title: Mechanism of inhibition of protein-tyrosine phosphatases by disodium aurothiomalate.
abstract: Disodium aurothiomalate (AuTM) has been used successfully in the treatment of various autoimmune and inflammatory disorders; however, the molecular target(s) for this agent remains unknown. The aim of this study was to investigate whether the activity of CD45, a protein-tyrosine phosphatase (PTP, EC 3.1.3.48) essential for antigen-receptor-mediated lymphocyte signaling, was modified by AuTM exposure. The effects of AuTM on the activities of CD45 and other PTPs were monitored in vitro by a continuous assay using the substrate fluorescein diphosphate. In addition, the inhibition of PTP1B by AuTM was determined using a novel binding assay that employed an optical biosensor (BIAcore). The experimental results are summarized here: AuTM inhibited CD45 activity with an IC50 of 1.2 +/- 0.1 microM, and inhibition was competitive with substrate. The effect of AuTM, however, was not restricted to CD45, as the cytoplasmic PTP (PTP1B) was also inhibited, with an IC50 of 3.6 +/- 0.2 microM. AuTM also blocked the binding of GST-PTP1B to an immobilized active site inhibitor: a non-hydrolyzable difluorophosphonomethyl phenylalanine-containing biotinylated hexapeptide. AuTM-inhibited CD45 could be reactivated by the addition of excess dithiothreitol. These findings indicate that AuTM may interact with the essential active site cysteine residue involved in the catalytic mechanism of PTPs. Thus, it is possible that some of the cellular effects of gold result from the inhibition of these important cell signaling molecules.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck-Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada. qingping_wang@merck.com
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: N
authors.initials: N
authors.lastname: Janzen
authors.forename: C
authors.initials: C
authors.lastname: Ramachandran
authors.forename: F
authors.initials: F
authors.lastname: Jirik
mesh_terms: D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000235:genetics; D006052:Gold Sodium Thiomalate / Q000494:pharmacology*; D017493:Leukocyte Common Antigens / Q000187:drug effects; D010206:Papain / Q000037:antagonists & inhibitors; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors
chemical_list: D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D006052:Gold Sodium Thiomalate; D005982:Glutathione Transferase; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; D010206:Papain