Inhibition of p56lckTyrosine Kinase by Isothiazolones

Abstract 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 IC 50 = 1–7 μM. 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 2α. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC 50 = 35 μM) and blocked T cell proliferation in response to alloantigen (IC 50 = 14 μM) and CD3/CD28-induced IL-2 secretion (IC 50 = 2.2 μM) 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 β-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.