Fab-Induced Stabilization of an Ion Channel Receptor Enables Mechanistic Characterization of Small-Molecule Therapeutics.

Developing small-molecule (SM) therapeutics that target membrane proteins (MPs) is often challenging, because few biophysical methods can handle the detergents required to maintain target stability. Here, we report a surface plasmon resonance (SPR)-based methodology that enables the characterization of interactions between SMs and an ion channel receptor (MP1) in complex with a stabilizing antibody fragment (Fab) and surfactant. Briefly, a stable MP1-Fab complex was formed by coimmobilizing MP1 with an anti-MP1-Fab within the hydrogel film to study the interactions of MP1 with SMs. The critical micelle concentration (CMC) is the concentration at which 50% of the surfactant monomers are assembled into micelles. Micelles readily absorb compounds resulting in compound-loaded micelles that generate high nonspecific binding and hinder resolution of SM binding responses. This micelle-induced interference was avoided by utilizing a weak detergent at a concentration below its CMC, allowing for the resolution of compound binding to a solvent-exposed pocket. Additional Fab stabilization was required to rescue binding at a second pocket buried within the transmembrane region of MP1. The resulting SPR-based assay proved invaluable during hit-to-lead optimization by progressing structure-activity relationship (SAR) studies and resolving the mechanism of action (MoA).