title: An enzyme-linked immunosorbent assay to measure insulin receptor dephosphorylation by PTP1B.
abstract: Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.
authors.affiliation: Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, MA 02140, USA.
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: May
authors.initials: M
authors.lastname: Tam
authors.forename: Steve
authors.initials: S
authors.lastname: Kirincich
authors.forename: Zhao-Kui
authors.initials: ZK
authors.lastname: Wan
authors.forename: Doug
authors.initials: D
authors.lastname: Wilson
authors.forename: Jun Jun
authors.initials: JJ
authors.lastname: Wu
authors.forename: Jinbo
authors.initials: J
authors.lastname: Lee
authors.forename: James F
authors.initials: JF
authors.lastname: Tobin
authors.forename: David V
authors.initials: DV
authors.lastname: Erbe
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases