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

Abstract

Summary 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. Objectives 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.

RETRACTED: Role of Rac1 Pathway in Epithelial-to-Mesenchymal Transition and Cancer Stem-like Cell Phenotypes in Gastric Adenocarcinoma

“…1F). NSC23766 is a chemical inhibitor of the binding and activation of Rac1 GTPase, although it is not entirely specific for Rac1 (22;23). NSC23766 at a dose of 100 μM completely abolished Rac1 activity in AGS cells grown as spheroids (Suppl.…”

Tiam1/Vav2-Rac1 axis: A tug-of-war between islet function and dysfunction

“…Interestingly, NSC23766 [50 μM] also inhibited carbachol-induced RhoA activation and muscarinic acetylcholine-induced inotropic response in isolated neonatal rats requiring the activation of Rho-dependent kinases. More recent studies by Dutting and associates [45] have reported critical off-target effects of NSC23766 and EHT1864 [a known inhibitor of Rac1; ref. 46] in mouse platelets.…”

Rac1 Modulates Endothelial Function and Platelet Aggregation in Diabetes Mellitus

“…Under normal glucose condition, the dose of 30 μmol/L, NSC23766 was sufficient to reduce Rac1 activity, modulating, in turn, platelet function. Although the dose used in our study was significantly lower compared with the one used elsewhere,42 we cannot completely exclude that the potential off‐target effects of NSC23766 might affect our results. Because the effect of NSC23766 on platelet aggregation, under high glucose condition, is already present for lower NSC23766 doses (15 μmol/L), this raised the question of how to separate the effect of hyperglycemia and the effect of NSC23766 on platelet aggregation.…”