A Targeted Library of Small-Molecule, Tyrosine, and Dual-Specificity Phosphatase Inhibitors Derived from a Rational Core Design and Random Side Chain Variation

Abstract

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.

Small molecule inhibitors of dual specificity protein phosphatases

“…We have re-synthesized both the quinoline-3-carboxylate and the naphthyridine-3-carboxylate, which were reported to have anti-Cdc25 activity (El-Subbagh et al, 1999) but were unable to demonstrate inhibition with our assay conditions (Rice et al, 1997;unpublished results). A broad screening project allowed Fritzen et al (2000) to identify isoquinolines with weak in vitro inhibitory activity against Cdc25B.…”

“…Dephosphorylation of cdk1 by nuclear Cdc25C activates the kinase. The chk1 pathway is activated by DNA damage leading to Cdc25C phosphorylation on Ser-216 and cytoplasmic sequestration due to 14-3-3 protein binding (Ducruet et al, 2000);¯uorescein diphosphate (FDP) (Rice et al, 1997) or; p-nitrophenyl phosphate (pNPP) (Koufaki et al, 1996). The choice of substrate, however, can a ect the enzyme kinetics, as can the domains of the enzyme used.…”

“…Natural products have also been used in the design of pharmacophore platforms for combinatorial synthetic approaches (Rice et al, 1997). Thus, SC-aad9 is a competitive inhibitor of all 3 human Cdc25 isoforms in vitro with low mM K i values (Rice et al, 1997).…”

Protein kinase CK2 regulates CDC25B phosphatase activity

“…The precipitates were washed twice with lysis buffer without NP40 in the absence of phosphatase inhibitors (NaF and Na 3 VO 4 ) and once with phosphatase buffer (30 mm Tris-HCl, pH 8.2, 75 mm NaCl, 0.67 mm EDTA, 0.033% bovine serum albumin, 1 mm DTT). CDC25B phosphatase activity was monitored using fluorescein diphosphate, FDP (Molecular Probes, Inc.), as substrate at a final concentration of 20 mm for 30 min at room temperature, as described by Rice et al (1997). Fluorescence emission from the reaction product was measured with a multiwell plate reader (Fluoroskan Ascent, Labsystems, excitation filter 485 nm; emission filter 538 nm).…”

“…We therefore examined whether CK2 modulated the intrinsic phosphatase activity of CDC25B in vitro. 35 S-methionine-labelled in vitrotranslated CDC25B3 was immunoprecipitated with an anti-CDC25B antibody, incubated or not with purified recombinant CK2, and then subjected to a phosphatase assay in the presence of an exogenous substrate, fluorescein diphosphate (FDP) (Rice et al, 1997). As shown in Figure 6a, CDC25B phosphatase activity was moderately but reproducibly upregulated (average of 1.3-fold of activation) after incubation with recombinant CK2, and this increase in activity paralleled the phosphorylation level of CDC25B (Figure 6a).…”