A multidisciplinary research program is designed to understand the structural basis of substrate specificity of the protein-tyrosine phosphates (PTPases) and to develop specific, high-affinity low molecular weight nonpeptidic PTPase inhibitors. PTPases constitute a large family of enzymes that can be collectively categorized into two groups: receptor-like and intracellular PTPases. The receptor-like PTPases, exemplified by PTP-alpha, generally have an extracellular domain, a single transmembrane region, and one or two cytoplasmic PTPase domains. The intracellular PTPases, exemplified by PTP1B, contain a single catalytic domain and various amino or carboxyl terminal extensions including SH2 domains that may have targeting or regulatory specificity phosphatases. VHR is a representative of the dual specificity phosphatases that can utilize substrates containing phosphotyrosine as well as phosphoserine/phosphothreonine. Although ligand binding and localization/targeting domains are believed to be involved in the regulation of PTPase activity, the role of the PTPase catalytic site in determining substrate specificity has not been fully investigated. This proposal will be focused on PTP1B, PTP-alpha and VHR. The substrate sequence specificity of these enzymes will be evaluated via combinatorial phosphopeptide libraries in combination with detailed kinetic analyses of individual pure peptides with systematically altered chemical properties. Active site-directed inhibitors will be identifies by screening synthetic libraries of low molecular weight aromatic phosphonates. The in vivo efficacy of selected inhibitors will be assessed in a Chinese hamster cell line that overexpresses human insulin receptor. Since the elucidation of the fundamentals of molecular recognition events requires an understanding of the atomic details of how molecules interact, the three-dimensional structures of PTPases complexed with high affinity substrates/inhibitors will be determined. PTP1B, PTP-alpha and VHR are ideal in this regard, since their crystal structures have been solved. Thus the crystal structures of these PTPases complexed with tight-binding substrates/ inhibitors can be solved by molecular replacement techniques.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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Ikeda, Richard A
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Albert Einstein College of Medicine
Schools of Medicine
United States
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