NSC 23766 trihydrochloride (Rac1 Inhibitor) is an inhibitor of Rac GTPase targeting Rac activation by GEFs (IC50: ~50 μM); no inhibitory for the closely related targets, RhoA or Cdc42.

Rac GTPase:50 μM

NSC23766被确认能够适配于Rac1的表面沟槽，这一沟槽对于GEF的特异性是关键。NSC23766有效地以剂量依赖的方式抑制由特异性GEF Trio或Tiam1引起的Rac1的结合和激活，同时不干扰与Cdc42或RhoA的密切相关的结合或激活以及它们各自的GEFs，也不干扰Rac1与BcrGAP或效应蛋白PAK1的相互作用。[1] NSC 23766在调控Rac GTPase在细胞骨架和许多细胞功能上发挥作用，包括细胞周期、细胞生长、黏附、迁移和基因转录。NSC 23766（50 μM）强效阻断血清或血小板衍生的生长因子引起的Rac1激活和假足形成，而不影响NIH 3T3细胞内源性Cdc42或RhoA的活性。 NSC 23766减少了Trio或Tiam1引起的但不影响Vav、Lbc、Intersectin或一个构成活性的Rac1变种刺激的NIH 3T3细胞生长，并抑制了Trio、Tiam1或Ras引起的细胞转化。NSC23766剂量依赖地抑制PC-3细胞的增殖和无锚生长。25μM NSC23766通过85%抑制PC-3细胞通过Matrigel的侵袭。[1] 50 μM NSC 23766抑制在人类血小板中由凝血酶引起的Rac1和Rac2的激活以及血小板的聚集。[2] NSC23766阻止在swAPP-HEK293细胞中Aβ40和Aβ42的产生，但不影响Notch和sAPPα。NSC23766在细胞中阻止γ-分泌酶活性，但不直接充当γ-分泌酶抑制剂。NSC23766剂量依赖地减少分泌和细胞内Aβ40的水平，IC50为48.94μM。50μM NSC 23766 抑制Aβ42的释放57.97%。[3]

NSC23766通过促进造血干细胞/前体的动员起作用。将NSC23766（2.5 mg/kg）经腹腔注射至“动员能力差”的C57Bl/6小鼠模型中，可在注射后6小时使循环中的造血干细胞/前体数量增加两倍。[2] NSC23766还能缓解小鼠脂多糖诱导的急性肺损伤。以1或3 mg/kg的剂量处理NSC23766，不仅减少了炎症细胞的浸润和髓过氧化物酶（MPO）活性，也抑制了促炎介质、肿瘤坏死因子-α和白细胞介素-1β的mRNA表达。NSC23766还减少了在LPS挑战的肺中Evans蓝和白蛋白的积累。[6]

Rho GTPase activity assay: Cells are grown in log phase in a 10-cm dish, and are starved in 0.5% serum medium or indicated otherwise for 24 h before lysis in a buffer containing 20 mM Tris HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40, 10% glycerol, and 1× protease inhibitor mixture. Lysates are clarified, the protein concentrations are normalized, and the GTP-bound Rac1 in the lysates is measured by an effector domain pull-down assay. For the His6-PAK1 PBD pull-down assay, cell lysates are incubated with Ni2+-agarose-immobilized His6-PAK1 PBD domain (～1 μg each) purified from E. coli for 30 min. The Ni2+-agarose co-precipitates are washed twice in the wash buffer and analyzed by immunoblotting with anti-Rac1 monoclonal antibody.

Cells (1.5 × 104/mL) are seeded in each well of 96-well tissue culture plates with 200 μL of medium. After 24 hours of plating, the medium is replaced with 200 μL of fresh medium containing NSC23766 at the indicated concentrations. At the end of the treatment period 20 μL of MTS solution are added to each well and incubated at 37 ℃ for 2 hours. Absorbance at 490 nm is read on a 96-well plate reader.(Only for Reference)