High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

Novel inhibitors of Trypanosoma cruzi dihydrofolate reductase.

There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Leishmania major.

Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K(m) value for dUTP (2.11 microM), a k(cat) of 49 s(-1), strict Michaelis-Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K(m) for dUTP increases with the dissociation of ionizable groups showing pK(a) values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K(m) is regulated by an enzyme residue exhibiting a pK(a) of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog alpha-beta-imido-dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.

New crystal forms of Trypanosoma cruzi dUTPase.

The dUTPase from Trypanosoma cruzi has been crystallized in two crystal forms, both belonging to space group P6(3)22, with unit-cell parameters a = b = 134.67, c = 148.66 A (form I, two molecules per asymmetric unit) and a = b = 136.43, c = 68.71 A (form II, one molecule per asymmetric unit). Single-wavelength data have been collected using synchrotron radiation to 3.0 A for crystal form I and to 2.4 A for crystal form II and structure solution is under way. T. cruzi dUTPase is a potential target for anti-protozoan drug design.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease.

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas' disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of approximately 1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids.

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i). dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.

The structure-based design and synthesis of selective inhibitors of Trypanosoma cruzi dihydrofolate reductase.

This paper describes the design and synthesis of potential inhibitors of Trypanosoma cruzi dihydrofolate reductase using a structure-based approach. A model of the structure of the T. cruzi enzyme was compared with the structure of the human enzyme. The differences were used to design modifications of methotrexate to produce compounds which should be selective for the parasite enzyme. The derivatives of methotrexate were synthesised and tested against the enzyme and intact parasites.

Crystallization and preliminary X-ray diffraction of Trypanosoma cruzi dUTPase.

Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 A. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.

Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli.

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.

3-Hydroxy-3-methyl-glutaryl-CoA reductase in Trypanosoma (Schizotrypanum) cruzi: subcellular localization and kinetic properties.

The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated in Trypanosoma cruzi epimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30 degrees C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis-Menten behavior with Km,app (HMG-CoA) 28 +/- 3 microM, Km,app (NADPH) 37 +/- 4 microM, and Vm,app 3.9 +/- 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA with Kis 23 nM and a noncompetitive inhibitor toward NADPH with Kii 29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.

Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major.

A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

Regulation of mevalonate 5-pyrophosphate decarboxylase in HeLa cells. Inhibition of enzymatic protein synthesis by serum lipoproteins.

Mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) has been considered as a secondary site of regulation of cholesterogenesis. Because of this, we have studied the regulation of decarboxylase in HeLa cells by serum lipoproteins in the cell culture medium. A first group of experiments was performed with cells grown in Eagle's medium with 10% foetal calf serum. The specific activity of decarboxylase was increased when whole foetal calf serum was replaced with lipoprotein-poor serum. This increase was clearly reduced in the presence of cycloheximide. Addition of serum lipoproteins to a medium containing lipoprotein-poor serum led to a clear decrease in the decarboxylase activity. An identical decrease was observed after the addition of lipoproteins alone or in combination with cycloheximide. These results suggest for the first time that the effect of serum lipoproteins on decarboxylase activity should be a decrease in the rate of enzymatic protein synthesis, and corroborate the important role of reactions other than those catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase in the regulation of cholesterogenesis.

Thermodynamic characterization of the binding of dCMP to the Asn229Asp mutant of thymidylate synthase.

Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2'-deoxycytidine 5'-monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15 degrees C to 35 degrees C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild-type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild-type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp-229 is increased upon dCMP binding to the mutant enzyme. At 25 degrees C, although the binding of dCMP to wild-type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature-dependent, because of the enthalpy-entropy compensation arising from a negative heat capacity change of binding equal to -0.83 +/- 0.02 kJ K(-1) per mol of dCMP bound.

A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi.

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain.

We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.

Thermodynamic analysis of the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate to thymidylate synthase over a range of temperatures.

The binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16-35 degrees C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases. A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16-35 degrees C. The enthalpy changes become more negative as the temperature increases, with delta Cp = -170 +/- 20 J.K-1.(mol FdUMP bound)-1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer.

Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.

Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species.

Amplification of the H locus in Leishmania infantum.

We have selected for a Leishmania infantum cell line resistant to high levels of methotrexate (MTX). The resulting cells were 1233-fold more resistant than wild-type and contained amplified H-region circles. Homologous genes to the antifolate resistant ltdh gene and to the P-glycoprotein ltpgpA gene of Leishmania tarentolae were observed to be contained within the amplicon. In order to invoke additional mechanisms of resistance, we examined possible variations in MTX accumulation. Resistance was not correlated with a decreased uptake of MTX. On the contrary, the resistant line presented a 3-fold increase in the steady-state accumulation of drug with regard to the wild-type line. Northern blot analysis using gene specific probes, showed that the ltdh probe and the ltpgpA probe recognized single transcripts of 1 kb and 5 kb respectively which were both overexpressed only approx. 5-fold in resistant cells. We propose that amplification of the antifolate resistance gene, homologue to the ltdh gene of L. tarentolae, is apparently the only mechanism involved in resistance to the cytotoxic drug MTX in L. infantum resistant to 1000 microM of MTX.