In silico studies and evaluation of antiparasitic role of a novel pyruvate phosphate dikinase inhibitor in Leishmania donovani infected macrophages.

The present work deals with the identification and characterization of a novel inhibitor Z220582104, specific to pyruvate phosphate dikinase, for leishmanicidal activities against free promastigotes and intracellular amastigotes. We have used structure-based drug designing approaches and performed homology modelling, virtual screening and molecular dynamics studies. Primary mouse macrophages and macrophage cell line J774A1 were infected with promastigotes of Leishmania donovani. Both promastigotes and infected macrophages were subjected to treatment with the varying concentrations of Z220582104 or miltefosine for assessment of leishmanicidal activity. The novel inhibitor Z220582104 demonstrated growth inhibitory potential and reduced the viability of the free promastigotes in a concentration- and time-dependent manner. Z220582104 was also effective against the intracellular form of the parasites and reduced the number of amastigotes in macrophages and also lowered the parasite index, compared with the untreated infected macrophages. Although less effective compared with the miltefosine, Z220582104 is well tolerated by the dividing cells and normal human lymphocytes and monocytes with no adverse effects on the growth kinetics or viability. Our in silico and in vitro studies suggested that Leishmania donovani pyruvate phosphate dikinase could be a potential new drug target.

In Vitro Testing of Potential Entamoeba histolytica Pyruvate Phosphate Dikinase Inhibitors.

Adverse effects and resistance to metronidazole have motivated the search for new antiamoebic agents against Entamoeba histolytica. Control of amoeba growth may be achieved by inhibiting the function of the glycolytic enzyme and pyruvate phosphate dikinase (PPDK). In this study, we screened 10 compounds using an in vitro PPDK enzyme assay. These compounds were selected from a virtual screening of compounds in the National Cancer Institute database. The antiamoebic activity of the selected compounds was also evaluated by determining minimal inhibitory concentrations (MICs) and IC50 values using the nitro-blue tetrazolium reduction assay. Seven of the 10 compounds showed inhibitory activities against the adenosine triphosphate (ATP)/inorganic phosphate binding site of the ATP-grasp domain. Two compounds, NSC349156 (pancratistatin) and NSC228137 (7-ethoxy-4-[4-methylphenyl] sulfonyl-3-oxido-2, 1, 3-benzoxadiazol-3-ium), exhibited inhibitory effects on the growth of E. histolytica trophozoites with MIC values of 25 and 50 µM, and IC50 values of 14 and 20.7 µM, respectively.

Design, synthesis, and evaluation of inhibitors of pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the phosphorylation reaction of pyruvate that forms phosphoenolpyruvate (PEP) via two partial reactions: PPDK + ATP + P(i) → PPDK-P + AMP + PP(i) and PPDK-P + pyruvate → PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogues are potent inhibitors of the Clostridium symbiosum PPDK. In silico modeling studies suggested that placement of a 3­6 carbon-tethered ammonium substituent at the 3'- or 4'-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady-state kinetic analysis of these substances demonstrates that the 4'-aminohexyl-5,7-dyhydroxyflavone 10 is a potent competitive PPDK inhibitor (K(i) = 1.6 ± 0.1 µM). Single turnover experiments were conducted using 4'-aminopropyl-5,7-dihydroxyflavone 7 to show that this flavone specifically targets the ATP binding site and inhibits catalysis of only the PPDK + ATP + P(i) → PPDK-P + AMP PP(i) partial reaction. Finally, the 4'-aminopbutyl-5,7-dihydroxyflavone 8 displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD.

Eusynstyelamides A, B, and C, nNOS inhibitors, from the ascidian Eusynstyela latericius.

Eusynstyelamides A-C (1-3) were isolated from the Great Barrier Reef ascidian Eusynstyela latericius, together with the known metabolites homarine and trigonelline. The structures of 1-3, with relative configurations, were elucidated by interpretation of their spectroscopic data (NMR, MS, UV, IR, and CD). The NMR data of 1 were found to be virtually identical to that reported for eusynstyelamide (4), isolated from E. misakiensis, indicating that a revision of the structure of 4 is needed. Eusynstyelamides A-C exhibited inhibitory activity against neuronal nitric oxide synthase (nNOS), with IC(50) values of 41.7, 4.3, and 5.8 microM, respectively, whereas they were found to be nontoxic toward the three human tumor cell lines MCF-7 (breast), SF-268 (CNS), and H-460 (lung). Compounds 1 and 2 displayed mild inhibitory activity toward Staphylococcus aureus (IC(50) 5.6 and 6.5 mM, respectively) and mild inhibitory activity toward the C(4) plant regulatory enzyme pyruvate phosphate dikinase (PPDK) (IC(50) values of 19 and 20 mM, respectively).

Current and future perspectives on the chemotherapy of the parasitic protozoa Trichomonas vaginalis and Entamoeba histolytica.

Trichomonas vaginalis and Entamoeba histolytica are clinically important protozoa that affect humans. T. vaginalis produces sexually transmitted infections and E. histolytica is the causative agent of amebic dysentery. Metronidazole, a compound first used to treat T. vaginalis in 1959, is still the main drug used worldwide to treat these pathogens. It is essential to find new biochemical differences in these organisms that could be exploited to develop new antiprotozoal chemotherapeutics. Recent findings associated with T. vaginalis and E. histolytica biochemistry and host-pathogen interactions are surveyed. Knowledge concerning the biochemistry of these parasites is serving to form the foundation for the development of new approaches to control these important human pathogens.

Molecular modeling on pyruvate phosphate dikinase of Entamoeba histolytica and in silico virtual screening for novel inhibitors.

Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.

Screening marine fungi for inhibitors of the C4 plant enzyme pyruvate phosphate dikinase: unguinol as a potential novel herbicide candidate.

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.

Translation of in vitro inhibition by marine natural products of the C4 acid cycle enzyme pyruvate P(i) dikinase to in vivo C4 plant tissue death.

Marine organism derived extracts, previously identified as containing compounds that inhibited the C4 acid cycle enzyme pyruvate P(i) dikinase (PPDK), were assessed for their ability to exhibit an effect on the C4 plants Digitaria ciliaris and Echinochloa crus-galli. Oxygen electrode studies revealed that over half of these extracts inhibited C4 acid driven photosynthesis in leaf slices. Seventeen extracts had a deleterious effect on C4 plants in vivo within 24 h, whereas 36 caused an observable phytotoxic response in one or both of the C4 plants used for in vivo testing. None of the extracts inhibited PPDK metabolism of pyruvate via a directly competitive mechanism, instead hindering the enzyme by either mixed or uncompetitive means. This screening strategy, using a suite of assays, led to the isolation and identification of the herbicidal marine natural product ilimaquinone.

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides.

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.

Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica.

The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate.

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.

Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase.

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.

Activation and inactivation of an enzyme catalyzed by a single, bifunctional protein: a new example and why.

Recent studies have shown that the light-dark mediated regulation of the leaf photosynthetic enzyme pyruvate, Pi dikinase results from interconversion between an active nonphosphorylated form of the enzyme and an inactive form phosphorylated on a threonine residue. These phosphorylation and dephosphorylation reactions are apparently catalyzed by a single protein termed the pyruvate, Pi dikinase regulatory protein and, notably, both reactions are mechanistically unique. We consider the evidence that this regulatory protein belongs to a group of unusual bifunctional enzymes that catalyze opposing reactions, apparently at separate catalytic sites on the same polypeptide. In three of the four known cases these bifunctional enzymes interconvert the active and inactive forms of another enzyme. The possible advantages of such opposing reactions being catalyzed by the same protein are considered.

Inhibition and inactivation of pyruvate phosphate dikinase with Cr(III) complexes of adenosine 5'-triphosphate and inorganic pyrophosphate.

The exchange inert complexes beta,gamma-bidentate Cr(H2O)4ATP and P1,P2-bidentate Cr(H2O)4PP were found to bind to the Bacteriodes symbiosus pyruvate phosphate dikinase ATP and PP binding sites, respectively. The inactivation of the enzyme that was observed with these complexes was shown to involve covalent attachment of the entire complex to the enzyme via insertion of enzyme amino acid side chains into the coordination sphere of the Cr(III). Incubation of Cr(H2O)4ATP with other proteins also resulted in covalent attachment.

Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate,Pi dikinase.

Pyruvate,Pi dikinase regulatory protein (PDRP) has been highly purified from maize leaves, and its role in catalyzing both ADP-mediated inactivation (due to phosphorylation of a threonine residue) and Pi-mediated activation (due to dephosphorylation by phosphorolysis) of pyruvate,Pi dikinase has been confirmed. These reactions account for the dark/light-mediated regulation of pyruvate,Pi dikinase observed in the leaves of C4 plants. During purification to apparent homogeneity the ratio of these two activities remained constant. The molecular weight of the native PDRP was about 180,000 at pH 8.3 and 90,000 at pH 7.5. Its monomeric molecular weight was 45,000. It was confirmed that inactive pyruvate,Pi dikinase free of a phosphate group on a catalytic histidine was the preferred substrate for activation. Michaelis constants for orthophosphate and the above form of active pyruvate,Pi dikinase were determined, as well as the mechanism of inhibition of the PDRP-catalyzed reaction by ATP, ADP, AMP, and PPi. For the inactivation reaction, Km values were 1.2 microM for the active pyruvate,Pi dikinase and 52 microM for ADP. CDP and GDP but not UDP could substitute for ADP. The inactivation reaction is inhibited by inactive pyruvate,Pi dikinase competitively with respect to both active pyruvate,Pi dikinase and ADP. Both the activation and inactivation reactions catalyzed by PDRP have a broad pH optimum between 7.8 and 8.3. The results are discussed in terms of the likely mechanism of dark/light regulation of pyruvate,Pi dikinase in vivo.

Regulation of C4 photosynthesis: catalytic phosphorylation as a prerequisite for ADP-mediated inactivation of pyruvate,Pi dikinase.

Evidence is provided that the role of ATP in the ADP plus ATP-dependent inactivation of pyruvate,Pi dikinase is to catalytically phosphorylate the enzyme. Only this phosphorylated form of the enzyme is susceptible to inactivation by reacting with ADP. Phosphoenolpyruvate, which also phosphorylates pyruvate,Pi dikinase during catalysis, can replace the ATP-requirement for inactivation.

Probes of the structure of phosphoenolpyruvate synthetase: effects of a transition state analogue on enzyme conformation.

Phosphoenolpyruvate synthetase of Escherichia coli is strongly inhibited by oxalate. The magnitude of the inhibition constant for oxalate suggests that this compound acts to produce a transition state analogue, in keeping with the suggestion of others that oxalate mimics the structure of enolpyruvate, a presumed catalytic intermediate in the enzymatic reaction. The addition of oxalate together with ATP results in a dramatic shielding of sensitive amino acid residues from reaction with both N-ethyl maleimide and phenylglyoxal. Thus, under conditions otherwise giving rise to almost complete inactivation by either reagent, no loss of activity is detectable in the presence of oxalate and ATP. These results indicate the formation of an enclosed structure during catalysis in which reactive groups are rendered quite inaccessible to solvent.