B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades.

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.

The role of liprin-α1 phosphorylation in its liquid-liquid phase separation: regulation by PPP2R5D/PP2A holoenzyme.

Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-ß, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.

Quantum-based modeling implies that bidentate Arg89-substrate binding enhances serine/threonine protein phosphatase-2A(PPP2R5D/PPP2R1A/PPP2CA)-mediated dephosphorylation.

PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M1/M2), an associated bridge hydroxide [W1(OH-)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein's seryl/threonyl phosphate coordinates the M1/M2 system, W1(OH-) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M1 is also expected to bind the substrate's phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg89) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg89 engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg89. The interaction of Arg89 with B:Glu198 in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan's Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg89-substrate binding and when Arg89 is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH‡ ≈ ΔE‡ = +15.5 kcal/mol for the former case, versus +18.8 kcal/mol for the latter, indicating that bidentate Arg89-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu198 sequestration of C:Arg89 under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.

Extended regulation interface coupled to the allosteric network and disease mutations in the PP2A-B56δ holoenzyme.

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

Cardioprotection by mild hypothermia during ischemia involves preservation of ERK activity.

Cooling the ischemic heart by just a few degrees protects it from infarction without affecting its mechanical function, but the mechanism of this protection is unknown. We investigated whether signal transduction pathways might be involved in the anti-infarct effect of mild hypothermia (35°C). Isolated rabbit hearts underwent 30 min of coronary artery occlusion/2 h of reperfusion. They were either maintained at 38.5°C or cooled to 35°C just before and only during ischemia. Infarct size was measured. The effects of the protein kinase C inhibitor chelerythrine, the nitric oxide synthase inhibitor N (ω)-nitro-L: -arginine methyl ester (L: -NAME), the phosphatidylinositol 3-kinase antagonist wortmannin, or either of the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors PD98059 or U0126 on cooling's protection were examined. Myocardial ATP assays were performed and the level of phosphorylation of extracellular signal-regulated kinase (ERK) and MEK was examined by western blotting. To investigate an effect of cooling on protein phosphatase (PPase), a PPase inhibitor cantharidin was tested in the infarct model and the effect of mild hypothermia on PP2A activity in vitro was measured. Infarct size was 34.4 ± 2.2% of the ischemic zone in normothermic (38.5°C) hearts, but only 15.6 ± 8.7% in hearts cooled to 35°C during ischemia. Mechanical function was unaffected. Neither chelerythrine, L: -NAME, nor wortmannin had any effect, but both PD98059 and U0126 completely eliminated protection. Ischemia rather than reperfusion was the critical time when ERK had to be active to realize protection. Phosphorylation of ERK and MEK fell during normothermic ischemia, but during hypothermic ischemia phosphorylation of ERK remained high while that of MEK was increased. Cooling only slightly delayed the rate at which ATP fell during ischemia, and ERK inhibition did not affect that attenuation suggesting ATP preservation was unrelated to protection. Cantharidin, like cooling, also protected during ischemia but not at reperfusion, and its protection was dependent on ERK phosphorylation. However, mild hypothermia had a negligible effect on PP2A activity in an in vitro assay. Hence, mild hypothermia preserves ERK and MEK activity during ischemia which somehow protects the heart. While a PPase inhibitor mimicked cooling's protection, a direct effect of cooling on PP2A could not be demonstrated.

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Total synthesis and evaluation of phostriecin and key structural analogues.

Full details of the total synthesis of phostriecin (2), the assignment of its relative and absolute stereochemistry, and the resultant structural reassignment of the natural product previously represented as sultriecin (1), a phosphate versus sulfate monoester, are detailed. Studies with authentic material confirmed that phostriecin, but not sultriecin, is an effective and selective inhibitor of protein phosphatase 2A (PP2A) defining a mechanism of action responsible for its antitumor activity. The extension of the studies to the synthesis and evaluation of a series of key synthetic analogues is disclosed that highlights the importance of the natural product phosphate monoester (vs sulfate or free alcohol, both inactive and >250-fold), the α,ß-unsaturated lactone (12-fold), and the hydrophobic Z,Z,E-triene tail (C12-C22, ca. 200-fold) including the unique importance of its unsaturation (50-fold, and no longer PP2A selective).

Structure-activity relationship studies of fostriecin, cytostatin, and key analogs, with PP1, PP2A, PP5, and( beta12-beta13)-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 side-by-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 site-directed 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 beta12-beta13 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.

Inhibitors of Serine/Threonine Protein Phosphatases: Biochemical and Structural Studies Provide Insight for Further Development.

BACKGROUND: The reversible phosphorylation of proteins regulates many key functions in eukaryotic cells. Phosphorylation is catalyzed by protein kinases, with the majority of phosphorylation occurring on side chains of serine and threonine residues. The phosphomonoesters generated by protein kinases are hydrolyzed by protein phosphatases. In the absence of a phosphatase, the half-time for the hydrolysis of alkyl phosphate dianions at 25º C is over 1 trillion years; knon ~2 x 10-20 sec-1. Therefore, ser/thr phosphatases are critical for processes controlled by reversible phosphorylation. METHODS: This review is based on the literature searched in available databases. We compare the catalytic mechanism of PPP-family phosphatases (PPPases) and the interactions of inhibitors that target these enzymes. RESULTS: PPPases are metal-dependent hydrolases that enhance the rate of hydrolysis ([kcat/kM]/knon ) by a factor of ~1021, placing them among the most powerful known catalysts on earth. Biochemical and structural studies indicate that the remarkable catalytic proficiencies of PPPases are achieved by 10 conserved amino acids, DXH(X)~26DXXDR(X)~20- 26NH(X)~50H(X)~25-45R(X)~30-40H. Six act as metal-coordinating residues. Four position and orient the substrate phosphate. Together, two metal ions and the 10 catalytic residues position the phosphoryl group and an activated bridging water/hydroxide nucleophile for an inline attack upon the substrate phosphorous atom. The PPPases are conserved among species, and many structurally diverse natural toxins co-evolved to target these enzymes. CONCLUSION: Although the catalytic site is conserved, opportunities for the development of selective inhibitors of this important group of metalloenzymes exist.

Development of a Synthetic 3-ketosteroid Δ1-dehydrogenase for the Generation of a Novel Catabolic Pathway Enabling Cholesterol Degradation in Human Cells.

Cholesterol is an essential component of membranes, which is acquired by cells via receptor-mediated endocytosis of lipoproteins or via de novo synthesis. In specialized cells, anabolic enzymes metabolize cholesterol, generating steroid hormones or bile acids. However, surplus cholesterol cannot be catabolized due to the lack of enzymes capable of degrading the cholestane ring. The inability to degrade cholesterol becomes evident in the development and progression of cardiovascular disease, where the accumulation of cholesterol/cholesteryl-esters in macrophages can elicit a maladaptive immune response leading to the development and progression of atherosclerosis. The discovery of cholesterol catabolic pathways in Actinomycetes led us to the hypothesis that if enzymes enabling cholesterol catabolism could be genetically engineered and introduced into human cells, the atherosclerotic process may be prevented or reversed. Comparison of bacterial enzymes that degrade cholesterol to obtain carbon and generate energy with the action of human enzymes revealed that humans lack a 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), which catalyzes the C-1 and C-2 desaturation of ring A. Here we describe the construction, heterologous expression, and actions of a synthetic humanized Δ1-KstD expressed in Hep3B and U-937 cells, providing proof that one of three key enzymes required for cholesterol ring opening can be functionally expressed in human cells.

The Antitumor Drug LB-100 Is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating with the Active-Site Catalytic Metals in PPP5C.

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

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).

Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z'=0.93; representative PP1Cα assay data: Z'=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5.

Total synthesis and evaluation of cytostatin, its C10-C11 diastereomers, and additional key analogues: impact on PP2A inhibition.

The total synthesis of cytostatin, an antitumor agent belonging to the fostriecin family of natural products, is described in full detail. The convergent approach relied on a key epoxide-opening reaction to join the two stereotriad units and a single-step late-stage stereoselective installation of the sensitive (Z,Z,E)-triene through a beta-chelation-controlled nucleophilic addition. The synthetic route provided rapid access to the C4-C6 stereoisomers of the cytostatin lactone, which were prepared and used to define the C4-C6 relative stereochemistry of the natural product. In addition to the natural product, each of the C10-C11 diastereomers of cytostatin was divergently prepared (11 steps from key convergence step) by this route and used to unequivocally confirm the relative and absolute stereochemistry of cytostatin. Each of the cytostatin diastereomers exhibited a reduced activity toward inhibition of PP2A (>100-fold), demonstrating the importance of the presence and stereochemistry of the C10-methyl and C11-hydroxy groups for potent PP2A inhibition. Extensions of the studies provided dephosphocytostatin, sulfocytostatin (a key analogue related to the natural product sultriecin), 11-deshydroxycytostatin, and an analogue lacking the entire C12-C18 (Z,Z,E)-triene segment, which were used to define the magnitude of the C9-phosphate (>4000-fold), C11-alcohol (250-fold), and triene (220-fold) contribution to PP2A inhibition. A model of cytostatin bound to the active site of PP2A is presented, compared to that of fostriecin, which is also presented in detail for the first time, and used to provide insights into the role of the key substituents. Notably, the alpha,beta unsaturated lactone of cytostatin, like that of fostriecin, is projected to serve as a key electrophile, providing a covalent adduct with Cys269 unique to PP2A, contributing to its potency (> or =200-fold for fostriecin) and accounting for its selectivity.

Structural basis for the catalytic activity of human serine/threonine protein phosphatase-5.

Serine/threonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp271-M1:M2-W1-His427-His304-Asp274 catalytic motif (where M1 and M2 are metals and W1 is a water molecule). The structure of PP5c provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

Fundamental role of the fostriecin unsaturated lactone and implications for selective protein phosphatase inhibition.

Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP beta12-beta13 active site loop accounting for PP2A/4 potency and selectivity.