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 p56lck, an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56lckkinase activity with IC50= 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-JunN-terminal kinase 2α. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50= 35 μM) and blocked T cell proliferation in response to alloantigen (IC50= 14 μM) and CD3/CD28-induced IL-2 secretion (IC50= 2.2 μM) in primary T cell cultures. Inhibition of p56lckby 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 p56lckkinase activity and TCR-dependent signal transduction. Incubation with thiols such as β-ME or DTT also blocked the ability of the isothiazolone to inhibit p56lckkinase activity. LC/MS analysis established the covalent modification of p56lckat cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56lckcatalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56lckenzyme. Loss of p56lckactivity 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 p56lckinhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.