Real-time PCR to quantify Leishmania donovani in hamsters.

Visceral leishmaniasis, a vector-borne disease caused by Leishmania donovani and Leishmania infantum , currently affects 12 million individuals in 88 countries. In the present study, a real-time PCR (rt-PCR) assay has been optimized and validated against 2 other routine methods, i.e., microscopy and limiting dilution culture assay, to estimate parasite load in the liver of infected Syrian hamsters (Mesocricetus auratus). A set of specific primers amplified a 116-bp target template of the kinetoplastid DNA of L. donovani in a SYBR® Green-based rt-PCR assay. To assess the methods, we tested 2 anti-leishmanial compounds belonging to the class of arylimidamides, DB745 (2,5-bis[2-ethoxy-4-(2-pyridylimino)aminophenyl]furan) and DB766 (2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan) for efficacy in vivo in Syrian hamsters infected with L. donovani promastigotes. Parasite load was quantified in liver by all 3 methods and was found comparable. Of the 3 methods, rt-PCR was the fastest and most convenient, sensitive, and reproducible method.

Novel arylimidamides for treatment of visceral leishmaniasis.

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC(50) < or = 0.12 microM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

Structural analysis of farnesyl pyrophosphate synthase from parasitic protozoa, a potential chemotherapeutic target.

Synthesis of farnesyl pyrophosphate (FPP), a key intermediate of the isoprenoid biosynthesis pathway, is catalyzed by FPP synthase (FPPS). Antiprotozoal properties of bisphosphonates, which target FPPS, have generated interest in FPPS as a potential antiprotozoal drug target. The genes encoding FPPS from parasitic protozoa were assessed to analyze structural and functional features of the enzyme. Comparisons of the FPPS from the parasitic protozoa and search for conserved motifs revealed that FPPS from both apicomplexan and trypanosomatid parasites show characteristic conserved regions for example first aspartate rich motif (FARM) contained within II conserved domain and the second aspartate rich motif (SARM) contained within VI conserved domain. Phylogenetic analysis of FPPS generated a tree with three distinct clusters. Overall topology of the phylogenic tree constructed with small subunit ribosomal RNA sequences was almost similar to that constructed with FPPS sequences. Comparative homology modeling and structural comparisons of FPPS from the parasitic protozoa provided significant insights into common and distinct characteristics of the enzyme. The critical interacting residues of the isopentenyl pyrophosphate binding site are conserved across the enzymes from the family except for malarial FPPS where the C-terminal residues from the BXB motif of helix J were missing. Variations noticed in aromatic residue pairs at the fourth and fifth position upstream of the FARM, which play important role in determination of chain length of the polyprenyl products, may produce functional differences among protozoan FPPSs. The structural comparison of protozoan FPPS may be useful in designing common or selective FPPS inhibitors as potential broad spectrum or selective antiprotozoal agents.

Probing the structures of leishmanial farnesyl pyrophosphate synthases: homology modeling and docking studies.

Leishmania donovani and Leishmania major farnesyl pyrophosphate synthase ( LdFPPS and LmFPPS) are potential targets for the development of antileishmanial therapy. The protein sequence for LdFPPS was recently elucidated in our laboratory. Highly refined homology models were generated using the protein sequences of LdFPPS and the closely related LmFPPS enzyme. A ligand-refined model of LmFPPS with a bound bisphosphonate ligand was generated using restraint-guided molecular mechanics followed by quantum mechanics/molecular mechanics refinement. The ligand-refined model of LmFPPS was further validated through extensive pose validation, enrichment, and other docking studies involving known bisphosphonate inhibitors. The model was able to explain the critical binding site interactions and site-directed mutagenesis data obtained from experimental studies on related FPPS enzymes. The ligand-refined model in conjunction with the validated docking protocol could be utilized in the future for structure-based virtual screening and rational drug design studies against these targets.

Effect of ceramide on the contractility of pregnant rat uterus.

Ceramide and other sphingolipid mediators have emerged as a novel class of lipid second messengers in cell signaling. We assessed the effect of C(2)-ceramide (a membrane permeable analog of ceramide) on spontaneous and agonist-induced contractile responses of uterus, isolated from 19-day pregnant rats. Ceramide (3, 10 microM) moderately, but significantly inhibited the amplitude of spontaneous rhythmic contractions. However, a variable effect was seen on agonist-induced contractions. While 5-HT-induced contractions were markedly inhibited at 3 and 10 microM ceramide, oxytocin and carboprost (a PGF(2)alpha analogue)-induced contractions were not affected by the sphingolipid. Ceramide (10 microM) also markedly inhibited CaCl(2)-induced contractions elicited in K(+)-depolarized tissues. Further, in rabbit portal vein myocytes, which display robust L-type calcium channel current, ceramide inhibited the I(Ba) in a dose-dependent manner. Therefore, it is suggested that the inhibitory effect of ceramide on uterine contractility may involve a decrease in the influx of Ca(2+) through voltage-dependent L-type Ca(2+) channels, such that contractile responses that are primarily dependent on extracellular Ca(2+), like rhythmic and serotonin contractions, were inhibited by ceramide. Further study is required to establish the role of endogenous ceramide and other sphingolipids in regulating uterine tone during gestation and at term.

Identification of novel parasitic cysteine protease inhibitors by use of virtual screening. 2. The available chemical directory.

The incidence of parasitic infections such as malaria, leishmaniasis, and trypanosomiasis has been steadily increasing. Since the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based antiparasitic agents against these diseases. Cysteine proteases have been established as valid targets for this purpose. The Available Chemical Directory consisting of nearly 355,000 compounds was screened in silico against the homology models of plasmodial cysteine proteases, falcipain-2, and falcipain-3, to identify structurally diverse non-peptide inhibitors. The study led to identification of 22 inhibitors of parasitic cysteine proteases out of which 18 compounds were active against falcipain-2 and falcipain-3. Eight compounds exhibited dual activity against both enzymes. Additionally, four compounds were found to inhibit L. donovani cysteine protease. While one of the cysteine protease inhibitors also exhibited in vitro antiplasmodial activity with an IC50 value of 9.5 microM, others did not show noticeable antiplasmodial activity up to 20 microM. A model identifying important pharmacophoric features common to the structurally diverse falcipain-2 inhibitors has also been developed. Very few potent non-peptide inhibitors of the parasitic cysteine proteases have been reported so far, and identification of these novel and chemically diverse inhibitors should provide leads to be optimized into candidates to treat protozoal infections.

Identification of novel parasitic cysteine protease inhibitors using virtual screening. 1. The ChemBridge database.

Trypanosomiasis, leishmaniasis, and malaria are major parasitic diseases in developing countries. The existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance. Cysteine proteases are exciting novel targets for antiparasitic drug design. Virtual screening was performed in an attempt to identify novel druglike nonpeptide inhibitors of parasitic cysteine proteases. The ChemBridge database consisting of approximately 241 000 compounds was screened against homology models of falcipain-2 and falcipain-3 in three consecutive stages of docking. A total of 24 diverse inhibitors were identified from an initial group of 84, of which 12 compounds appeared to be dual inhibitors of falcipain-2 and falcipain-3. Four compounds showed inhibition of both the malarial cysteine proteases as well as Leishmania donovani cysteine protease.