Exploring the binding kinetics of intracellular allosteric antagonists for CC chemokine receptor 2 — a key to improve insurmountable antagonism

CC chemokine receptor 2 (CCR2), a class A G protein-coupled receptor (GPCR), represents a promising therapeutic target for inflammatory and autoimmune diseases. Although several CCR2 antagonists have advanced to clinical trials, none have reached the market, largely due to limited efficacy. In recent decades, binding kinetics has emerged as a key consideration in early drug discovery, complementing traditional equilibrium parameters to enhance clinical efficacy. In parallel, targeting allosteric sites offers unique advantages, i.e. insurmountablility, further improving therapeutic safety and efficacy. However, the interplay between binding kinetics and allosterism has received little attention in drug discovery as a strategy to optimize insurmountablility. As such, we developed a kinetic competition association assay, employing the intracellular allosteric radioligand 3 HLUF7482, which displayed high affinity in both equilibrium and kinetic binding assays, with consistent nanomolar K D values. After validation of the kinetic assay with unlabeled LUF7482, we profiled five known intracellular CCR2 antagonists, including the covalent binder LUF7834 as a positive control, which displayed an apparent long residence time (RT) of 576 min. Functional cAMP assays further revealed a strong correlation between binding kinetics and the extent of insurmountable antagonism Together, this study established the competition association assay as a robust approach to characterize the kinetic profiles of intracellular CCR2 antagonists, and revealed a clear correlation between binding kinetics and insurmountability. These findings may guide the selection and optimization of next-generation allosteric antagonists targeting CCR2, or the other GPCRs, in drug discovery pipelines, with the aim of improving insurmountability and, consequently, clinical efficacy.