Structure-Activity Relationship Studies of Fostriecin, Cytostatin, and Key Analogs, with PP1, PP2A, PP5, and ( 12– 13)-Chimeras (PP1/PP2A and PP5/PP2A), Provide Further Insight into the Inhibitory Actions of Fostriecin Family Inhibitors

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) 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/PP2APP5/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 This work was supported in part by the National Institutes of Health [Grants CA42056, CA60750, MD002314]. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program [Grant C06-RR11174] from the National Center for Research Resources. M.R.S. and L.A. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.155630. ABBREVIATIONS: PP2A, Ser/Thr protein phosphatase 2A; PP2Ac, Ser/Thr protein phosphatase 2A catalytic subunit; PP1, Ser/Thr protein phosphatase 1; PP1c, Ser/Thr protein phosphatase 1 catalytic subunit; PP5c, Ser/Thr protein phosphatase 5 catalytic subunit; PPase, Ser/Thr phosphatase; SAR, structure-activity relationship; PAGE, polyacrylamide gel electrophoresis; PKA, protein kinase A; PIPES, 1,4-piperazinediethanesulfonic acid. 0022-3565/09/3311-45–53$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 155630/3513262 JPET 331:45–53, 2009 Printed in U.S.A. 45 at A PE T Jornals on Jne 9, 2017 jpet.asjournals.org D ow nladed from review, see de Jong et al., 1997; Lewy et al., 2002; Honkanen, 2005). To evaluate its potential for use as an antitumor agent in humans, fostriecin entered National Cancer Institutesponsored clinical trials (Lê et al., 2004). Although limited, the data obtained from the phase 1 trials suggest that plasma levels of fostriecin shown to have antitumor activity in animals can be achieved in humans (Leopold et al., 1984; Susick et al., 1990; Lê et al., 2004). Unfortunately, the trials were discontinued before the maximal tolerated dose was established when concerns related to the storage stability of the naturally produced material surfaced (Lê et al., 2004). The biological actions of fostriecin were initially ascribed to its ability to inhibit topoisomerase II; however, its cell-cycle effects and potency are inconsistent with this target of action (for review, see Lewy et al., 2002; Honkanen, 2005). Subsequently, fostriecin (Walsh et al., 1997; Buck et al., 2003), cytostatin (Bialy and Waldmann, 2004; Lawhorn et al., 2006), and structurally related natural products [phospholine, leustroducsin, and phoslactomycins (Usui et al., 1999; Kawada et al., 2003); Fig. 1] have all been shown to inhibit a subset of PPP-family serine/threonine protein phosphatases. Fostriecin acts as a potent inhibitor of PP2A/PP4 (IC50 0.2–4 nM) and a weak inhibitor of PP1 and PP5 (PP2A/PP4 versus PP1/PP5 selectivity 10) (Walsh et al., 1997; Buck et al., 2003). Cytostatin is also a potent and selective inhibitor of PP2A (PP2A IC50 20–400 nM; PP2A versus PP1/PP5 10) (Bialy and Waldmann, 2004; Lawhorn et al., 2006). Phospholine, leustroducsin H, and phoslactomycins are weaker inhibitors of PP2A (Usui et al., 1999; Kawada et al., 2003) and have not been examined using other phosphatases. Synthetic efforts have provided methods for producing fostriecin and related PP2A-inhibitors. Following the first total synthesis of fostriecin (Boger et al., 2001), at least nine total or formal syntheses have been reported (Chavez and Jacobsen, 2001; Esumi et al., 2002; Reddy and Falck, 2002; Miyashita et al., 2003; Maki et al., 2005; Trost et al., 2005). The total synthesis of cytostatin (Bialy and Waldmann, 2004; Lawhorn et al., 2006; Jung et al., 2008) and cytostatin analogs (Lawhorn et al., 2006; Jung et al., 2008) have also been reported. These studies have sparked renewed interest in the potential for development of more stable derivatives that may have utility as novel antitumor drugs. In efforts to identify the features of fostriecin-family compounds required for its potent and selective inhibition of PP2A, we have reported the synthesis of structural derivatives of fostriecin (Buck et al., 2003; Lawhorn et al., 2006) and, more recently, cytostatin (Lawhorn et al., 2006) that share distinctive phosphate monoester, (Z,Z,E)-triene, and , -unsaturated -lactone structural units (Fig. 1). Key analogs that have been synthesized include dephosphofostriecin, dephosphocytostatin, the C10/C11-diastereomers of cytostatin, a cyclic phosphodiester of fostriecin, a partial structure of fostriecin lacking the entire lactone moiety, and derivatives of fostriecin from which the lactone ring is saturated or contained in other modifications that alter the electrophilic nature of C3 (Buck et al., 2003; Lawhorn et al., 2006). The diastereomers of cytostatin demonstrated the importance of the C11-hydroxyl for PP2A inhibition (Lawhorn et al., 2006). Compounds in which the lactone is modified suggest that the lactone contributes to potent inhibitory activity against PP2A (Buck et al., 2003; Lawhorn et al., 2006). Compounds lacking the phosphate were less active against PP2A and less cytotoxic against cultured cancer cell lines (Buck et al., 2003; Lawhorn et al., 2006). The crystal structures of PP1 (Goldberg et al., 1995), PP2A (Xing et al., 2006; Cho and Xu, 2007), and PP5 (Swingle et al., 2004) have been solved. Superpositions of PP1c, PP2Ac, and PP5c (Swingle et al., 2004) gives pairwise -carbon root mean square deviations of 1.3 to 1.5 Å within the 285-residue aligned region. This indicates that the similarity between the catalytic domains of PP1, PP2A, and PP5 is striking. Notably, the active site of all three PPases is located at the base of a shallow depression on the surface that is formed by the interstrand loops of a common central -sandwich. When inhibition studies with fostriecin (Buck et al., 2003) and cytostatin (Lawhorn et al., 2006) are considered along with structural studies of PP2A (Buck et al., 2003; Xing et al., 2006), we predicted that the unsaturated lactone reacts with a cysteine near the active site of PP2A (Buck et al., 2003) and a hydrogen bond occurs between the C11-hydroxyl and Arg, which is a conserved binding feature of the nonselective PP1 pharmacophore (Colby and Chamberlin, 2006). The comparison of PP1, PP2A, and PP5 still reveals sequence and conformational differences near the active site, suggesting the feasibility of developing type-specific inhibitors. Notably, differences occur in the 12– 13 loop, a region immediately adjacent to the active site and known to participate in okadaic acid and microcystin-mediated inhibition of PP1 (Goldberg et al., 1995) and PP2A (Xing et al., 2006). Here, we performed site-directed mutagenesis of PP1c and PP5c, altering domains predicted to be important for inhibitor binding in PP2Ac. Head-to-head dose-response studies then were conducted with PP1c, PP2Ac, PP5c, and chimeras (PP1/PP2A and PP5/PP2A), testing key compounds for inhibitory activity. Materials and Methods Synthesis and Characterization of Inhibitors. The synthesis and structural characterization of fostriecin, cytostatin, and structural analogs (compounds 1, 2, and 6-15) has been described previously (Boger et al., 2001; Buck et al., 2003; Lawhorn et al., 2006). Preparation of Phosphohistone Substrate and Determination of Phosphatase Activity. Phosphohistone, with a specific activity of 4.5 10 dpm/nmol of incorporated phosphate, was prepared by the phosphorylation of bovine brain histone with cAMPdependent protein kinase (PKA) from rabbit muscle in the presence of [P]ATP. Histone (type-2AS) was phosphorylated with PKA in a reaction containing 10 mg/ml histone, 2 mg/ml PKA, 6 mCi/ml [ -P]ATP (200 mM ATP), 0.4 mM cAMP, 40 mM PIPES, pH 6.8 (at 37°C), 7 mM MgCl2, 0.1 mM EDTA, and 5 mM dithiothreitol as described previously (Honkanen et al., 1990; Walsh et al., 1997; Swingle et al., 2007). Protein phosphatase activity against phosphohistone was measured by the quantification of [P] liberated from phosphohistone, using established protocols (Honkanen et al., 1990; Swingle et al., 2007). In brief, dephosphorylation reactions were conducted for 10 min at 30°C. For all reactions, the dephosphorylation of substrate was kept to less than 10% of the total phosphorylated substrate, and the reactions were linear with respect to enzyme concentration and time. For inhibition studies, compounds were added to the enzymes 10 min before the initiation of the reaction by the addition of substrate. In the literature, the reported strength of PP2A inhibition for fostriecin and cytostatin varies considerably (e.g., IC50 values range from 0.2 to 40 nM for fostriecin). This probably reflects differences in the amount of enzyme used in the assay, the choice of substrate, 46 Swingle et al. at A PE T Jornals on Jne 9, 2017 jpet.asjournals.org D ow nladed from