Trevillyan J M, Chiou X G, Ballaron S J, Tang Q M, Buko A, Sheets M P, Smith M L, Putman C B, Wiedeman P, Tu N, Madar D, Smith H T, Gubbins E J, Warrior U P, Chen Y W, Mollison K W, Faltynek C R, Djurić S W
Abbott Laboratories, Immunological Disease Research, Abbott Park, Illinois, 60064-6119, USA.
Arch Biochem Biophys. 1999 Apr 1;364(1):19-29. doi: 10.1006/abbi.1999.1099.
Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.
Lck编码一种56 kDa的蛋白酪氨酸激酶，主要在T淋巴细胞中表达，对启动T细胞抗原受体（TCR）信号转导途径至关重要，最终导致T细胞细胞因子基因表达和效应功能。作为对p56(lck)的选择性新型抑制剂进行高通量筛选的结果，鉴定出一种异噻唑酮化合物，即甲基-3-(N-异噻唑酮)-2-噻吩羧酸酯（A-125800），它以IC50 = 1-7 microM的浓度抑制p56(lck)激酶活性。在类似的检测条件下，该异噻唑酮化合物在阻断ZAP-70酪氨酸激酶活性方面具有同等效力，但对p38 MAP激酶和c-Jun N末端激酶2α的催化活性的效力要低50至100倍。A-125800阻断了Jurkat T细胞中依赖激活的TCR酪氨酸磷酸化和细胞内钙动员（IC50 = 35 microM），并在原代T细胞培养中阻断了T细胞对同种异体抗原的增殖反应（IC50 = 14 microM）以及CD3/CD28诱导的IL-2分泌（IC50 = microM）。A-125800对p56(lck)的抑制作用具有剂量和时间依赖性，且是不可逆的。将该化合物异噻唑酮环中的硫原子替换为亚甲基完全消除了其抑制p56(lck)激酶活性和TCR依赖性信号转导的能力。与β-巯基乙醇或二硫苏糖醇等硫醇一起孵育也阻断了异噻唑酮抑制p56(lck)激酶活性的能力。液相色谱/质谱分析确定了p56(lck)在半胱氨酸残基378、465和476处的共价修饰。这些数据共同支持了一种抑制机制，即p56(lck)催化结构域内的半胱氨酸-SH基团与异噻唑酮环反应，导致环打开并与p56(lck)酶形成二硫键。有人提出，由于-SH氧化导致p56(lck)活性丧失在艾滋病病理学中起作用。因此，本报告中描述的导致p56(lck)抑制的类似巯基氧化机制可能在完整的T细胞中发生，并且可能是某些T细胞病理学的基础。
