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Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp(181), Lys(120), and Tyr(46). PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.