GPCRs, over the past two decades, have been shown to form higher-order physical complexes termed homomers (complexes of similar receptors) or heteromers (complexes of different receptors). The discovery of these receptor complexes has provided a new paradigm, as previous GPCR drug discovery efforts have been focused only on monomeric receptors. The goal of Blue Therapeutics is to utilize our deep knowledge in GPCR biology and develop potent analgesics that lack addictive potential. Opioids are the standard of care for treatment of moderate to severe pain, but their widespread use has caused a national health crisis centered on prescription opioid abuse, misuse, and addiction. To tackle this, we are currently advancing our lead molecule, Blue181 – a selective activator of HM2K (HeteroMer-Mu-Kappa) as a non-addictive, non-narcotic pain therapeutic through IND studies. In this presentation, Dr. Yekkirala will describe efforts on the development of Blue181 as a case study on utilizing GPCR heteromer biology for targeted drug development.

The understanding and modulation of the opioid receptors' pharmacological profiles is still considered as one of the highest priorities in order to develop safer pain-relieving agents. We used a structure-based approach to develop a small library of selective G protein-biased agonists, which aimed at targeting simultaneously both the orthosteric and allosteric sodium-binding pocket site of the 
μ-opioid receptor. Structure activity relationships were validated using mutational and structural analysis.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited by the requirement for injection. Here we describe the discovery and characterization of PF-06882961, an effective and orally bioavailable small molecule GLP-1R agonist drug candidate. This work opens the door to a new era of oral small molecule therapies that target the GLP-1R pathway for metabolic health.

To advance early stage drug discovery, developing relevant HTS assays for the target is paramount. It requires speed, robustness and reproducibility.  For such purpose, as a CRO and research tools provider, we at Multispan have built a long successful track record in assay development, high-throughput screening and cell engineering.  In this presentation, we will share details on our approach and introduce our new efforts in BacMam and HSV viral delivery of MultiScreen™ Arrestin-Sensor for native GPCRs, CRISPR knock-out and SARS-CoV2 assays.  We will also share a few case studies highlighting how we helped our partners accomplish their project goals by running projects in our hands, driving chemistry efforts with high quality and reliable data generated at Multispan.

To elucidate the contribution of APJ/beta-arrestin signalling, we generated a transgenic mouse harbouring a point mutation (APJ I107A) that maintains full G protein activity, but fails to recruit b-arrestin following receptor activation. APJ I107A-mutant mice did not alter cardiac function at rest, following exercise challenge or in response to pressure overload-induced cardiac hypertrophy.  Additionally, APJ I107A mice have comparable body weights, plasma glucose, and lipid levels to WT mice when fed a chow diet. However, APJ I107A mice showed significantly lower body weight, blood insulin levels, improved glucose tolerance, and greater insulin sensitivity when fed a high-fat diet. Furthermore, loss of APJ b-arrestin signalling also affected fat composition and the expression of lipid metabolism-related genes in adipose tissue from high-fat fed mice. Taken together, our results suggest that G protein-biased APJ activation may be more effective for certain disease indications given that loss of APJ-mediated, b-arrestin signalling appears to mitigate several aspects of diet-induced metabolic dysfunction.

Our models of G protein-coupled receptor (GPCR) signaling have evolved from cell surface receptors activating a single specific pathway in a linear fashion, to one that is highly complex, exhibiting extensive signal crosstalk and diversity via a number of mechanisms. Untangling the receptor signaling wires to understand how cells decode this complexity into downstream specific responses remains a current outstanding question. This question has driven research into how membrane trafficking, a system deeply integrated with cell signaling, not only regulates signal diversity, but also signal specificity. Studies over the past decade have demonstrated that membrane trafficking of GPCRs is critical for spatial and temporal control of signaling by directly facilitating G protein signaling from distinct intracellular compartments, including endosomes. Our studies with the gonadotrophin hormone receptors, key in reproduction and pregnancy, have revealed that GPCR activity can be spatially regulated at a complex multi-endosomal level through the identification of a new intracellular compartment, the very early endosome (VEE). In this session, I will discuss how different GPCRs, not only important in reproduction but also in carbohydrate sensing in the gut, employ this trafficking system to create highly regulated and specific signaling profiles for pleiotropically coupled GPCRs, and the physiological impact of such 'location-bias'. Application of these evolving models to therapeutic strategies suggests new mechanisms that could be exploited in GPCR-directed pharmacotherapy.

G protein-coupled receptors are important transmembrane proteins which account for more than 30% of direct clinical drug targets. Two main signaling pathways, either mediated by different G protein subtype or arrestins, underlie most of 800 GPCR functions in human genome. Selective ligands targeting to one of the G protein- or arrestin-signaling through specific receptor, which is also called biased ligands, may have beneficial effects and delete the unwanted side effects compared with traditional full agonists or antagonists.

Recent structural studies unveiled that both receptor seven transmembrane core and phospho-C-tail engaged with arrestin interactions. Using unnatural amino acid incorporation and genetic expanding technologies, combined with biochemical and crystallographic approaches, we have provided direct experimental evidences that the receptor phosphorylation barcodes in their C-tails could be read by the phospho-pattern readers located in the arrestin, which is the combination of the phosphoate or negative-charged amino acids binding sites. Binding of each site allosterically produced unique arrestin conformations and correlated to selective arrestin downstream functions. Our results indicated that operation of receptor phospho-barcode could be an important strategy to modulate selective receptor functions, serving as an important aspect for developing biased receptor agonists as new therapies.

G protein-coupled receptors (GPCRs) are important drug targets. For GPCR drug discovery, protein production and 3D molecular structures of GPCRs are essential. I will share strategies and tricks which we have been using for GPCR protein production and introduce the emerging atomic-resolution cryoEM approach for GPCR structure determination.

G protein-coupled receptors (GPCRs), which constitute the largest family of proteins targeted by approved drugs, still represent a huge opportunity to develop new drugs for “old” targets or orphan receptors. Significant progresses have been made in the field of fragment screening against those challenging membrane proteins. Recent results obtained with the adenosine receptor using NMR and Microscale thermophoresis (from NanoTemper Technologies) will be discussed.

Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multi-pass transmembrane proteins using three case studies: the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO; the second being a modest stabilization of SERCA2; and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane-bound targets.