Promiscuous Ligands from Experimentally Determined Structures, Binding Conformations, and Protein Family-Dependent Interaction Hotspots

[Image: see text] Compound promiscuity is often attributed to nonspecific binding or assay artifacts. On the other hand, it is well-known that many pharmaceutically relevant compounds are capable of engaging multiple targets in vivo, giving rise to polypharmacology. To explore and better understand promiscuous binding characteristics of small molecules, we have searched X-ray structures (and very few qualifying solution structures) for ligands that bind to multiple distantly related or unrelated target proteins. Experimental structures of a given ligand bound to different targets represent high-confidence data for exploring promiscuous binding events. A total of 192 ligands were identified that formed crystallographic complexes with proteins from different families and for which activity data were available. These “multifamily” compounds included endogenous ligands and were often more polar than other bound compounds and active in the submicromolar range. Unexpectedly, many promiscuous ligands displayed conserved or similar binding conformations in different active sites. Others were found to conformationally adjust to binding sites of different architectures. A comprehensive analysis of ligand–target interactions revealed that multifamily ligands frequently formed different interaction hotspots in binding sites, even if their bound conformations were similar, thus providing a rationale for promiscuous binding events at the molecular level of detail. As a part of this work, all multifamily ligands we have identified and associated activity data are made freely available.