Structural Characterization and Computer-Aided Optimization of a Small-Molecule Inhibitor of the Arp2/3 Complex, a Key Regulator of the Actin Cytoskeleton

摘要

CK 666 (1) is a recently discovered small molecule inhibitor of the actin-related protein 2 3 (Arp2/3>complex, a key actin cytoskeleton regulator with roles in bacterial pathogenesis and cancer cell motility. Although 1 is commercially available, the crystal structure of Arp2/3 complex with 1 bound has not been reported, making its mechanism of action uncertain. Fur-thermore. its relatively low potency increases its potential for off-target effects in vivo, complicating interpretation of its in fluence in cell biological studies and precluding its clinical use. Herein we report the crystal structure of 1 bound to Arp2/3 complex, which reveals that 1 binds between the Arp2 andArp3 subunits to stabilize the inactive conformation of the complex. Based on the crystal structure, we used computation al docking and free-energy perturbation calculations of mono-substituted derivatives of 1 to guide optimization efforts. Biochemical assays of ten newly synthesized compounds led to the identification of compound 2, which exhibits a threefold increase in inhibitory activity in vitro relative to 1. In addition, our computational analyses unveiled a surface groove at the interface of the Arp2 and Arp3 subunits that can be exploited for additional structure-based optimization.