ML-7 hydrochloride (ML-7 HCl) is a cell-permeable, reversible, effective, ATP-competitive, and specific inhibitor of myosin light chain kinase (Ki: 300 nM); also inhibits smooth-muscle myosin light chain kinase, PKA, and PKC.

PKC:42 μM, PKA:21 μM, MLCK:0.3 μM(Ki)

ML-7通过抑制MLCK激活粘附性MCF-10A及其Ras变异细胞中的caspase-3[2]。ML-7处理以剂量依赖的方式降低MLC20磷酸化水平，并相应增加平滑肌细胞(SMC)的死亡率。ML-7对MLCK的抑制作用具有高度选择性，其对MLCK的Ki为0.3 μM，而对蛋白激酶A的Ki为21 μM，对蛋白激酶C的Ki为42 μM。过表达Bcl-2可以保护细胞免受ML-7引起的凋亡[4]。

ML7通过调节高脂饮食喂养的兔子中的MLCK和MLC磷酸化作用，影响紧密连接（TJ）蛋白区域桥蛋白（ZO)-1和闭锁蛋白的表达，从而改善血管内皮功能障碍（VED）和动脉粥样硬化（AS）。ML7能降低高脂饮食兔子动脉壁中MLCK和MLC的磷酸化表达，并减少AS兔子中的脂质沉积损伤[1]。

IKK-2 Assay: Recombinant human IKK-2 (residues 1-756) is expressed in baculovirus as an N-terminal GST-tagged fusion protein, and its activity is assessed using a time-resolved fluorescence resonance energy transfer assay. In brief, IKK-2 (5 nM final) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01% w/v BSA) is added to wells containing various concentrations of compound or dimethyl sulfoxide (DMSO) vehicle (3% final). The reaction is initiated by the addition of GST-IκBα substrate (25 nM final)/ATP (1 μM final), in a total volume of 30 μL. The reaction is incubated for 30 min at room temperature, then terminated by the addition of 15 μL of 50 mM EDTA. Detection reagent (15 μL) in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) containing antiphosphoserine- IκBα-32/36 monoclonal antibody 12C2, labeled with W-1024 europium chelate, and an allophycocyanin-labeled anti-GST antibody is added, and the reaction is further incubated for 60 min at room temperature. The degree of phosphorylation of GST- IκBαis measured as a ratio of specific 665-nm energy transfer signal to reference europium 620-nm signal, using a Packard Discovery plate reader.

Cells are treated with vehicle alone, Blebbistatin, ML-7, ML-9, latrunculin A or Y-27632 in media without serum at varying concentrations and time points before being fixed or prepared for FACS analysis. Untreated cells are also serum starved for the duration of the inhibitor treatment. Stock solutions of each agent are made in DMSO (50 μM Blebbistatin, 5 mM LA), 50% ethanol (10 mM ML-7), 70% ethanol (20 mM ML-9) or water (20 mM Y-27632) and are maintained at -20°C.(Only for Reference)