Inhibition of p56(lck) tyrosine kinase by isothiazolones

• Published in: Archives of biochemistry and biophysics 1999-04, Vol.364 (1), p.19-29
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Binding Sites - drug effects; Catalysis - drug effects; Cell Line; Cysteine - metabolism; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Humans; Jurkat Cells; Lymphocyte Activation - drug effects; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - antagonists & inhibitors; Molecular Sequence Data; Phosphorylation - drug effects; Receptors, Antigen, T-Cell - metabolism; Signal Transduction - drug effects; Sulfhydryl Compounds - pharmacology; Thiazoles - metabolism; Thiazoles - pharmacology; Time Factors
• ISSN: 0003-9861
• 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.