At BioAscent, our scientists have a wealth of experience in GPCR drug discovery. We spoke with one of BioAscent’s GPCR pharmacology experts, Kyle Thompson, who shared his insights from years of hands-on experience working across diverse GPCR targets.

Can you describe your role at BioAscent and your background working with GPCRs?

I’ve been part of the team at BioAscent for just over 3 years. Prior to this I completed my PhD at the University of Aberdeen, where I studied the signalling and regulation of the M5 muscarinic receptor.

Since joining BioAscent, I’ve worked extensively on GPCR-focused client projects and internal R&D projects. I’ve characterised a range of GPCRs and developed suites of cell-based assays across multiple technology platforms to investigate their function and pharmacology.

GPCRs are fascinating to me because of their complexity; despite their structural diversity, there is significant conserved homology between many GPCRs that makes receptor selectivity a significant challenge. The ability of GPCRs to couple to multiple cellular signalling pathways adds additional layers of complexity, and this can be influenced by receptor expression levels, tissue/cell type expression, and different ligand-induced conformations (biased ligands).

BioAscent have the capabilities to run a wide range of GPCR assay technologies. How do you decide which assay format is right for a given project?

Ultimately, assay selection depends on the scientific question we need to answer. We usually begin by validating the known canonical receptor signalling pathways in recombinant expression systems using tool compounds, then develop and optimise robust assay formats to address the project’s goals.

At BioAscent, we routinely adapt and optimise commercial technologies that quantify second messenger levels to investigate G-protein signalling and measure β-arrestin signalling – the key pathways downstream of GPCR activation. Because GPCR signalling is complex, often a single assay does not reveal the full story. Therefore, we typically optimise and run multiple assay formats sequentially or in parallel to fully understand their dynamic pharmacology.

We also employ xCELLigence cellular impedance assays to monitor whole-cell responses in real time. Using pathway inhibitors/modulators in isolation or in combination with one another can provide insights into ligand bias, and relative contributions of individual pathways to observed whole-cell responses.

What are some of the most common challenges you encounter when developing GPCR assays?

Typical GPCR assay approaches involve the generation of stable cell lines or transiently-transfected cell lines, in which the target is overexpressed. Overexpression can often lead to overestimation of ligand activity, making inactive compounds appear to be active, or partial agonists appear as full agonists. While this phenomenon can be useful in some scenarios, such as revealing low intrinsic agonist activity of ligands, it may not reflect the true activity of the ligand in endogenous systems or in vivo.

Another common challenge faced when characterising GPCRs arises from recombinant expression in commonly used cell lines such as HEK293 and CHO-K1. These cell lines endogenously express a myriad of GPCRs that can produce non-specific or background signals in functional assays. In addition, differences in levels of effector/signalling proteins between cell lines can lead to apparent differences in the activity of ligands through cell type-dependent pathway coupling.

To mitigate these issues, we generate a panel of cell lines for GPCR projects, where a range of GPCR expression levels can be selected to clarify whether observed ligand activity is likely to be the result of overexpression or cell line-dependent signalling profiles.

How does BioAscent’s approach differ from a typical CRO when it comes to GPCR projects?

BioAscent scientists have deep expertise in GPCR biology and pharmacology, it is a key strength of ours. We’ve encountered many of the common pitfalls in GPCR work and have a good understanding of where to begin to tackle these problems and address the underlying questions posed when working with these challenging targets.

We have invested in a broad range of assay technologies, from rapid receptor activation readouts such as cAMP accumulation or Ca2+ release, to more dynamic and complex readouts like β-arrestin signalling and real-time, whole-cell responses. This enables us to not just determine whether a ligand binds to a target or is active at the receptor, but to provide comprehensive mechanistic, kinetic data involving functional activity, potency and pathway bias(es).

What advice would you give to a biotech company starting a GPCR drug discovery programme?

• Understand the importance of orthogonal cell-based assay formats – your cell line, receptor expression level and even assay conditions can produce artefacts that can artificially inflate or obscure compound activity. Additional assay formats can deconvolute assay artefacts and highlight the need for further assay optimisation/development.

• Always consider the use of de-selection assays/selectivity counter-screens immediately after initial hit finding. Off-target GPCR activity is a major cause of clinical side effects.

• When possible, obtain kinetic data for your compounds. Many GPCRs have different signalling kinetics so choosing a specific time point as an assay readout that works well for one target may not work for another.

• Don’t be afraid to question the literature – often tool compounds may have been characterised in a single assay format that does not show all potential activities. We have found, for example, that reported antagonists behave as inverse agonists in certain assay formats.