A Potent and Selective Inhibitor of Cdc42 GTPase

Cdc42, a member of the Rho family of GTPases, has been shown to play a role in cell adhesion, cytoskeletal arrangement, phagocytosis and cell motility and migration, in addition to a host of other diverse biological processes. The function of Rho-family GTPases in disease pathogenesis has been well established and identification of small, cell permeable molecules that selectively and reversibly regulate Rho GTPases is of high scientific and potentially therapeutic interest. There has been limited success in identifying inhibitors that specifically interact with small Rho family GTPases. The identified probe, ML141 (CID-2950007), is demonstrated to be a potent, selective and reversible non-competitive inhibitor of Cdc42 GTPase suitable for in vitro assays, with low micromolar potency and selectivity against other members of the Rho family of GTPases (Rac1, Rab2, Rab7). Given the highly complementary nature of the function of the Rho family GTPases, Cdc42 selective inhibitors such as those reported here should help untangle the roles of the proteins in this family

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.This work was supported by National Science Foundation (NSF) Grant MCB0956027 and National Institutes of Health Grant R03 MH081231-01 from the Molecular Libraries Program (to A. W. N.); University of New Mexico Center for Molecular Discovery Molecular Libraries Probe Production Centers (UNMCMD MLPCN) National Institutes of Health Grants U54MH084690 and R01HL081062 (to L. A. S.); UNM National Center for Research Resources (NCRR) Grant 5P20RR016480 (to L. G. H.); National Institutes of Health Grant R21 CA170375-01 through the NCI (to A. W. N., L. G. H., and J. E. G.); National Institutes of Health Grants NS066429 and AI092130 (to T. B.); and University of Kansas Specialized Chemistry Center (KUSCC) MLPCN National Institutes of Health Grant U54HG005031 (to J. A.)