Awanish Kumar

Publications of Awanish Kumar

• Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani.

  Authors: Awanish Kumar, Brijesh Sisodia, Pragya Misra, Shyam Sundar, Ajit Kumar Shasany, Anuradha Dube

  British journal of clinical pharmacology. 10/2010; 70(4):609-17.

  This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patientsThis study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine-leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.

• 16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide from Polyalthia longifolia: a safe and orally active antileishmanial agent.

  Authors: Pragya Misra, Koneni V Sashidhara, Suriya Pratap Singh, Awanish Kumar, Reema Gupta, Shailendra S Chaudhaery, Souvik Sen Gupta, H K Majumder, Anil K Saxena, Anuradha Dube

  British journal of pharmacology. 02/2010; 159(5):1143-50.

  New antileishmanials from natural products are urgently needed due to the emergence of drug resistance complicated by severe cytotoxic effects. 16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olideNew antileishmanials from natural products are urgently needed due to the emergence of drug resistance complicated by severe cytotoxic effects. 16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide (Compound 1) from Polyalthia longifolia was found to be a potential antileishmanial and non-cytotoxic, as evidenced by long-term survival (>6 months) of treated animals. This prompted us to determine its target and, using molecular modelling, identify the interactions responsible for its specific antileishmanial activity. In vitro activity of compound was assessed using intracellular transgenic green fluorescent protein-stably expressed Leishmania donovani parasites. In vivo activity and survival of animals post-treatment were evaluated in L. donovani-infected hamsters. Known property of clerodane diterpenes as potent human DNA topoisomerase inhibitors led us to evaluate the inhibition of recombinant L. donovani topoisomerase I using relaxation assay. Mode of cell death induced by Compound 1 was assessed by phosphotidylserine exposure post-treatment. Molecular modelling studies were conducted with DNA topoisomerase I to identify the binding interactions responsible for its activity. Bioassay-guided fractionation led to isolation of Compound 1 as a non-cytotoxic, orally active antileishmanial. Compound 1 inhibited recombinant DNA topoisomerase I which, ultimately, induced apoptosis. Molecular docking studies indicated that five strong hydrogen-bonding interactions and hydrophobic interactions of Compound 1 with L. donovani DNA-topoisomerase are responsible for its antileishmanial activity. The data reveal Compound 1 is a potent and safe antileishmanial. The study further exploited the structural determinants responsible for its non-cytotoxic and potent activity, to raise the feasibility of specifically targeting the target enzyme responsible for its activity through rational drug design.

• Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms.

  Authors: Awanish Kumar, Vijay Raju Boggula, Pragya Misra, Shyam Sundar, Ajit Kumar Shasany, Anuradha Dube

  Acta tropica. 10/2009;

  The Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis anThe Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis an attempt was made to differentiate Leishmania major; Leishmania tropica and Leishmania donovani genetically for the first time using amplified fragment length polymorphism (AFLP)-a high throughput DNA fingerprinting technique. The objective of this research work was to identify DNA markers of CL and VL. Ten combinations of selective primers detect a total of 1487 informative AFLP marker. Percentage of polymorphism was 45.12%. Three hundred and thirty-seven unique AFLP markers were also identified in three species of Leishmania. A clear distinction was revealed between L. major and L. donovani. It was inferred by AFLP analysis that a higher rate of polymorphisms occurred among Leishmania species which indicate the distinguished pattern of the disease cause by Leishmania, i.e. VL and CL. Analysis based on polymorphic AFLP markers revealed considerably high genetic variation among the genome of these species which was sufficient to distinguish between CL and VL.

• A pro-apoptotic effect of Landrace of Piper betle- Bangla Mahoma on Leishmania donovani may be due to high content of eugenol.

  Authors: Pragya Misra, Awanish Kumar, Prashant Khare, Swati Gupta, Nikhil Kumar, Anuradha Dube

  Journal of medical microbiology. 07/2009;

  In the absence of effective and safe treatment of Kala-azar (VL) - a most devastating parasitic disease caused by Leishmania donovani a need for the search of antileishmanial from natural resources,In the absence of effective and safe treatment of Kala-azar (VL) - a most devastating parasitic disease caused by Leishmania donovani a need for the search of antileishmanial from natural resources, in common use, is imperative. Recently, a comparative in vitro antileishmanial activity of methanolic extract of two landraces of Piper betle- Bangla Mahoba (PB-BMM) and Kapoori Vellaikodi (PB-KVM) has been reported. Herein, the putative pathway responsible for death induced by the effective extract of PB-BMM in promastigotes as well as intracellular amastigotes form of L. donovani was assessed using various biochemical approaches. It was observed that PB-BM is capable of selectively inhibiting both the stages of Leishmania parasites by accelerating apoptotic events by generation of ROS targeting mitochondrion without any cytotoxicity to macrophages. Study was extended to reason out the presence/absence of activity in PB-BMM and PB-KVM on the basis of differences in essential oil composition present in methanolic extract assessed by gas chromatography and mass spectra. The essential oil from PB-BM was found to be rich in eugenol as compared to PB-KV. The antileishmanial efficacy of PB-BMM mediated through apoptosis is probably due to the higher content of eugenol in active landrace. This observation emphasizes the need of extending the studies related to traditional medicines from bioactive plants below species to gender/landrace level for better efficacy and reproducibility.

• Identification of genetic markers in sodium antimony gluconate (SAG) sensitive and resistant Indian clinical isolates of Leishmania donovani through amplified fragment length polymorphism (AFLP).

  Authors: Awanish Kumar, Vijay Raju Boggula, Shyam Sundar, Ajit Kumar Shasany, Anuradha Dube

  Acta tropica. 05/2009; 110(1):80-5.

  Sodium Antimony Gluconate (SAG) is currently used worldwide as the first-line drugs for the treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) since 1940s. Unfortunately, theSodium Antimony Gluconate (SAG) is currently used worldwide as the first-line drugs for the treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) since 1940s. Unfortunately, the resistance of Leishmania parasite to this drug is increasing in several parts of the world. The mechanism of drug resistance in clinical isolates is still not very clear. Earlier, we have established a differentiation between six clinical isolates as sensitive and resistant on the basis of their sensitivity to SAG in vitro and in vivo as well as expression of proteophosphoglycan contents. In this preliminary study, we have further analyzed these isolates on the basis of their genetic diversity, molecular variance and phylogenetic structure using for the first time, a fingerprinting approach--amplified fragment length polymorphism (AFLP). Altogether 2338 informative AFLP bands were generated using 10 selective primer combinations. Percentage of polymorphism was 55.35%. A number of unique AFLP markers (217) were also identified in these strains. It was deduced that a higher rate of variations occurred among Leishmania clinical isolates which indicate the shifting of drug sensitive nature of parasite towards resistant condition.

• Discovery of novel vaccine candidates and drug targets against visceral leishmaniasis using proteomics and transcriptomics.

  Authors: Shraddha Kumari, Awanish Kumar, Mukesh Samant, Neeloo Singh, Anuradha Dube

  Current drug targets. 12/2008; 9(11):938-47.

  Among the three clinical forms (cutaneous, mucosal and visceral) of leishmaniasis visceral (VL) one is the most devastating type caused by the invasion of the reticuloendothelial system of human byAmong the three clinical forms (cutaneous, mucosal and visceral) of leishmaniasis visceral (VL) one is the most devastating type caused by the invasion of the reticuloendothelial system of human by Leishmania donovani, L. infantum and L. chagasi. India and Sudan account for about half the world's burden of VL. Current control strategy is based on chemotherapy, which is difficult to administer, expensive and becoming ineffective due to the emergence of drug resistance. An understanding of resistance mechanism(s) operating in clinical isolates might provide additional leads for the development of new drugs. Further, due to the lack of fully effective treatment the search for novel immune targets is also needed. So far, no vaccine exists for VL despite indications of naturally developing immunity. Therefore, an urgent need for new and effective leishmanicidal agents and for this identification of novel drug and vaccine targets is imperative. The availability of the complete genome sequence of Leishmania has revolutionised many areas of leishmanial research and facilitated functional genomic studies as well as provided a wide range of novel targets for drug designing. Most notably, proteomics and transcriptomics have become important tools in gaining increased understanding of the biology of Leishmania to be explored on a global scale, thus accelerating the pace of discovery of vaccine/drug targets. In addition, these approaches provide the information regarding genes and proteins that are expressed and under which conditions. This review provides a comprehensive view about those proteins/genes identified using proteomics and transcriptomic tools for the development of vaccine/drug against VL.

• Antileishmanial activity mediated by apoptosis and structure-based target study of peganine hydrochloride dihydrate: an approach for rational drug design.

  Authors: Pragya Misra, Tanvir Khaliq, Anshuman Dixit, Souvik Sengupta, Mukesh Samant, Shraddha Kumari, Awanish Kumar, Pramod K Kushawaha, H K Majumder, Anil K Saxena, T Narender, Anuradha Dube

  The Journal of antimicrobial chemotherapy. 09/2008;

  Objectives The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural andObjectives The aim of this study was to resolve the putative pathway responsible for death induced by peganine hydrochloride dihydrate isolated from Peganum harmala seeds at cellular, structural and molecular level in Leishmania donovani, a causative agent of fatal visceral leishmaniasis. Methods The mode of action was assessed using various biochemical approaches including phosphatidylserine exposure, estimation of mitochondrial transmembrane potential and in situ dUTP nick end labelling staining of nicked DNA in the parasite. Molecular modelling and molecular dynamics studies were conducted with DNA topoisomerase I to identify the target of peganine hydrochloride dihydrate mediating apoptosis. Further, DNA topoisomerase I inhibition by peganine hydrochloride dihydrate was also assessed using an L. donovani topoisomerase I relaxation assay. Results Peganine hydrochloride dihydrate, besides being safe, was found to induce apoptosis in both the stages of L. donovani via loss of mitochondrial transmembrane potential. Molecular docking studies suggest that a binding interaction with DNA topoisomerase I of L. donovani (binding energy of -79 kcal/mol) forms a stable complex, indicating a possible role in apoptosis. The compound also inhibits L. donovani topoisomerase I. Conclusions The compound induces apoptosis in L. donovani and inhibits DNA topoisomerase I.

• Proteomic approaches for discovery of new targets for vaccine and therapeutics against visceral leishmaniasis.

  Authors: Shraddha Kumari, Awanish Kumar, Mukesh Samant, Shyam Sundar, Neeloo Singh, Anuradha Dube

  Proteomics. Clinical applications. 03/2008; 2(3):372-86.

  Visceral leishmaniasis (VL) is the most devastating type caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi. The therapeutic mainstay is still based on the antiquatedVisceral leishmaniasis (VL) is the most devastating type caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi. The therapeutic mainstay is still based on the antiquated pentavalent antimonial against which resistance is now increasing. Unfortunately, due to the digenetic life cycle of parasite, there is significant antigenic diversity. There is an urgent need to develop novel drug/vaccine targets against VL for which the primary goal should be to identify and characterize the structural and functional proteins. Proteomics, being widely employed in the study of Leishmania seems to be a suitable strategy as the availability of annotated sequenced genome of Leishmania major has opened the door for dissection of both protein expression/regulation and function. Advances in clinical proteomic technologies have enable to enhance our mechanistic understanding of virulence/pathogenicity/host-pathogen interactions, drug resistance thereby defining novel therapeutic/vaccine targets. Expression proteomics exploits the differential expression of leishmanial proteins as biomarkers for application towards early diagnosis. Further using immunoproteomics efforts were also focused on evaluating responses to define parasite T-cell epitopes as vaccine/diagnostic targets. This review has highlighted some of the relevant developments in the rapidly emerging field of leishmanial proteomics and focus on its future applications in drug and vaccine discovery against VL.

• Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis.

  Authors: Nasib Singh, Awanish Kumar, Prasoon Gupta, Kailash Chand, Mukesh Samant, Rakesh Maurya, Anuradha Dube

  Parasitology research. 03/2008; 102(3):561-5.

  The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in newThe chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in new drug developments from traditionally used medicinal plants which offers unprecedented diversity in structures and bioactivity. With this rationale, ethanolic extract of Tinospora sinensis Linn and its four fractions were tested in vitro against promastigotes and intracellular amastigotes and in vivo in Leishmania donovani infected hamsters. Ethanolic extract exhibited an appreciable activity against promastigotes (IC(50) 37.6+/-6.2 microg/ml) and intracellular amastigotes (IC(50) 29.8+/-3.4 microg/ml). In hamsters, it resulted in 76.2+/-9.2% inhibition at 500 mg/kg/day x 5 oral dose level. Among fractions, n-butanol imparted highest in vitro and in vivo activities. Ethanolic extract and butanol fraction also enhances reactive oxygen species (ROS) and nitric oxide (NO) release. The results indicate that T. sinensis may provide new lead molecules for the development of alternative drugs against VL.

• Age-influenced population kinetics and immunological responses of Leishmania donovani in hamsters.

  Authors: Nasib Singh, Mukesh Samant, Shraddha K Gupta, Awanish Kumar, Anuradha Dube

  Parasitology research. 10/2007; 101(4):919-24.

  Susceptibility of animals to infections depends upon various factors including sex and age of the host, which plays a pivotal role. In this communication, we have investigated the "intake" ofSusceptibility of animals to infections depends upon various factors including sex and age of the host, which plays a pivotal role. In this communication, we have investigated the "intake" of Leishmania donovani infection in young (3-4 weeks old) and adult (15-16 weeks old) golden hamsters. The splenic parasite load in young hamsters on day 15 post infection (p.i.) was 54 +/- 4 amastigotes/100 macrophage nuclei and increased to 106.3 +/- 3.5 on day 30 p.i. However, adult group showed 2.2-(P < 0.001) and 1.75-fold (P < 0.001) lesser parasite burden on these days, respectively. But as the disease progresses further, differences in parasite burden become less significant, as revealed by comparable levels of parasite loads at 2 months p.i. Spleen weight measurements correspond to the above observations. In the young group, the levels of antileishmanial antibody rise two and 4.5 times on days 15 and 30 p.i., respectively, as compared to only 1.3 and 2.3 times increase in their respective adult counterparts. However, after 2 months of infection both groups recorded analogous (12-fold) rise in antibody levels. Both mitogenic and antigenic responses in adult hamsters were less suppressed compared to young hamsters on days 15 and 30 p.i. However, both groups exhibited highly suppressed cell-mediated immune (CMI) responses after 2 months of infection. These findings implicate that age of the host may influence the susceptibility and resistance to Leishmania infection.