Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.     

Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase

Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PPi-dependent metabolism. Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes. We characterized in Trypanosoma brucei a single-copy gene encoding a PPi-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in Escherichia coli. Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of T. brucei, indicating a differential expression. Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies. Expression and localization of recombinant PPDKs in procyclic forms of T. brucei showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting. PPDK was detected in every trypanosomatid tested-Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Phytomonas, Crithidia and Leishmania-with a good correlation between amount of protein and enzymatic activity. The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified.     

In vitro and in vivo activities of trybizine hydrochloride against various pathogenic trypanosome species

Trybizine hydrochloride [O,O'-bis(4,6-diamino-1,2-dihydro-2, 2-tetramethylene-s-triazine-1-yl)-1,6-hexanediol dihydrochloride] was active in vitro against the sleeping sickness-causing agents Trypanosoma brucei subsp. rhodesiense and T. brucei subsp. gambiense; against a multidrug-resistant organism, T. brucei subsp. brucei; and against animal-pathogenic organisms Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma congolense; but not against the intracellular parasites Trypanosoma cruzi and Leishmania donovani. Cytotoxic effects against mammalian cells were observed at approximately 10(6)-fold higher concentrations than those necessary to inhibit T. brucei subsp. rhodesiense. Trybizine hydrochloride was able to eliminate T. brucei subsp. rhodesiense and T. brucei subsp. gambiense in an acute rodent model with four intraperitoneal doses of 0.25 mg kg of body weight-1 or four doses of 1 mg kg-1, respectively, or with four oral doses of 20 mg kg-1. The compound expressed activity against suramin-resistant T. evansi strains in mice. However, these concentrations were not sufficient to cure mice infected with multidrug-resistant T. brucei subsp. brucei. A late-stage rodent model with central nervous system involvement could not be cured, indicating that trybizine may not pass the blood-brain barrier in sufficient quantities.     

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.     

Cloning of a cdc2-related protein kinase from Trypanosoma cruzi that interacts with mammalian cyclins

Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.     

Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.     

Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan

A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.     

Odontogenic tumors. A demographic study of 759 cases in a Chinese population

Hybrid molecules were constructed with either polyclonal antibodies against Trypanosoma cruzi antigens or monoclonal antibody against Trypanosoma brucei brucei low-density lipoprotein (LDL)-receptor conjugated with chlorambucil. Physical-chemical analysis of the hybrid molecule showed four chlorambucil coupling sites in each IgG and a binding constant in the order of 10(4). Maintenance of IgG integrity was indicated by its circular dichroism pattern. Biologic activity of the hybrid molecule was shown by its inhibitory effect on the mobility and proliferation of the parasite. An IgG-chlorambucil conjugate, produced with monoclonal antibody anti-T. b. brucei LDL-receptor, led to the immobilization of the T. cruzi forms, albeit at a much lesser level than that obtained with a mouse polyclonal anti-T. cruzi IgG linked to the drug. Targeting experimental T. cruzi infection with a specific IgG-chlorambucil conjugate resulted in consistent reduction of parasitemia and mortality, thus showing its potential usefulness in controlling the acute form of the disease.     

The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei

The in-vitro activities of four anticancer alkyllysophospholipids, ET-18-OCH3 (edelfosine), hexadecylphosphocholine (miltefosine), ilmofosine and SRI 62-834, were determined with ED50 values for Leishmania donovani and Trypanosoma cruzi amastigotes between 0.2 and 5.0 microM and for Trypanosoma brucei trypomastigotes between 7.0 and 50.8 microM. In BALB/c mice miltefosine and ilmofosine were the most active compounds with ED50 values of 2.9 and 14.5 mg/kg x 5 doses against L. donovani and a suppressive effect on T. cruzi infections at 30 mg/kg x 5 doses.     

Trypanosoma brucei gamma-glutamylcysteine synthetase. Characterization of the kinetic mechanism and the role of Cys-319 in cystamine inactivation

The parasitic protozoan Trypanosoma brucei utilizes a conjugate of glutathione and spermidine, termed trypanothione, in place of glutathione to maintain cellular redox balance. The first committed step in the biosynthesis of glutathione and thereby trypanothione, is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS). We have determined the kinetic mechanism for T. brucei gamma-GCS. The kinetics are best described by a rapid equilibrium random ter-reactant mechanism, in which the model derived Kd values for the binding of L-Glu, L-alpha-aminobutyrate, and ATP to free enzyme are 2.6, 5.1, and 1.4 mM, respectively. However, significant dependences exist between the binding of some of the substrate pairs. The binding of either ATP or L-Glu to the enzyme increases the binding affinity of the other by 18-fold, whereas the binding of L-Glu or L-alpha-aminobutyrate decreases the binding affinity of the other by 6-fold. Similarly to the mammalian enzyme, cystamine is a time-dependent, irreversible inhibitor of T. brucei gamma-GCS. It has been suggested by several studies that cystamine labels an active site Cys residue essential for catalysis. Among the enzymes reported to be inactivated by cystamine, only one Cys residue is invariant (Cys-319 in T. brucei gamma-GCS). Mutation of Cys-319 to Ala in T. brucei gamma-GCS renders the enzyme insensitive to cystamine inactivation without significantly affecting the enzyme's catalytic efficiency, kinetic mechanism, or substrate affinities. These studies suggest that cystamine inactivates the enzyme by blocking substrate access to the active site and not by labeling an essential active site residue.     

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.     

Interferon-gamma activation of a mitogen-activated protein kinase, KFR1, in the bloodstream form of Trypanosoma brucei

KFR1, a mitogen-activated protein (MAP) kinase identified in the African trypanosome, Trypanosoma brucei, is a serine protein kinase capable of phosphorylating the serine residues in histone H-1, myelin basic protein, and beta-casein. It phosphorylates four proteins with estimated molecular masses of 22, 34, 46, and 90 kDa from the T. brucei bloodstream-form lysate in vitro. KFR1 bears significant sequence similarity to the yeast MAP kinases KSS1 and FUS3 but cannot functionally complement the kss1/fus3 yeast mutant. It is encoded by a single-copy gene in the diploid T. brucei, and only one of the two alleles can be successfully disrupted, suggesting an essential function of KFR1 in T. brucei. KFR1 activity is present at a much enhanced level in the bloodstream form of T. brucei when compared with that in the insect (procyclic) form. This enhanced activity can be eliminated in vitro by the treatment with protein phosphatase HVH2 known to act specifically on MAP kinases. It can also be decreased in the bloodstream form of T. brucei by serum starvation but induced specifically by interferon-gamma. The production of interferon-gamma in the mammalian host is known to be triggered by T. brucei infection, and this cytokine, as has been reported, promotes the proliferation of T. brucei in the mammalian blood. Since none of these phenomena can be observed in the procyclic form of T. brucei, activation of KFR1 is most likely involved in mediating the interferon-gamma-induced proliferation of T. brucei in the mammalian host.     

Preliminary crystallographic study of 6-phosphogluconate dehydrogenase from Trypanosoma brucei

We have obtained well-ordered single crystals of 6-phosphogluconate dehydrogenase, an enzyme of the oxidative branch of the pentose phosphate pathway, from Trypanosoma brucei. The crystals are trigonal rhombs with unit cell dimensions a = b = 135.1 A, c = 116.7 A and belong to one of the enantiomorphic pair of space groups P3(1)21/P3(2)21. X-ray diffraction to better than 2.8 A has been recorded using a rotating anode CuK alpha source. Elucidation of the three-dimensional structure of the T. brucei enzyme will, by indicating structural differences from the known sheep enzyme structure, aid the design of mutants to probe the active site. Knowledge of the structure will also assist in assessing the potential use of rationally designed compounds to inhibit this enzyme specifically.     

Expansions of T-cell subsets expressing particular T-cell receptor variable regions in chronic beryllium disease

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.     

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.     

Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages

This study explores the role of mevalonate inhibitors in the activation of NF-kbeta and the induction of inducible nitric oxide synthase (iNOS) and cytokines (TNF-alpha, IL-1beta, and IL-6) in rat primary astrocytes, microglia, and macrophages. Lovastatin and sodium phenylacetate (NaPA) were found to inhibit LPS- and cytokine-mediated production of NO and expression of iNOS in rat primary astrocytes; this inhibition was not due to depletion of end products of mevalonate pathway (e.g., cholesterol and ubiquinone). Reversal of the inhibitory effect of lovastatin on LPS-induced iNOS expression by mevalonate and farnesyl pyrophosphate and reversal of the inhibitory effect of NaPA on LPS-induced iNOS expression by farnesyl pyrophosphate, however, suggests a role of farnesylation in the LPS-mediated induction of iNOS. The inhibition of LPS-mediated induction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase, suggests that farnesylation of p21(ras) or other proteins regulates the induction of iNOS. Inhibition of LPS-mediated activation of NF-kbeta by lovastatin, NaPA, and FPT inhibitor II in astrocytes indicates that the observed inhibition of iNOS expression is mediated via inhibition of NF-kbeta activation. In addition to iNOS, lovastatin and NaPA also inhibited LPS-induced expression of TNF-alpha, IL-1beta, and IL-6 in rat primary astrocytes, microglia, and macrophages. This study delineates a novel role of the mevalonate pathway in controlling the expression of iNOS and different cytokines in rat astrocytes, microglia, and macrophages that may be important in developing therapeutics against cytokine- and NO-mediated neurodegenerative diseases.     

Phosphonate and bisphosphonate analogues of farnesyl pyrophosphate as potential inhibitors of farnesyl protein transferase

Several phosphonate and bisphosphonate analogues of farnesyl pyrophosphate have been prepared for an examination of their ability to inhibit farnesyl protein transferase (FPTase). A Horner-Wadsworth-Emmons condensation of farnesal or geranial with tetraethyl methylenediphosphonate gave the desired vinyl phosphonates, while alkylation of the dimethyl methylphosphonate anion with a terpenoid bromide gave the corresponding saturated phosphonates. Alkylation of tetraethyl methylenediphosphonate with farnesyl bromide gave the expected alkyl bisphosphonate, which was converted to its alpha, beta-unsaturated derivative by preparation of the phenyl selenide, oxidation to the selenoxide, and elimination. In a similar fashion, triethyl phosphonoacetate was converted to a farnesyl pyrophosphate analogue by reaction with farnesyl bromide. After preparation of the respective acids, each compound was tested for inhibition of FPTase at concentrations ranging up to 10 microM. The effect of these compounds on FPTase activity varied substantially, ranging from depressed to surprisingly enhanced enzymatic activity.     

Antiparasitic effects of the intra-Golgi transport inhibitor megalomicin

The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC50) of 0.2 microg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 microg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC50, 2 microg/ml) and Leishmania donovani and Leishmania major promastigotes (IC50, 3 and 8 microg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 microg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.     

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.