Brief reports from Industry 1

Frank Cao,

Domain Therapeutics, Montreal, QC, Canada, and Strasbourg, France

Large-scale profiling of physiologically relevant naturally occurring rare GPCR variants using the bioSensAll® technology

GPCR signaling can be differentially modulated by specific ligands to selectively promote the engagement of different subsets of signaling pathways, an outcome known as biased signaling. Although dysregulation of GPCR activity has been linked to pathological consequences, the involvement of their signaling pathways toward specific clinical repercussions remain unresolved. Thus, linking the role of GPCR signaling pathways to distinct physiological processes is not only important for understanding GPCR biology, but also essential for successfully targeting these receptors as potential therapeutic avenues. Here, to exploit the potential of naturally occurring GPCR variants emerging from large scale exome and genome sequencing efforts, we assessed the signaling profiles of rare variants across different GPCRs found in human populations with documented clinical phenotypes. Signaling profiles were assessed through an automated platform using our bioSensAll® technology, which measures the activity of various signaling pathways using a panel of 16 selective bioluminescence resonance energy transfer (BRET)-based biosensors that monitor the activation of heterotrimeric G proteins (Gαs, Gαi1, Gαi2, GαoA, GαoB, Gαz, Gαq, Gα11, Gα14, Gα15/16, Gα12, Gα13) or recruitment of β-arrestins to the plasma membrane (β arrestin 1, and β-arrestin 2) upon agonist stimulation. To achieve the large-scale screen in a high throughput system, bioSensAll® assays were first miniaturized and adapted to a 384-well format. Subsequently, HEK293 cells were co-transfected with receptors and biosensors, and a scheduling software was used to coordinate the interaction between various instruments via a robotic microplate mover. Analysis was performed through an automated tool to generate and compile signaling profiles for each variant normalized to the wild-type receptor, revealing gain- or loss-of-function properties induced by the variants across different signaling pathways, thus also allowing us to identify genetic alterations that impart biased signaling profiles. By pairing the multiparametric signaling profiles of variants found in human populations generated through our automated bioSensAll® platform with information on their associated clinical phenotypes, we can uncover associations between altered receptor pharmacology and clinical outcomes to accelerate the discovery of disease-relevant targets and improve translation from bench to the clinic.

Maria Majellaro,

Celtarys Research S.L., Santiago de Compostela, Spain

How to improve GPCRs drug screening and characterization using novel fluorescent probes

During the last decades, fluorescence-based assays emerged as an attractive alternative to methodologies such as radioactivity. Among the noticeable advantages, fluorescent probes demonstrated to be suitable for qualitative and quantitative measurements by a variety of experiments such as fluorescence microscopy, high content screening, FRET, HTRF and BRET in both native and living cells. However,

fluorescent ligands have not yet been widely adopted due to the complexity of their development process and the consequent lack of optimal fluorescent probes available for the majority of the targets of interest. In Celtarys Research, we developed a rational and versatile synthetic strategy enabling to rapidly identify optimal fluorescent probes to tag diverse molecular targets. The key role of GPCRs in drug discovery led us to start applying our technology in this field, developing fluorescent probes for different receptor families and applications. Herein we document some representative case studies in collaboration with different research groups, evidencing the potential and robustness of this core technology in the context of GPCR research.

Brief reports from Industry 2

Sophie Bradley


Chris de Graaf,

Sosei Heptares, Cambridge, UK

From structure to clinic: taking muscarinic drug discovery to the next level

The M1 muscarinic acetylcholine receptor mAChR represents an established therapeutic target for improving cognitive decline in Alzheimer’s disease (AD). However, previous clinical trials targeting muscarinic receptors have been hampered by significant adverse effects. Through a two-part presentation, we will demonstrate our stepwise approach, from structure through to clinical studies, to the development of a next-generation muscarinic ligand with improved pharmacological properties and the potential to treat memory loss in AD.