Proton-sensing GPCRs, including GPR4, GPR65, and GPR68, can be activated by low pH in the tissue microenvironments under various pathological conditions. Recent studies demonstrate that proton-sensing GPCRs play important roles in cancer and inflammation. I will present our research findings on the roles of GPR4 and GPR65 using mouse genetic and pharmacological approaches. Particularly, my presentation will focus on colorectal cancer, inflammatory bowel disease, and COVID-19.

GPR61 is an orphan GPCR linked to BMI phenotypes. Pfizer undertook a GPR61-targeted inverse agonist program to treat cachexia, and the team used extensive protein engineering, aided by AlphaFold2, to enable structures of active and inactive GPR61 for SBDD and rationalization of SAR. Co-elucidation of GPR61 with the inverse agonist series revealed a novel G protein-competitive inverse agonist mechanism, offering key functional insights and new molecular tools for studying GPR61.

Members of the proteinase-activated receptor (PAR) subfamily are G protein-coupled receptors that play critical roles in processes such as inflammation, wound healing, and cancer progression. Using structural snapshots, we reveal how tethered ligands activate PAR1 and PAR2, uncovering a conserved orthosteric binding mechanism. We also highlight the structure of PAR2 bound to GB88, showcasing how pathway-selective inhibition mimics tethered ligand interactions, advancing our understanding of PAR signaling and therapeutic targeting.

Orphan GPCRs are understudied receptors and a potential source of druggable targets for treating diverse human diseases. GPR52 is an orphan brain receptor primarily expressed in the human striatum and recently identified as a schizophrenia risk gene. We synthesized and pharmacologically evaluated multiple novel GPR52 agonist series, including the first G protein biased agonists. An advanced novel GPR52 agonist lead shows robust activity in preclinical models relevant to schizophrenia and substance use disorders.

The µ-opioid receptor (µOR) is the target of opioids that are critical analgesics for pain management. The molecular understanding of the µOR behavior in the presence of drugs will facilitate the development of better therapeutics. Using methods of single-molecule fluorescence resonance energy transfer (smFRET) and double electron-electron resonance (DEER), we show how ligand-specific conformational dynamics of the µOR translate into a broad range of intrinsic efficacies at the transducer level.

Phosphorylation of residues in the C-terminal tail of GPCRs has been strongly implicated in promoting receptor interactions with β-arrestins, which are proteins that modulate G protein coupling, receptor internalization, and perhaps also serve as signaling modules. We used proteomic methods to identify C-tail residues that are phosphorylated in the glucagon family of GPCRs (GLP-1R, GCGR, and GIPR) following agonism. We also examine the impact of these modifications on cell signaling.

GPCRs present unique challenges to drug discovery due to their low expression, complexity, and poor stability. Detergent extraction from their native environment is commonplace but can prohibit basic biochemical characterization and render false positives in downstream screening campaigns. To address this problem, we have implemented membrane mimics to generate detergent-free GPCRs to facilitate their characterization, enable Hit ID screening campaigns, and validation follow-up.

We present new NMR-based methodology for the characterization and quantification of GPCR drug efficacies and illustrate this method with the human A2A adenosine receptor.  The method is particularly valuable for compounds where precise information is needed, for example, accurate characterization of inverse agonists and partial agonists.  This new approach relates drug efficacy directly to receptor structural changes, rather than the detection of secondary metabolite production in traditional cell-based approaches.

GPCRs are intracellular receptors that mediate physiological functions through ligand binding. There are numerous GPCRs that remain classified as orphan GPCRs because their native ligands remain unidentified. GPCRs are attractive targets for numerous indications ranging from brain injury to obesity, but structural study has been hindered by their innate qualities. Here we present and compare novel structures of a class A orphan GPCR with bound ligand to inform mechanism and drug discovery.

Guided by 19F-qNMR, we resolved the first intermediate GPCR-G protein complex structure, and elucidated its function. This advance will facilitate structure-based drug development by screening drugs targeting individual conformations of GPCR and its complex with different signaling partners.

This talk introduces a novel label-free, charge-sensitive method for multiplexed quantification of small-molecule binding kinetics to virion-displayed membrane proteins via wide-field surface plasmon resonance microscopy imaging of affinity barcoded nanocomposite oscillators. Virion display maintains native membrane protein environments. Affinity-resolved multi-state DNA barcoding efficiently addresses individual nano-oscillators and enables measurement of a library of G protein-coupled receptors on a single chip.