CK 666 is a cell-permeable actin-related protein Arp2/3 complex inhibitor (IC50=12μM)[1]. CK 666 binds to Arp2/3 complex, stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation[2]. CK 666 is routinely used in studies of cytoskeletal dynamics such as cell migration, phagocytosis and cytokinesis[3][4].
In vitro, CK 666 (100μM; 24h) inhibited proliferation in KYSE-30 esophageal, T24 bladder, MDA-MB-231 breast, FaDu hypopharyngeal and SKOV3 ovarian cancer cells, elevated LC3-II, decreased p62 to promote autophagy, and increased release of CD63⁺exosome-like vesicles[5]. Pretreatment of human lung microvascular endothelial cells (HLMVEC) and pulmonary artery endothelial cells (HPAEC)with CK 666 (50μM) 1h before sphingosine-1-phosphate (S1P)-induction significantly reduced trans-endothelial electrical resistance (TER), attenuated S1P-induced barrier enhancement, and markedly decreased individual lamellipodium depth[6].
In vivo, CK 666 (30mg/kg; i.p.; once 24h and once 2h before ischemia) markedly decreased serum creatinine and blood urea nitrogen levels, reduced renal malondialdehyde (MDA) content, attenuated tubular necrosis, and significantly lowered TUNEL-positive apoptotic cells in a mouse renal ischemia–reperfusion injury model[7].
References:
[1] Hetrick B, Han MS, Helgeson LA, Nolen BJ. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem Biol. 2013;20(5):701-712.
[2] Nolen BJ, Tomasevic N, Russell A, et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature. 2009;460(7258):1031-1034.
[3] Liu CS, Cheung PW, Dinesh A, et al. Actin-related protein 2/3 complex plays a critical role in the aquaporin-2 exocytotic pathway. Am J Physiol Renal Physiol. 2021;321(2):F179-F194.
[4] Efremov YM, Dokrunova AA, Efremenko AV, Kirpichnikov MP, Shaitan KV, Sokolova OS. Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells. Biochim Biophys Acta. 2015;1853(11 Pt B):3117-3125.
[5] Li L, Cai S, Chen J, et al. CK-666 exerts anticancer effects by regulating autophagy, tunneling nanotubes and extracellular vesicles formation. Biomed Pharmacother. 2025;183:117825.
[6] Belvitch P, Brown ME, Brinley BN, et al. The ARP 2/3 complex mediates endothelial barrier function and recovery. Pulm Circ. 2017;7(1):200-210.
[7] Hu Q, Zhao Y, Sun WY, et al. CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism. J Biol Chem. 2024;300(12):107942.
CK 666是一种可穿透细胞的肌动蛋白相关蛋白Arp2/3复合物抑制剂（IC50=12μM）[1]。CK 666通过与Arp2/3复合物结合，稳定其非活化构象，阻断Arp2与Arp3亚基向活化丝状（短螺距）构象的移动[2]。CK 666常被用于细胞迁移、吞噬和胞质分裂等细胞骨架动力学研究[3][4]。
体外实验中，CK 666（100μM；24h）可抑制KYSE-30食管癌细胞、T24膀胱癌细胞、MDA-MB-231乳腺癌细胞、FaDu下咽癌细胞和SKOV3卵巢癌细胞的增殖，上调LC3-II、下调p62以促进自噬，并增加CD63⁺外泌体样囊泡的释放[5]。在鞘氨醇-1-磷酸（S1P）诱导前1h用CK 666（50μM）预处理人肺微血管内皮细胞（HLMVEC）和肺动脉内皮细胞（HPAEC），可显著降低跨内皮电阻（TER），减弱鞘氨醇-1-磷酸（S1P）诱导的屏障增强效应，并显著减小单个板状伪足的深度[6]。
体内实验中，CK 666（30mg/kg；腹腔注射；分别在缺血前24h和2h给药一次）在小鼠肾缺血-再灌注损伤模型中显著降低血清肌酐和尿素氮水平，减少肾组织丙二醛（MDA）含量，减轻肾小管坏死，并显著降低TUNEL阳性凋亡细胞数量[7]。