Structure-Activity Relationship Studies of Fostriecin, Cytostatin, and Key Analogs, with PP1, PP2A, PP5, and (β12–β13)-Chimeras (PP1/PP2A and PP5/PP2A), Provide Further Insight into the Inhibitory Actions of Fostriecin Family Inhibitors

Abstract

Fostriecin and cytostatin are structurally related natural inhibitors of serine/threonine phosphatases, with promising antitumor activity. The total synthesis of these antitumor agents has enabled the production of structural analogs, which are useful to explore the biological significance of features contained in the parent compounds. Here, the inhibitory activity of fostriecin, cytostatin, and 10 key structural analogs were tested in sideby-side phosphatase assays to further characterize their inhibitory activity against PP1c (Ser/Thr protein phosphatase 1 catalytic subunit), PP2Ac (Ser/Thr protein phosphatase 2A catalytic subunit), PP5c (Ser/Thr protein phosphatase 5 catalytic subunit), and chimeras of PP1 (Ser/Thr protein phosphatase 1) and PP5 (Ser/Thr protein phosphatase 5), in which key residues predicted for inhibitor contact with PP2A (Ser/Thr protein phosphatase 2A) were introduced into PP1 and PP5 using sitedirected mutagenesis. The data confirm the importance of the C9-phosphate and C11-alcohol for general inhibition and further demonstrate the importance of a predicted C3 interaction with a unique cysteine (Cys 269 ) in the ␤12-␤13 loop of PP2A. The data also indicate that additional features beyond the unsaturated lactone contribute to inhibitory potency and selectivity. Notably, a derivative of fostriecin lacking the entire lactone subunit demonstrated marked potency and selectivity for PP2A, while having substantially reduced and similar activity against PP1 and PP1/PP2A-PP5/PP2A-chimeras that have greatly increased sensitivity to both fostriecin and cytostatin. This suggests that other features [e.g., the (Z,Z,E)-triene] also contribute to inhibitory selectivity. When considered together with previous data, these studies suggest that, despite the high structural conservation of the catalytic site in PP1, PP2A and PP5, the development of highly selective catalytic inhibitors should be feasible.Fostriecin and cytostatin are structurally related phosphate monoesters produced by Streptomyces pulveraceus and Streptomyces sp. MJ654-Nf4, respectively, that display cytotoxicity and antitumor activity (for review, see Lewy et al., 2002). Cytostatin has cytotoxic activity toward melanoma and leukemia cell lines and has been shown to inhibit lung tumor metastasis (Masuda et al., 1995;Kawada et al., 1999). The antitumor activity of fostriecin (also called CI-920, NSC 339638, or PD 110,161) has been evaluated extensively (for review, see de Jong et al., 1997;Lewy et al., 2002;Honkanen, 2005). It demonstrates marked cytotoxicity against many cancer cell lines and potent antitumor activity in animals (for

Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells

“…Ser/Thr phosphatase activity was assessed using the commercially available Fluorimetric SensoLyte FDP Protein Phosphatase Assay Kit (AnaSpec, Fremont, CA) as we have previously described (35,36).…”

Synthetic Strategies Employed for the Construction of Fostriecin and Related Natural Products

“…208 The diastereomeric analogues 6b – 6d all possessed at least 2 orders of magnitude lower activity (for the natural substrate phosphohistone) than the natural product, highlighting the importance of the central stereotriad. Analogue 290 , lacking the phosphate group, did not bind PP2A well, as expected from previous results obtained for fostriecin.…”

“…This challenge is met by a myriad of different approaches, including asymmetric Michael addition, 202,203 enzymatic acylation, 204 chiral auxiliary directed Diels–Alder cyclization, 205–207 and enantioselective allylic alkylation. 208 …”

Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

“…Fostriecin, a natural product produced by Streptomyces sp., is a highly selective inhibitor of PP2A/PP4 (IC 50 < 2 nM) and a weak inhibitor of PP1 and PP5 (IC 50 > 70 mM). 8 Fostriecin demonstrated sufficient antitumor activity in animals to warrant Phase I human clinical trials. 9,10 The other afore-mentioned natural products are strong inhibitors (IC 50 ; low nM) of PP1/PP2A/PP4/PP5/PP6 that demonstrate modest or no selectivity.…”