We present a new application of NMR spectroscopy to quantitatively determine GPCR drug efficacy. NMR data quantitatively correlate with results from cellular signaling assays. We highlight examples where the NMR method proves particularly advantageous, especially in accurately quantifying efficacy for compounds with lower potency. Additionally, we present a new approach that leverages NMR to identify weakly interacting small molecule fragments for GPCR drug discovery.

Despite the increasing availability of high-resolution GPCR structures, finding allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. I present a novel strategy to site-specifically covalently tether drug fragments adjacent to allosteric sites in engineered GPCRs. In proof-of-concept studies, reactive noncanonical amino acids were introduced in CCR5 near the binding site for its allosteric drug maraviroc. In cellular assays, maraviroc analogues containing reactive bioorthogonal moieties displayed an increase in potency for CCR5 harboring reactive noncanonical amino acids adjacent to the maraviroc binding site. The strategy should prove useful to probe allosteric or cryptic binding-site functionality in fragment-based GPCR-targeted drug discovery.

We present novel approaches for the selection of molecules from DNA-encoded libraries (DEL) using enzymatic tags on target proteins. We apply these assays for DEL discovery with GPCRs in live cells for both the discovery of ligands and for specific discovery of biased agonists.

Therapeutic options for treating chronic pain are limited by low efficacy and adverse effects. Delta opioid receptor (DOR) agonists show potential in relieving chronic pain symptoms; however, they can exhibit adverse effects like seizures and tolerance. Our studies aim to understand pharmacological and behavioral profiles of DOR full agonists, partial agonists, and allosteric modulators to develop a structural platform for designing the next generation of pain relievers.

The mu-opioid receptor (MOR) is a well-established GPCR target for analgesia, yet conventional orthosteric opioid agonists suffer from serious adverse effects, notably respiratory depression and addiction. We screened a large DNA-encoded chemical library and discovered new positive and negative allosteric modulators of the receptor. We then used cryoEM to determine detailed structural mechanisms of allosteric modulation and demonstrated in vivo efficacy for a new class of "Narcan-boosters."

This presentation will focus on the development of small molecule agonist versions of peptide binding GPCRs such as GLP-1R.

Formyl Peptide Receptor 2 (FPR2) agonism stimulates resolution of inflammation and promotes wound healing. During our drug discovery efforts, FPR2 agonists with biased GPCR signaling profiles were identified through a combination of screening and rational design. A detailed evaluation of the agonists in cellular assays and in vivo models demonstrated that a specific signaling profile afforded efficacy in rodent heart failure models. Optimization of an arylpiperidinone chemotype with the preferred GPCR signaling profile led to the identification of BMS-986331, which was progressed into Phase I clinical trials.

Allosteric ligands modulate receptor responses without completely blocking their functions, an attractive property for drug development. However characterizing allosteric modulators is challenging. We studied allosteric modulator binding of the CB1 receptor by developing a fluorescent allosteric probe which differentiated effects from orthosteric ligands. We combined this with G protein recruitment and ModuMelt™ assays to understand the pharmacological activity and cooperativity of orthosteric and allosteric ligand binding. This toolbox can be applied to other GPCRs.

Agonism of the Orexin type 2 receptor has emerged as an attractive approach for the treatment of narcolepsy. Here, we describe the identification of an agonist series [PC1] using a new screening assay based on ConfoBodies, the elaboration of the chemical series and its puzzling structure-activity relationship. Determination of the active-state structure with a series representative revealed the cause of these unexpected activity cliffs.