Project Summary/Abstract Separate from its roles in cellular energy metabolism, the metabolite lactate influences multiple physiological processes and contributes to cancer progression by acting as a signaling molecule. Cancer cells often undergo metabolic reprogramming which results in increased lactate production and secretion. Extracellular lactate activates hydroxycarboxylic acid receptor 1 (HCAR1), an understudied G protein-coupled receptor that promotes tumor growth by driving cancer cell proliferation, stimulating tumor vascularization, and inhibiting immune surveillance. Inhibiting HCAR1 activity represents a promising strategy for cancer treatment, but the lack of selective pharmacological tools targeting HCAR1 hampers our ability to target this receptor in disease and limits our understanding of HCAR1 function in physiology. Our long-term goal is to understand the molecular basis for ligand recognition by HCAR1 and to use this information to design selective small molecules targeting this receptor. This project will lay the foundation for future structure-based drug discovery efforts by determining the ability of known HCAR1 ligands to promote binding between purified HCAR1 and engineered G proteins (mini-G proteins) or camelid single-chain antibody fragments (nanobodies). In Aim 1, we will examine the allosteric coupling between currently available HCAR1 agonists and a panel of mini-G proteins and investigate the mechanism of action of potential HCAR1 antagonists. In Aim 2, we will identify conformationally selective nanobodies that recognize HCAR1 and determine their selectivity and binding mode at HCAR1. In both aims, we will determine conditions to form stable HCAR1 complexes for future structural studies of HCAR1 in multiple states. This work will provide insight into the molecular pharmacology of currently available HCAR1 ligands, discover novel nanobodies to probe HCAR1 function, and enable future structure-based design of HCAR1 ligands for cancer treatment.

Project Narrative In many types of cancer, cells change their metabolism and produce large amounts of the metabolite lactate, which in turn can promote cancer progression by activating hydroxycarboxylic acid receptor 1 (HCAR1), an understudied G protein-coupled receptor. The lack of pharmacological tools for HCAR1 limits our understanding of HCAR1 function and hampers our ability to target this receptor in disease, therefore this project will explore the pharmacology of molecules known to bind HCAR1, discover new antibody-based tools to study HCAR1 function, and identify conditions for determining the structure of HCAR1. The results of this work will lay the foundation needed to enable future drug screening and structure-based drug design efforts, which will provide new molecules to study the physiological role of HCAR1 and strategies to block HCAR1 signaling in cancer.