Discovery of Protein Phosphatase 2C Inhibitors by Virtual Screening

Abstract

Protein phosphatase 2C (PP2C) is an archetype of the PPM Ser/Thr phosphatases, characterized by dependence on divalent magnesium or manganese cofactors, absence of known regulatory proteins, and resistance to all known Ser/Thr phosphatase inhibitors. We have used virtual ligand screening with the AutoDock method and the National Cancer Institute Diversity Set to identify small molecule inhibitors of PP2Cα activity at a protein substrate. These inhibitors are active in the micromolar range, and represent the first non-phosphate-based molecules found to inhibit a type 2C phosphatase. The compounds docked to three recurrent binding sites near the PP2Cα active site and displayed novel Ser/Thr phosphatase selectivity profiles. Common chemical features of these compounds may form the basis for development of a PP2C inhibitor pharmacophore and may facilitate investigation of PP2C control and cellular function.

Small molecule activators of pre-mRNA 3′ cleavage

“…6, lane 13), a concentration close to the threshold at which L-83633 showed activity (Fig. 2C) and close to the approximate IC 50 of L-83633 for PP2C (Rogers et al 2006). Compound 2's threshold for activating cleavage is therefore 50-fold lower than the 10 mM concentration required by CP and 8 for comparable activity (Fig.…”

“…If an inauthentic cleavage context, such as the absence of transcription, causes the putative phosphatase to dephosphorylate one of the cleavage factors and thereby inhibit its activity, then including a phosphatase inhibitor in the reaction may preserve the phospho form of the cleavage factor and enable cleavage to occur in vitro. We have shown here that two phosphatase inhibitors, fluoride/EDTA, a general S/T phosphatase inhibitor, and NCI 83633 (leucine-2-napthylamide), a PP2C inhibitor (Rogers et al 2006), stimulate 39 cleavage in vitro.…”

“…The discovery of inhibitors of the PPM family of phosphatases has lagged behind that of the PPP family, but several PP2C inhibitors were recently identified (Rogers et al 2006). Two of these, NCI 83633 and NCI 401366 (Fig.…”

Inhibitors of the Ca2+/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

“…For PPM phosphatases, however, only two compounds have been so far reported as specific inhibitors. One is a compound obtained by a virtual screening, reported by Rogers et al [26], while the other is a peptide inhibitor developed by Yamaguchi et al [27]. Unfortunately, the former inhibitor showed only modest specificity to PP2C with substantial inhibitory activity toward PPP family phosphatases [26].…”

“…One is a compound obtained by a virtual screening, reported by Rogers et al [26], while the other is a peptide inhibitor developed by Yamaguchi et al [27]. Unfortunately, the former inhibitor showed only modest specificity to PP2C with substantial inhibitory activity toward PPP family phosphatases [26]. The latter inhibitor is potent and highly specific to a PPM phosphatase, Wip1, but it is thought to have poor cell permeability because it is a peptide-based inhibitor with multiple negative charges, which may prevent permeation of the cell membrane.…”