Puvvada Kalpana Murthy

Integrated dataset of screening hits against multiple neglected disease pathogens.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A-D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold. In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

Secondary amines as new pharmacophores for macrofilaricidal drug design.

Several secondary amines exhibit promising macrofilaricidal response in vivo through oral route of administration against Acanthocheilonema viteae in which N-hexylcyclohexylamine (1) shows 100% macrofilaricidal activity while a tertiary amine such as 9 elicits predominantly microfilaricidal (93%) response.

In vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines: a new class of antifilarial agents.

A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of these, six compounds (4a-f) possessed improved in vitro anti-filarial activity and examples 4d and 4f were also found to be active in the in vivo experiments. These results demonstrate that 3,6-epoxy [1,5]dioxocines exhibits potent antifilarial activity and might be developed into a new class of antifilarial drug.

Solid phase synthesis of quinolones

Solid phase syntheses of ethyl 6-carboxyquinol-4(1H-)-one-3-carboxylate (5) and N-substituted 6-carboxyquinol-4(1H)-one-3-carboxamides 7a-d have been described. Antifilarial in vitro activities of 5,7a-d against Brugia malayi have also been delineated.

In Vitro, In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuters ‘Metadrug’ tool screening predictions.

Inflammatory mediator release by Brugia malayi from macrophages of susceptible host Mastomys coucha and THP-1 and RAW 264.7 cell lines

OBJECTIVE To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages. METHODS The human macrophage/monocyte cell line THP-1, the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages (PM) from the rodent host Mastomys coucha (M. coucha) were incubated at 37 °C in 5% CO(2) atmosphere with extracts of microfilariae (Mf), third stage infective larvae (L(3)) and adult worms (Ad) of Brugia malayi. After 48 hr post exposure, IL-1β, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated. RESULTS Extracts of all the life stages of the parasite were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines in both the cell lines and peritoneal macrophages of M. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L(3) and Ad; however, Ad was a strong stimulator of IL-10 release. Mf was found to have potential to modulate LPS-induced NO release in RAW cells. Ad-induced NO release was concentration dependent with maximum at 20 μg/mL in both RAW and PMs. CONCLUSIONS The results show that parasites at all life stages were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines and NO release from macrophages of susceptible host M. coucha, human and mouse macrophage cell lines. Mf can suppress the LPS-induced NO release in RAW cells. The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.

Leishmania donovani molecules recognized by sera of filaria infected host facilitate filarial infection

We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both filarial and leishmanial infections. In the present study, we identified a 52.9–93.6 kDa fraction (Ld1) of L. donovani that cross-reacted with sera of B. malayi infected animals and investigated effect of Ld1 on filarial infection. Immunization of BALB/c mice with Ld1 facilitated B. malayi infection with remarkable increase in parasite burden. Facilitation of filarial infection was associated with downregulated cell proliferation, IL-5, IL-13, IFN-γ, TNF-α, and IL-2 levels and upregulated IL-4 and TGF-β. Ld1 exposure also suppressed MHC class-I, MHC class-II, and FcεR1 expression, and phagocytosis in naive mouse macrophages, and CD4+, CD8+, and CD19+ cell population in mouse spleen. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization–time of flight-mass spectrometry revealed eight proteins in Ld1: putative heat shock protein (HSP) 70-related protein 1, HSP70 mitochondrial precursor, alanine aminotransferase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, protein disulfide isomerase, putative ATPase beta subunit, trypanothione reductase, and a hypothetical protein. HSP70 protein mitochondrial precursor and trypanothione reductase showed homology with Trypanosoma cruzi and L. donovani, respectively, and the rest 6 proteins including hypothetical protein bear homology with L. infantum. In conclusion, the present study for the first time shows that immunization with filarial cross-reactive Ld1 fraction of L. donovani facilitates filarial infection by modulating Th1 and Th2 responses. Ld1 molecules may therefore facilitate filarial infection in filaria-leishmania co-infection.