Antiparasitic Meroterpenoids Isolated from Memnoniella dichroa CF-080171.

Memnoniella is a fungal genus from which a wide range of diverse biologically active compounds have been isolated. A Memnoniella dichroa CF-080171 extract was identified to exhibit potent activity against Plasmodium falciparum 3D7 and Trypanosoma cruzi Tulahuen whole parasites in a high-throughput screening (HTS) campaign of microbial extracts from the Fundación MEDINA's collection. Bioassay-guided isolation of the active metabolites from this extract afforded eight new meroterpenoids of varying potencies, namely, memnobotrins C-E (1-3), a glycosylated isobenzofuranone (4), a tricyclic isobenzofuranone (5), a tetracyclic benzopyrane (6), a tetracyclic isobenzofuranone (7), and a pentacyclic isobenzofuranone (8). The structures of the isolated compounds were established by (+)-ESI-TOF high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Compounds 1, 2, and 4 exhibited potent antiparasitic activity against P. falciparum 3D7 (EC50 0.04-0.243 µM) and T. cruzi Tulahuen (EC50 0.266-1.37 µM) parasites, as well as cytotoxic activity against HepG2 tumoral liver cells (EC50 1.20-4.84 µM). The remaining compounds (3, 5-8) showed moderate or no activity against the above-mentioned parasites and cells.

Cytotoxycity and antiplasmodial activity of phenolic derivatives from Albizia zygia (DC.) J.F. Macbr. (Mimosaceae).

BACKGROUND: The proliferation and resistance of microorganisms area serious threat against humankind and the search for new therapeutics is needed. The present report describes the antiplasmodial and anticancer activities of samples isolated from the methanol extract of Albizia zygia (Mimosaseae). MATERIAL: The plant extract was prepared by maceration in methanol. Standard chromatographic, HPLC and spectroscopic methods were used to isolate and identify six compounds (1-6). The acetylated derivatives (7-10) were prepared by modifying 2-O-ß-D-glucopyranosyl-4-hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside, previously isolated from A. zygia (Mimosaceae). A two-fold serial micro-dilution method was used to determine the IC50s against five tumor cell lines and Plasmodium falciparum. RESULTS: In general, compounds showed moderate activity against the human pancreatic carcinoma cell line MiaPaca-2 (10 < IC50 < 20 µM) and weak activity against other tumor cell lines such as lung (A-549), hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7and A2058) (IC50 > 20 µM). Additionally, the two semi-synthetic derivatives of quercetin 3-O-α-L-rhamnopyranoside exhibited significant activity against P. falciparum with IC50 of 7.47 ± 0.25 µM for compound 9 and 6.77 ± 0.25 µM for compound 10, higher than that of their natural precursor (IC50 25.1 ± 0.25 µM). CONCLUSION: The results of this study clearly suggest that, the appropriate introduction of acetyl groups into some flavonoids could lead to more useful derivatives for the development of an antiplasmodial agent.

Quantitation of deoxynucleoside triphosphates by click reactions.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

DCTPP1 prevents a mutator phenotype through the modulation of dCTP, dTTP and dUTP pools.

To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.

Base excision repair plays an important role in the protection against nitric oxide- and in vivo-induced DNA damage in Trypanosoma brucei.

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes.

Antiprotozoan sesterterpenes and triterpenes isolated from two Ghanaian mushrooms.

Bioassay-guided compound isolation led to the discovery of two new scalarane sesterterpenes (1 and 2) and two new triterpenes (3 and 4) from two mushroom species, Pleurotus ostreatus (edible) and Scleroderma areolatum, collected from Ghana. Their structures, including absolute stereochemistry, were established by spectroscopic methods, particularly (+)-ESI-TOF mass spectrometry and 1D and 2D NMR. The four compounds exhibited IC50 values of 1.65-7.63 µM against Plasmodium falciparum 3D7 and 5.04-13.65 µM against Trypanosoma cruzi Tulahuen C4 parasites and were also non-cytotoxic against HepG2 tumoral human liver cells. This is the first report describing the isolation of sesterterpenes belonging to the scalarane structural class from a terrestrial source.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine.

Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major.

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 µM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.

MDN-0104, an antiplasmodial betaine lipid from Heterospora chenopodii.

Bioassay-guided fractionation of the crude fermentation extract of Heterospora chenopodii led to the isolation of a novel monoacylglyceryltrimethylhomoserine (1). The structure of this new betaine lipid was elucidated by detailed spectroscopic analysis using one- and two-dimensional NMR experiments and high-resolution mass spectrometry. Compound 1 displayed moderate in vitro antimalarial activity against Plasmodium falciparum, with an IC50 value of 7 µM. This betaine lipid is the first monoacylglyceryltrimethylhomoserine ever reported in the Fungi, and its acyl moiety also represents a novel natural 3-keto fatty acid. The new compound was isolated during a drug discovery program aimed at the identification of new antimalarial leads from a natural product library of microbial extracts. Interestingly, the related fungus Heterospora dimorphospora was also found to produce compound 1, suggesting that species of this genus may be a promising source of monoacylglyceryltrimethylhomoserines.

Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase.

8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

Structures of adenosine kinase from Trypanosoma brucei brucei.

Trypanosoma brucei is a single-cellular parasite of the genus Kinetoplastida and is the causative agent of African sleeping sickness in humans. Adenosine kinase is a key enzyme in the purine-salvage pathway, phosphorylating adenosine to AMP, and also activates cytotoxic analogues such as cordycepin and Ara-A by their phosphorylation. The structures of T. brucei brucei adenosine kinase (TbAK) in its unliganded open conformation and complexed with adenosine and ADP in the closed conformation are both reported to 2.6 Šresolution. The structures give insight into the binding mode of the substrates and the conformational change induced upon substrate binding. This information can be used to guide the improvement of cytotoxic substrate analogues as potential antitrypanosomal drugs.

Kinetic analyses and inhibition studies reveal novel features in peptide deformylase 1 from Trypanosoma cruzi.

In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on all nascent polypeptides and methionine aminopeptidase (MAP), which exscinds methionine specifically and depends on the previous removal of the N-formyl group. In Trypanosoma cruzi two genes encoding bacterial PDF homologues have been identified and referred to as TcPDF-1 and TcPDF-2. Here we report the biochemical characterization of a truncated soluble version of TcPDF-1 lacking the hydrophobic N-terminal domain that is active with the bacterial PDF substrate formyl-methionyl-alanyl-serine but, in contrast to other PDFs, is not inhibited by actinonin. The enzyme is strongly activated by Cu(2+) and inhibited by Ni(2+). Our results show that T. cruzi PDF exhibits unique features thus providing a new avenue for the design of potential inhibitors for use in the treatment of diseases caused by trypanosomatid parasites.

Structural basis for the efficient phosphorylation of AZT-MP (3'-azido-3'-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase.

Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3'-azido-3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP-ADP and AZT-MP-ADP) and a binary complex with the transition state analogue AP5dT [P1-(5'-adenosyl)-P5-(5'-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.

Depletion of dimeric all-alpha dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei.

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-alpha NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.

Delta24(25)-sterol methenyltransferase: intracellular localization and azasterol sensitivity in Leishmania major promastigotes overexpressing the enzyme.

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.

Plasmodium falciparum dUTPase: studies on protein stability and binding of deoxyuridine derivatives.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.

Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption.

We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-1-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices analysis and pharmacophore modeling studies of farnesyl diphosphate synthase (FPPS) inhibition, human Vgamma2Vdelta2 T cell activation and bone resorption inhibition. The most potent molecules have high activity against an expressed FPPS from Leishmania major, in Dictyostelium discoideum growth inhibition, in gammadelta T cell activation and in an in vitro bone resorption assay. As such, they represent useful new leads for the discovery of new bone resorption, antiinfective and anticancer drugs.

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa.

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.

Novel azasterols as potential agents for treatment of leishmaniasis and trypanosomiasis.

This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites. Azasterols are a known class of (S)-adenosyl-L-methionine: Delta24-sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa. The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp. and Trypanosoma spp. The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT. The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed. When an acetate was attached to the 3beta-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism. Thus, an acetate group on the 3beta-OH may have application as a prodrug. However, there may be an additional mode(s) of action for these acetate derivatives. These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum. The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis.