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An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

ABSTRACT: Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [³²P/³³P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

SUBMITTER: Swingle M

PROVIDER: S-EPMC8041090 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In all, 15 aryl-containing phosphonates have been synthesized and tested for their effect on protein-tyrosine phosphatase (PTPase) activity. Two compounds, (naphth-2-yl) difluoromethylphosphonic acid (12) and (napthy-1-yl) difluoromethylphosphonic acid (13) have been found to inhibit dephosphorylation of [32P]insulin receptors by PTP-1B, a protein tyrosine phosphatase (PTPase), with IC50 values of 40-50 microM. Compound 12 competitively inhibited insulin-receptor dephosphorylation by PTP-1B. Compound 12 also inhibited PTP-1B-catalysed dephosphorylation of a synthetic tyrosine phosphorylated substrate poly(Glu80-Tyr20) at the same potency, indicating that 12 acted via interaction with the PTPase. Additionally, 12 inhibited insulin-receptor PTPase(s) and epridermal-growth-factor-receptor PTPase(s) present in solubilized membranes from CHO (Chinese-hamster ovary)/HIRc and A431 cells respectively. IC50 values of 40-50 microM were obtained in all cases with compound 12. Of note is the fact that these compounds did not have any effect on insulin-receptor autophosphorylation. Nine out of the 15 compounds potently inhibited serine/threonine phosphatase PP-2A activity without any effect on serine/threonine phosphatase PP-1 when tested at a concentration as high as 675 microM. The most potent compounds acting toward PP-2A had IC50 values of 45-50 microM. These PP-2A inhibitors could be useful tools for studying serine/threonine-phosphatase-mediated signal transduction. Two compounds, 12 and 13, inhibited both tyrosine phosphatase PTP-1B and serine/threonine phosphatase PP-2A with similar potency; IC50 values being 40-50 microM in both cases. Details of the synthesis of compounds 10, 11 and 13 are given in Supplementary Publication SUP 50177 (6 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1995) 305, 9.

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An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

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