project will seek to identify approaches that could enable structure determination of inhibitor-bound GPCRs using the class A vasopressin V1a and/or V2 receptors as exemplars. Vasopressin receptor inhibitors have potential for the treatment of many clinical conditions. This project will use a selection of commercially available non-peptide vasopressin receptor antagonists that are either FDA approved, or have been used in clinical trials.

The project will address different aspects of the biochemistry to support structure determination, including incorporation of fusion proteins, or rigid extensions (e.g. a-helices) within the receptors, as stand-alone fiducial markers or in combination with antibodies (Fab/Nb or scFv) that rigidly target the fusion proteins. There is also abundant information from existing class A GPCR structures on interactions that can stabilise inactive states (e.g. so called “ionic lock”) that could be targeted to increase stability of inactive states (thus reducing receptor dynamics) that may be explored, in isolation or in combination with fusion protein approaches. In parallel, studies on the performance of different reconstitution technologies that may provide smaller or more rigid “scaffolding” of lipid encapsulated GPCRs will be explored (e.g. nanodiscs, peptidiscs, saposins). These approaches will be systematically explored in the presence and absence of inhibitors. Constructs will be assessed biochemically for efficiency of expression, solubilisation, purification and reconstitution using SDS-PAGE, western blot and SEC analyses. The impact of mutations and/or inhibitor binding on receptor stability will also be assessed in melting temperature assays. Sample homogeneity will also be assessed by negative stain EM on our Talos L120c. Well behaved samples will be used for vitrification and imaging. Grid type, and glow discharge conditions will be varied to identify optimal conditions for partitioning of particles into thin vitreous ice and orientation distribution. Cryo-EM data will be collected on 200kV or 300kV instruments and assessed for particle homogeneity, 2D and 3D classification, and 3D reconstruction). This latter component will include assessment of the most recent improvements in particle picking, alignment and 3D reconstruction software. The project will yield improved understanding of the parameters that support cryo-EM of inactive, inhibitor-bound GPCRs.

Structure determination of GPCR heteromers

Prof Patrick Sexton, A/Prof Denise Wootten and Dr Matthew Belousoff

Industry partner: Dimerix Bioscience

 

For many receptors, no structures currently exist, or the available structures are confined to a single receptor state (most also with only a single ligand). In the case of oligomeric GPCR complexes, only those from class C subfamily GPCRs that form obligate dimers have been resolved structurally. Within this project area, novel receptor structures will be determined, along with analysis of conformational dynamics of individual ligand receptor complexes. Specifically, the project will focus on determination of structures of heteromers involved in inflammation and chemotaxis.

The project will provide biochemical and structure insight into the formation of heteromeric receptor complexes and the binding of ligands to these complexes. In parallel, structures of the individual receptors will be determined in monomeric or oligomeric states. To achieve this, expression constructs for receptors will be designed that will enable purification of individual receptors and receptor complexes through the incorporation of specific purification tags. Further modifications may also be made to individual receptors to support efficient G protein coupling, including C-terminal G protein fusions. Preliminary work will also be performed to assay efficiency of dimer formation with different ratios of receptors, and in the presence of ligands that may promote dimer stability. Additional experiments will be performed to characterise G protein coupling to individual and heterodimeric receptor complexes in response to agonists for each of the receptors. These experiments will inform the design of biochemical experiments to assess the expression and stability of receptors for structure determination. Constructs for characterisation of dimerization and pharmacology will be supplied by the PO. Complex stability will be assessed by techniques including native PAGE and SEC, and will be assessed under different conditions of solubilisation and reconstitution. Stable complexes will be visualised by negative stain TEM and, if suitable, proceed to structure determination by cryo-EM. The project will yield novel biochemical approaches for purification of stable heteromeric GPCR complexes and provide novel structural information that can support future drug discovery and development.

Structural characterisation of CXCR3 activation and inhibition

Dr Natalie Diepenhorst, Dr Greg Stewart and Dr Chris Langmead

Industry partner: Servier, France

 

CXCR3 is a chemokine G protein-coupled (7TM) receptor that is implicated in a number of diseases, including cancer and auto-immunity. It is primarily activated by three different chemokines (CXCL9, CXCL10, and CXCL11), which are reported to engender different signalling events downstream of the receptor. It also reported to be activated, with lower potency, by other chemokines including CCL13 and CCL20. Its pharmacology is further complicated by the existence of two major splicing variants, CXCR3A and CXCR3B, which differ in the N-terminal sequences that engage chemokines and have different roles with respect to T-cell function in vivo.

The project will examine the agonist-bound active-states of CXCR3 isoforms with different agonist ligands by cryo-EM to determine particular epitopes that are involved in chemokine-specific activation mechanisms. The project will also involve supporting multi-pathway profiling pharmacology studies to complement structural studies.

The final part of the project will explore the pharmacological and structural basis of the inhibition of CXCR3 by monoclonal antibodies (mAbs), profiling commercially available (and potentially novel) mAbs in both aforementioned pharmacology assays and by cryo-EM in complex with the inactive, non-G protein-bound receptor.

A structural basis of subtype selectivity at muscarinic acetylcholine receptors

Dr David Thal

 

The aim of this project is to understand how different types of ligands (orthosteric or allosteric) selectively bind to muscarinic acetylcholine receptors (mAChRs). The five (M1-M5) human mAChRs are a highly conserved family of class A G protein-coupled receptors (GPCRs) that are activated by the neurotransmitter acetylcholine. The mAChRs are widely expressed throughout the central and peripheral nervous system, and there has been immense interest from the pharmaceutical sector to develop therapeutics that target mAChRs for the treatment of major central nervous system related disorders, including dementia, schizophrenia, and drug addiction. This project will use cryo-EM to determine the structures of mAChRs in complex with their cognate heterotrimeric G protein and subtype selective ligands. Structural work will be complemented with pharmacology and biophysical techniques to provide mechanistic insight into how ligands selectivity bind to and activate particular mAChR subtypes. The findings from this work are anticipated to aid the design of future mAChR ligands.