Chemical dissection of sterol and bile acid signaling via clickable photoaffinity probes

Sterols and bile acids, traditionally viewed as structural components and digestive surfactants, have emerged as critical signaling molecules involved in diverse physiological processes, including metabolism, immunity, cancer, and host-microbiota interactions. Despite their importance, the full spectrum of proteins that interact with these metabolites remains incompletely defined. Clickable photoaffinity probes have revolutionized our ability to systematically profile sterol and bile acid-binding proteins in living systems. These bifunctional probes, incorporating a photo-crosslinker and a bioorthogonal handle, enable covalent capture and enrichment of probe-interacting proteins for proteomic identification and binding site mapping. Here, we review the design principles and applications of sterol and bile acid probes in target identification and mechanistic studies. We highlight key discoveries that illuminate how cholesterol and bile acid derivatives engage diverse protein targets, including nuclear receptors, ion channels, transporters, and virulence regulators, to influence cellular and microbial physiology. Together, these advances underscore the power of chemical biology tools to decode metabolite signaling networks and identify therapeutic targets at the interface of metabolism, immunity, cancer, and microbial ecology