Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets.

BackgroundPlatelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested invivo for a variety of disorders. Two small-molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively. ObjectivesTo analyze the specificity of NSC23766 and EHT1864. MethodsPlatelet function was assessed in mouse wild-type and Rac1-deficient platelets by the use of flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed with differential interference contrast microscopy, and activation of effector molecules was analyzed with biochemical approaches. ResultsNSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100m in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoproteinIb-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors p21-activated kinase (PAK)1 and PAK2. ConclusionsOur results reveal critical off-target effects of NSC23766 and EHT1864 at 100m in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.