Identification and evaluation of inhibitors for the human cytoplasmic protein tyrosine phosphatase
Abstract
Transient phosphorylation of the cytoplasmic residues of integral membrane proteins is a common mechanism for the transmission of extracellular signals to intracellular components. The EphA2 Receptor Tyrosine Kinase (EphA2) regulates normal cell growth through maintenance of cell-cell contact mediated signaling. However, EphA2 is found under-phosphorylated in transformed cell culture, which is correlated with the disruption of normal cell-cell contacts. EphA2 is the cellular substrate for the Human Cytoplasmic Protein Tyrosine Phosphatase (HCPTP), which is found overexpressed in these transformed cell lines. General inhibition of phosphatases in cellular studies has restored EphA2 phosphorylation state and the cells to a non-transformed phenotype. Thus the selective inhibition of HCPTP presents a novel therapeutic route for the treatment of metastatic transformation. Initial attempts at inhibitor design have focused on both rationally designed inhibitors, which are based upon a cocrystal structure of a homologous low molecular weight phosphatase from yeast, and in silico screening of small molecule libraries. Low micromolar inhibitors, which are among the most effective small molecule inhibitors reported to date, have been identified and kinetically characterized. Crystallographic experiments were undertaken to provide a detailed atomic description of the binding of small molecules to HCPTP. Cocrystal complexes between HCPTP and small molecules have identified a secondary binding site distant from the active site, which presents the possibility of identifying interactions at either site. Solution state NMR experiments have been used to confirm the presence of this secondary site and attempt to characterize the interactions of the previously identified strong inhibitors with HCPTP. The kinetic results, cocrystallographic structures, and NMR experiments present more robust, dynamic models for micromolar inhibitors of HCPTP that may led to the next generation of potent, selective inhibitors.
Degree
Ph.D.
Advisors
Stauffacher, Purdue University.
Subject Area
Biochemistry|Biophysics
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