title: Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding.
abstract: Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Pointe-Claire, Quebec H9R 4P8, Canada.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Montalibet
authors.forename: Kathryn
authors.initials: K
authors.lastname: Skorey
authors.forename: Dan
authors.initials: D
authors.lastname: McKay
authors.forename: Giovanna
authors.initials: G
authors.lastname: Scapin
authors.forename: Ernest
authors.initials: E
authors.lastname: Asante-Appiah
authors.forename: Brian P
authors.initials: BP
authors.lastname: Kennedy
mesh_terms: D000495:Allosteric Site; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D017931:DNA Primers / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry*; D015723:Gene Library; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D015233:Models, Statistical; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology*; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D013997:Time Factors
chemical_list: C509920:7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile; D017931:DNA Primers; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009570:Nitriles; D063065:Organophosphonates; D015688:Oncogene Protein pp60(v-src); D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases