Preeti Vishwakarma

Discovery of a New Class of Natural Product-Inspired Quinazolinone Hybrid as Potent Antileishmanial agents

The high potential of quinazolinone containing natural products and their derivatives in medicinal chemistry led us to discover four novel series of 53 compounds of quinazolinone based on the concept of molecular hybridization. Most of the synthesized analogues exhibited potent leishmanicidal activity against intracellular amastigotes (IC50 from 0.65 ± 0.2 to 7.76 ± 2.1 μM) as compared to miltefosine (IC50 = 8.4 ± 2.1 μM) and nontoxic toward the J-774A.1 cell line and Vero cells. Moreover, activation of Th1 type and suppression of Th2 type immune responses and induction in nitric oxide generation proved that 8a and 8g induce murine macrophages to prevent survival of parasites. Compounds 8a and 8g exhibited significant in vivo inhibition of parasite 73.15 ± 12.69% and 80.93 ± 10.50% against Leishmania donovani /hamster model. Our results indicate that compounds 8a, 8g, and 9f represent a new structural lead for this serious and neglected disease.

Design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents.

An analogous library of 2-substituted quinoline compounds was synthesized with the aim to identify a potential drug candidate to treat visceral leishmaniasis. These molecules were tested for their in vitro and in vivo biological activity against Leishmania donovani. Metabolic stability of these compounds was also improved through the introduction of halogen substituents. Compound (26g), found to be the most active; exhibited an IC₅₀ value of 0.2 μM and >180 fold selectivity. The hydrochloride salt of (26g) showed 84.26 ± 4.44 percent inhibition at 50 mg/kg × 5 days (twice daily, oral route) dose in L. donovani/hamster model. The efficacy was well correlated with the PK data observed which indicating that the compound is well distributed.

Nitroimidazo-oxazole compound DNDI-VL-2098: an orally effective preclinical drug candidate for the treatment of visceral leishmaniasis

OBJECTIVES The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). METHODS A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining. The best antileishmanial candidates (racemate DNDI-VL-2001) and its R enantiomer (DNDI-VL-2098) were evaluated in vitro against a range of Leishmania strains. These candidates were further studied in a hamster model using various dose regimens. Cytokine and inducible nitric oxide synthase estimations by real-time PCR and nitric oxide generation by Griess assay were also carried out for DNDI-VL-2098. RESULTS In vitro screening of nitroimidazo-oxazole compounds identified the racemate DNDI-VL-2001 (6-nitroimidazo-oxazole derivative) and its enantiomers as candidates for further evaluation in in vivo models of VL. DNDI-VL-2098 (IC50 of 0.03 μM for the DD8 strain) showed excellent in vivo activity in both mouse and hamster models, with an ED90 value of 3.7 and <25 mg/kg, respectively, and was also found to be very effective against high-grade infection in the hamster model. Our studies revealed that, along with leishmanicidal activity, DNDI-VL-2098 was also capable of inducing host-protective immune cells to suppress Leishmania parasites in hamsters. CONCLUSIONS These studies led to the identification of compound DNDI-VL-2098 as a preclinical candidate for further drug development as an oral treatment for VL.

Synthesis, structure-activity relationships, and biological studies of chromenochalcones as potential antileishmanial agents.

Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, five compounds (11, 14, 16, 17, 22, and 24) exhibited better activity than the marketed drug miltefosine in in vitro studies against the intracellular amastigotes form of Leishmania donovani. Three promising compounds, 16, 17, and 22, were tested in a L. donovani/hamster model. Oral administration of chalcone 16, at a concentration of 100 mg/kg of body weight per day for 5 consecutive days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and immunological studies revealed that compound 16 has a dual nature to act as a direct parasite killing agent and as a host immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound 16 has the potential to be a candidate for the treatment of the nonhealing form of leishmaniasis.

Combination of liposomal CpG oligodeoxynucleotide 2006 and miltefosine induces strong cell-mediated immunity during experimental visceral leishmaniasis.

Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.

Enhancement in therapeutic efficacy of miltefosine in combination with synthetic bacterial lipopeptide, Pam3Cys against experimental Visceral Leishmaniasis

Existing drugs for visceral leishmaniasis (VL) are partially effective, toxic, having high cost and long term treatment. Their efficacies are also compromised due to suppression of immune function associated during the course of infection. Combination therapy including a potential and safe immunostimulant with lower doses of effective drug has proven as a significant approach which is more effective than immunotherapy or drug therapy alone. In the present study, we have used the combination of Pam3Cys (an in-built immunoadjuvant and TLR2 ligand) and miltefosine. Initially dose optimization of both the agents was carried out and after that, antileishmanial effect of their combination was evaluated. All experiments were done in BALB/c mouse model. The immunomodulatory role of Pam3Cys on the immune functions of the host receiving combination treatment was also determined using immunological and biochemical parameters viz. phagocytosis, Th1/Th2 cytokines and production of ROS, RNS and H(2)O(2). Combination group showed significant enhancement in parasitic inhibition as compared to groups receiving miltefosine and Pam3Cys separately. Enhanced production of Th1 cytokines as well as ROS, RNS and H(2)O(2) was witnessed during the study of immunological alterations. Remarkable increase in phagocytosis index was also observed. Thus, the risk of development of drug resistance against miltefosine can be resolved through using low doses of it and Pam3Cys (single-dose) in combination and also provide a promising alternative for cure of leishmaniasis, with a pronounced transformation of the host immune response.

CpG Oligodeoxynucleotide 2006 and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis

ABSTRACT In view of the severe immunosuppression in visceral leishmaniasis (VL), a rational approach to effectively combat the parasitic scourge would be to enhance the immune status of the host. Use of CpG oligodeoxynucleotide (CpG-ODN) against leishmaniasis has previously been reported, especially as an immunomodulator and adjuvant with various immunogens. In the present study, experiments were carried out with BALB/c mice and hamsters infected with Leishmania donovani. Immunostimulating class B bacterial CpG-ODN namely, ODN-2006, was administered at various doses by the intraperitoneal (i.p.) route. The dose of CpG-ODN-2006 (1 nM/single dose) showing the most antileishmanial activity was given as free and liposomal forms with different doses of miltefosine, namely, 5 and 10 mg/kg of body weight, for 5 days in mice and hamsters, respectively. Among the various groups, mice coadministered liposomal CpG-ODN and miltefosine (5 mg/kg) showed the best inhibitory effect (97% parasite inhibition) compared with free CpG-ODN plus miltefosine and miltefosine, free CpG-ODN, and liposomal CpG-ODN given separately. Similar responses were observed in the case of hamsters, where the combination of liposomal CpG-ODN with miltefosine (10 mg/kg) gave 96% parasite inhibition. Promising antileishmanial efficacy was observed in animals treated with liposomal CpG-ODN and miltefosine.

Novel β-carboline–quinazolinone hybrid as an inhibitor of Leishmania donovani trypanothione reductase: Synthesis, molecular docking and bioevaluation

Trypanothione reductase (TR) is a vital enzyme in the trypanothione based redox metabolism of trypanosomatid parasites. It is one of the few chemically validated targets for Leishmania. Herein, we report the synthesis of novel β-carboline–quinazolinone hybrids that are able to inhibit Leishmania donovani TR (LdTR) and subsequently inhibit cell growth. A molecular modeling approach based on docking studies and subsequent binding free energy estimation was performed in the active site of LdTR to understand their possible binding sites. With the enzymatic assay on LdTR with compounds, we were able to identify six hit compounds (8j–8o) that were all found to be the competitive inhibitors of TR with Ki in the range of 0.8–9.2 μM. The whole-cell screening assay highlighted the analogues 8k, 8l and 8n as the most active compounds with IC50 of 4.4, 6.0 and 4.3 μM, respectively, along with an adequate selectivity index (SI) of >91, 36 and 24, respectively.

Synthesis and biological evaluation of indolyl glyoxylamides as a new class of antileishmanial agents

A series of indolylglyoxylamide derivatives have been synthesized and evaluated in vitro against amastigote form of Leishmania donovani. Compound 8c has been identified as the most active analog of the series with IC(50) value of 5.17μM and SI value of 31.48, and is several folds more potent than the standard drugs sodium stilbogluconate and pentamidine.

Aminothiazoles: Hit to lead development to identify antileishmanial agents.

As part of Drugs for Neglected Diseases initiatives lead optimization program for the development of new chemical entities to treat visceral leishmaniasis (VL), a series of aminothiazoles were synthesized and screened for in vitro efficacy, solubility and microsomal stability. The primary aim of identifying a lead structure with sub-micromolar activity was achieved. Out of 43 compounds synthesized, 16 compounds showed in vitro activity at less than 1 μM against VL. Compound 32 showed excellent antileishmanial potency (IC50 = 3 nM) and had all the acceptable properties except for metabolic instability. Blocking the metabolic soft spots in compound 32, where the 4-methoxy pyridine substituent was replaced by 5-ethoxy group, led to compound 36 (IC50 = 280 nM) with improved stability. To understand the disposition of 36, in vivo pharmacokinetic study was conducted in a mouse model. Compound 36 showed high clearance (91 mL/min/kg); short half-life (0.48 h) after intravenous administration (1 mg/kg) and exposure (AUC0-24) following oral administration was 362 ng h/mL with absolute bioavailability of 8%. To summarize, 43 analogs were synthesized out of which 15 compounds showed very potent sub-nanomolar efficacy in in vitro systems but the liability of metabolic instability seemed to be the major challenge for this chemical class and remains to be addressed.