title: Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets.
abstract: BACKGROUND
  Platelet 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 in vivo 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.
  
  
  OBJECTIVE
  To analyze the specificity of NSC23766 and EHT1864.
  
  
  METHODS
  Platelet 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.
  
  
  RESULTS
  NSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100 μm in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoprotein Ib-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.
  
  
  CONCLUSIONS
  Our results reveal critical off-target effects of NSC23766 and EHT1864 at 100 μm in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.
authors: Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
authors: S
authors: S
authors: Dütting
authors: J
authors: J
authors: Heidenreich
authors: D
authors: D
authors: Cherpokova
authors: E
authors: E
authors: Amin
authors: S-C
authors: SC
authors: Zhang
authors: M R
authors: MR
authors: Ahmadian
authors: C
authors: C
authors: Brakebusch
authors: B
authors: B
authors: Nieswandt
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009479:Neuropeptides / Q000037:antagonists & inhibitors* / Q000235:genetics; D010766:Phosphorylation; D011743:Pyrimidines / Q000494:pharmacology*; D011753:Pyrones / Q000494:pharmacology*; D011804:Quinolines / Q000494:pharmacology*; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D000634:Aminoquinolines; C506907:EHT 1864; C487513:NSC 23766; D009479:Neuropeptides; D011743:Pyrimidines; D011753:Pyrones; D011804:Quinolines; C484602:Rac1 protein, mouse; D020830:rac1 GTP-Binding Protein
keywords: Rac1 GTP-binding protein; blood platelets; enzyme inhibitors; signal transduction; thrombosis