Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Abstract

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.Rho family proteins (20 human members) comprise a major branch of the Ras superfamily of small GTPases (1, 2). Like Ras, Rho GTPases function as GDP/GTP-regulated binary on-off switches. In resting, quiescent cells, Rho GTPases exist predominately in their inactive, GDP-bound state. Upon growth factor stimulation, Rho-specific guanine nucleotide exchange factors (RhoGEFs) 4 are activated, and they stimulate the intrinsic guanine nucleotide exchange activity to promote formation of the active GTP-bound protein. Rho-GTP binds preferentially to downstream effector proteins. Rho-specific GTPaseactivating proteins then stimulate the intrinsic GTP hydrolysis activity of Rho GTPases, returning the protein to its inactive state and terminating effector interaction.RhoA, Rac1, and Cdc42 are the best studied and understood members of the Rho GTPase family (1, 2). Rho GTPases are regulators of a diverse variety of cellular processes that include regulation of cell proliferation, actin cytoskeleton reorganization, and gene expression (3). Like Ras and other members of the Ras superfamily (4, 5), the aberrant activity of Rho GTPases has been implicated in cancer and other human diseases (6 -10). In particular, the Rac subfamily has also been linked to c...

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration

“…Because Paks are the effectors of Rac1 (40,41), but our results revealed that RhoA is downstream to Rac1 and mediates thrombin-induced Pak2 activation, we applied a pharmacological approach to confirm these unusual observations. EHT1864, a selective inhibitor of Rac1 (39), and Rho inhibitor I, a selective inhibitor of RhoA (42), also suppressed thrombin-induced Pak2 activity (Fig. 5I).…”

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity

“…Consistent with this specific mechanism of action, the inhibitory effect of DCAI is only toward SOS-catalyzed nucleotide exchange and does not affect the intrinsic nucleotide exchange by Ras. This mode of action is in sharp contrast to that of a Rac exchange inhibitor, EHT 1864, which inhibits both intrinsic and GEF-mediated nucleotide exchange for Rac (20 DCAI exhibits cellular activity in HEK-293Tcells; it attenuates the EGF-stimulated activation of Ras and blocks the recruitment of the cRaf RBD-CRD domain to the cytoplasmic membrane. Given that DCAI binds to Ras and acts as a competitive inhibitor against association of SOS with Ras, it is expected that its potency in cellular assays is in part determined by the cellular concentrations of SOS and Ras.…”

The role of Rac1 in the regulation of NF-kB activity, cell proliferation, and cell migration in non-small cell lung carcinoma

“…44 More recently, another Rac1 inhibitor (EHT 1864) has been developed which inhibits guanine nucleotide association, thus rendering Rac1 inactive. [45][46][47] The results of this study demonstrate the important roles of Rac1 in NSCLC cell proliferation and migration and compare the effects of Rac1-specific siRNA to that of Rac1 inhibition by NSC23766. One possible mechanism contributing to these multiple roles of Rac1 may be through the Rac1-dependent regulation of NFκB transcriptional activity in NSCLC.…”