Endogenous cellular lipids regulate GPCR activity but structural mechanisms of receptor-lipid interactions are not well understood. Using NMR spectroscopy, we show how specific lipids alter the conformation of GPCRs and how receptor-lipid interactions influence recognition of partner proteins, including native and engineered G proteins.  We compare data of GPCR complexes in different membrane mimetics and show how the selection of membrane mimetics influences the conformation of signaling complexes.

ß-Arrestins belong to a small family of globular proteins discovered for their ability to dampen G protein-coupled receptor (GPCR) signaling transmitted through the heterotrimeric G proteins. ß-arrestins critically regulate multiple signaling pathways and their ability to undergo dynamic conformational changes enables their protein interactions and existence in different intracellular pools. GPCR signal transmission through select transducers, i.e., G protein and/or ß-arrestin subtypes is influenced by a variety of codes (phosphorylation, ubiquitination etc.) and recent studies indicate that ligands that engender allostery along with bias could be beneficial in the therapeutic targeting of GPCR signaling.

GPCRs are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and ß-arrestins. Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. In this talk, I will present our recent work characterizing the naturally-occurring 7TMRs that selectively engage ß-arrestins, but not G-proteins, even in response to their endogenous agonists.

We are applying the "Innate Proximity" paradigm based on knowledge of constitutive or transient interactions between proteins that can be used to design molecules with better medicinal properties. I present several examples in the Opioid and Dopamine receptor interactomes. We are developing potent drugs in IBS-D and pain and have expanded the core receptor-interactomics platform to neurodegenerative disorders such as Parkinson's.

The adenosine A2A receptor (A2AR) is a prototypic GPCR and immuno-oncology (IO) target. Recent studies reveal that in the context of a lipid bilayer, both small molecule orthosteric ligands and G proteins are allosteric modulators of dynamic A2AR conformations.These results provide a mechanism for phenomenological observations of GPCRs including efficacy, signaling and allostery. This study has implications for the design of future experiments and the design of GPCR modulators.

Inhibition of either A2AR or CD73 alone has proven to be effective strategy in producing antitumor immunity in the clinic by inhibiting immunosuppressing adenosine signaling. However, the co-blockade of CD73 and A2AR results in a more pronounced anti-tumor activity than blockade of either, likely due to increased CD73 expression as compensation upon A2AR inhibition. Data of our strategically developed orally bioavailable dual inhibitor of CD73 and A2AR will be presented.

This talk will focus on structure-based approaches toward discovering, designing, and developing pathway-selective GPCR ligands. Using the aminergic GPCR subtypes as a starting point, I will detail progress toward designing new biased agonists at a host of other receptors. This talk will detail strategies to either design G protein or Arrestin-biased signaling by focusing on ligand interactions with common mechanisms of GPCR activation.

Traditional hybridoma and phage display methods have shown limited success in delivering therapeutic antibodies against difficult targets like most GPCRs and ion channels.  Amelie Kutschera will present case studies of how Genovac has overcome this challenge by leveraging their core genetic immunization technology and Berkeley Lights’ Beacon OptoFluidic system to rapidly discover antibodies against challenging targets, focusing on GPCRs, from a variety of different animal species.   

Recent discovery of the sodium-binding allosteric pocket, conserved in >600 of class A GPCR opened a new opportunity for selective modulation of GPCR signaling pathways. We have designed several series of bitopic ligands targeting this sodium pocket. This talk will describe design, molecular modeling, structural, functional, and behavioral characterization of these bitopic compounds, suggesting that such bitopic ligands can be designed to selectively modulate downstream signaling in many GPCRs.

G protein coupled receptors are valuable therapeutic targets activated by ligands that exert their action via the interactions with the receptor. We hypothesised specific atomic interactions determine ligand activity. We computationally docked ~2700 known β2AR ligands to multiple β2AR structures, generating 75,000 docking poses. Using Machine Learning we defined a very detailed 3D-SAR for β2AR. This approached can be applied to other drug targets.

Despite the surge in therapeutic antibodies against various target classes, the number of GPCR-targeting therapeutic antibodies is lagging behind, mainly due to the difficulty in generating suitable antigen formats for antibody discovery. Confo Therapeutics aims to overcome this bottleneck by using conformationally selective VHH – ConfoBodies – to stabilize GPCRs in their therapeutically relevant conformation(s). We will present how ConfoBody-stabilized GPCRs allow the identification of novel biologics with agonist pharmacology.

Sensitive protein stability assays for membrane proteins are crucial for developing purification protocols, structural and biophysical receptor characterisation, and drug discovery.  We will describe novel higher throughput 384-well ultrasensitive thermostability methodologies, ThermoFRET and ThermoBRET. These nanoscale methods measure resonance energy transfer between a donor-labelled GPCR and a thiol-reactive fluorescent dye that binds to cysteine residues exposed upon protein unfolding in response to thermal denaturation. Both methods are functional in crude solubilised membrane preparations and can detect receptor stabilising ligands. These new methods will uncover 'hits' for more challenging GPCR targets, such as orphan receptors, enabling future drug discovery.

Monoclonal antibodies are a promising therapeutic modality for GPCR engagement, yet complex cell surface receptors pose considerable challenges for traditional antibody discovery approaches. This talk will discuss how leveraging increased epitopic coverage of antibody repertoires with hyperimmune mice, coupled with flexible assay formats and information-dense functional screening of primary B cells, can overcome the barriers associated with antibody discovery against challenging membrane protein targets.

The first G-protein-biased allosteric agonist of CB1R, (+)-(R,S)-GAT1601 has been discovered using the "magic methyl effect" strategy. The initial demonstration of the preclinical efficacy and unprecedented long duration (12h) of action of this biased allosteric agonist for lowering intra-ocular pressure without producing significant adverse cannabimimetic effects reveals its potential therapeutic value in treating glaucoma.

GPCRs are highly druggable targets but many receptor families still present formidable challenges to established drug discovery techniques. We present a GPCR drug development platform that generates compounds with tunable signaling activity against GPCRs whose natural ligands are peptides and proteins. Our platform, at a high-throughput/rapid pace, enhances the potency of the natural ligands. We will also demonstrate our platform's functionality in engaging complex GPCR targets.