DOI: 10.1002/cbic.200600287 Cellular Inhibition of Protein Tyrosine Phosphatase 1B by Uncharged Thioxothiazolidinone Derivatives Matthew Stuible, [a] Liang Zhao, [b] Isabelle Aubry, [a] Dirk Schmidt-Arras, [c] Frank- D. Bçhmer, [c] Chao-Jun Li, [b] and Michel L. Tremblay* [a] Introduction The control of cellular tyrosine phosphorylation levels requires the precise balance of protein tyrosine kinase (PTK) and phos- phatase (PTP) activities. Similar to the expression of oncogenic PTKs, [1] nonspecific inhibition of PTPs results in a massive in- crease in cellular phosphotyrosine content. [2] The human PTP superfamily consists of over 100 cysteine-dependent en- zymes. [3] They contain a range of noncatalytic motifs and do- mains that typically mediate protein–protein interactions or target PTPs to particular subcellular compartments. [3] Individual PTPs play specific roles in cellular signal transduction, including the regulation of metabolic and mitogenic signaling, cell ACHTUNGTRENNUNGadhesion and migration, and gene transcription. The mutation or genetic ablation of PTPs in mice causes generally adverse phenotypes, often involving immune [4, 5] or neuronal develop- ment [6, 7] effects. However, one notable exception is PTP1B (EC 3.1.3.48). Mice lacking this enzyme are healthy and display resistance to diet-induced diabetes and obesity, [8, 9] this is likely to be due to its function as a negative modulator of insulin [10] and leptin [11] signaling. This phenotype generated interest in PTP1B as a drug target for type II diabetes. Indeed, numerous small-molecule PTP1B inhibitors with varying specificity and cell-permeability have been reported. [12, 13] The PTP catalytic mechanism involves nucleophilic attack by an anionic cysteine residue on the phosphotyrosyl phosphate group. [14] This cysteine, part of the PTP signature motif, HC(X)5R, is situated at the base of a flexible active site pocket. By crystallography, similar binding interactions have been re- ported for PTP1B in complex with phosphotyrosine and pep- tide substrates. [15–17] Specifically, the phenyl ring of the sub- strate phosphotyrosine forms aromatic–aromatic interactions with Y46, at one side of the pocket, and F182, which is part of the flexible WPD loop that closes over substrates upon bind- ing. In addition, extensive hydrogen bond interactions occur between the negatively charged phosphate group and resi- dues within the active site pocket. Rational design and library screening projects for PTP1B inhibitors have identified several nonhydrolyzable phosphotyrosine analogs or mimetics, includ- ing aryl difluoromethylenephosphonates (DFMP) [18] and N-aryl oxamic acid derivatives. [19] These compounds commonly bear one or more negative charges, and additional charged moiet- ies have been included to target a secondary phosphate bind- ing site unique to PTP1B and the closely related TCPTP. [15] De- spite achieving excellent (low nanomolar) potency in vitro, [20, 21] these charged compounds have poor cell permeability and often require additional strategies, such as prodrug esterifica- tion [22, 23] or fatty acid conjugation, [24, 25] to improve cellular ACHTUNGTRENNUNGactivity. Here, we report an uncharged, bis-aryl thioxothiazolidinone compound (compound 1) that acts as a competitive PTP inhib- As important regulators of cellular signal transduction, members of the protein tyrosine phosphatase (PTP) family are considered to be promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, thus limiting cellular permeability. Here, we have identified an un- charged thioxothiazolidinone derivative (compound 1), as a com- petitive inhibitor of a subset of PTPs. Compound 1 effectively in- hibited protein tyrosine phosphatase 1B (PTP1B) in two cell-based systems: it sensitized wild-type, but not PTP1B-null fibroblasts to insulin stimulation and prevented PTP1B-dependent dephosphor- ylation of the FLT3-ITD receptor tyrosine kinase. We have also tested a series of derivatives in vitro against PTP1B and proposed a model of the PTP1B–inhibitor interaction. These compounds should be useful in the elucidation of cellular PTP function and could represent a starting point for development of therapeutic PTP inhibitors. [a] M. Stuible, I. Aubry, Prof. M. L. Tremblay McGill Cancer Centre and Department of Biochemistry, McGill University 3655 Prom. Sir William Osler, MontrØal, QuØbec, H3G 1Y6 (Canada) Fax: (001)514-398-6769 E-mail: michel.tremblay@mcgill.ca [b] L. Zhao, Prof. C.-J. Li Department of Chemistry, McGill University MontrØal, QuØbec, H3A 2K6 (Canada) [c] D. Schmidt-Arras, Prof. F.-D. Bçhmer Institute of Molecular Cell Biology, Medical Faculty Friedrich Schiller University Drackendorfer Strasse 1, 07747 Jena (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. ChemBioChem 2007, 8, 179 – 186 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 179