Md. Yousuf Ansari

Computational Elucidation of Structural Basis for Ligand Binding with Leishmania donovani Adenosine Kinase

Enzyme adenosine kinase is responsible for phosphorylation of adenosine to AMP and is crucial for parasites which are purine auxotrophs. The present study describes development of robust homology model of Leishmania donovani adenosine kinase to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Docking calculation using reported organic small molecules and natural products revealed key active site residues such as Arg131 and Asp16 for ligand binding, which is consistent with previous studies. Molecular dynamics simulation of ligand protein complex revealed the importance of hydrogen bonding with active site residues and solvent molecules, which may be crucial for successful development of drug candidates. Precise role of Phe168 residue in the active site was elucidated in this report that provided stability to ligand-protein complex via aromatic-π contacts. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.

Computational prediction and analysis of potential antigenic CTL epitopes in Zika virus: A first step towards vaccine development.

The Zika virus disease is an Aedes mosquito-borne disease caused by the ZIKA virus. The unavailability of vaccines or proper chemotherapeutic treatment emphasizes the need for the development of preventive and therapeutic vaccines. T cell specific epitopes have been used as vaccine candidates to generate desired immune responses against a variety of viral pathogens. Herein, the immune-informatics approach was used for the screening of potential major histocompatibility complex class I restricted epitopes, which may be competent to generate a cell-mediated immune response in humans. A total of 63 epitopes were identified, which revealed a comprehensive binding affinity to the 42 different human leukocyte antigen class I supertypes: A01, A02, A08, A23, A24, A25, A26, A29, A30, A32, A66, A68, A69, A80, B07, B08, B14, B15, B27, B35, B39, B40, B42, B45, B46, B48, B51, B53, B54, B57, B58, B83, C12, C03, C04, C05, C06, C07, C08, C12, C14, and C15, and which had no homologs in humans. By combining the human leukocyte antigen binding specificity and population coverage, nine promiscuous epitopes located in Capsid 1 Protein (MVLAILAFL(P1)), Envelop Protein (RLKGVSYSL (P2) and RLITANPVI (P3)), NS2A (AILAALTPL (P4)), NS4B (LLVAHYMYL (P5) and LVAHYMYLI (P6)) and NS5 (SLINGVVRL (P7), ALNTFTNLV (P8) and YLSTQVRYL (P9)) were shortlisted. Most of these consensus epitopes revealed 100% conservancy in all Zika virus strains and were very less conserved against the human proteome. The combination of the selected epitopes accounted for an optimal coverage in the world wide population (>99%) independent of ethnicity. Structural analysis of these selected epitopes by the PatchDock web server showed their preferential mode of presentation to the T cell receptor. All these results recommended the possibility of a combined epitope vaccine strategy and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

Structure prediction of gBP21 protein of L. donovani and its molecular interaction

Visceral leishmaniasis (Kala-azar) is a fatal disease caused by the obligate intracellular parasite Leishmania donovani and the available drugs for the treatment are few, and are frequently associated with side effects and toxicity. RNA editing is one of the essential metabolic processes in the kinetoplastids, where the pre-mRNAs are edited post-transcriptionally by the guide RNAs with the addition or deletion of uridine residues. The aim is to block the gBP21 protein involved in RNA editing process thereby other direct and indirect protein activity is reduced and ultimately the editing process in L. donovani is disturbed and it will inhibit the growth. RNA editing factors are RNA-linked proteins essential for in vivo editing i.e. mitochondrial RNA binding protein1 (MRP 1) originally called as gBP21. The model of L. donovani gBP21 (gBP21Ldv) showed that this protein bears an anti-parallel ß sheet (segregated α and ß regions) with ß-ß-ß-ß-α-ß-ß-ß-ß-α-type topology (“whirly” transcription-factor fold). Each of the four ß strands within a given ß-ß-ß-ß-α repeats and form a curved anti-parallel ß-sheet that packs perpendicularly against the sheet from the other repeat. Among all of the computationally screened compounds by the GLIDE program (Schrödinger) and GOLD program hyperoside1a, posaconazole, quercetin, and pentanediol, 427 exhibited higher binding affinities with the modeled gBP21 protein of L. donovani. Ligandfit program (DSv2.5) revealed that DNA, RNA polymerase inhibitors acyclovir, mitomycin C, and daunorubicin have better binding affinity towards gBP21Ldv. These compounds may be given in combination with miltefosine (first line therapy) against patients with VL and other associated disorders like anemia.

Mining the proteome of Leishmania donovani for the development of novel MHC class I restricted epitope for the control of visceral leishmaniasis

Although, the precise host defence mechanism(s) is not completely understood, T cell‐mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8), and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1–P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8+ T cell response and increased IFN‐γ production. An upregulated NF‐κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1‐type cytokine profile (presence of IFN‐γ and absence of IL‐10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. J. Cell. Biochem. 119: 378–391, 2018.

Up-regulation of cytosolic tryparedoxin in Amp B resistant isolates of Leishmania donovani and its interaction with cytosolic tryparedoxin peroxidase

Leishmania is a unicellular protozoan parasite which causes leishmaniasis, a neglected tropical disease. It possess a unique thiol metabolism comprising of several proteins among which, tryparedoxin (cTXN) and tryparedoxin peroxidase (cTXNPx), function in concert as oxidoreductases, utilizing trypanothione as a source of electrons to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is unique and essential for the survival of Leishmania. Herein, we report the functional characterization of Leishmania donovani cTXN and its interaction with cTXNPx. The full length recombinant cTXN and cTXNPx proteins were purified in the native state and biochemical analysis showed that the cTXN-cTXNPx coupled system efficiently degraded hydrogen peroxide and tert-butyl hydroperoxide by transferring reducing equivalents from trypanothione. In silico investigation of the potential interaction between cTXN and cTXNPx proteins showed strong interaction of model structures with amino acids Ile109, Thr132, Glu107, Trp70, Trp39, Cys40 and His129 of Ld-cTXN and Thr54, Lys93, Arg128 and Asn152 of Ld-cTXNPx predicted to be involved in interaction. Moreover, co-purification, pull down assay and immunoprecipitation studies confirmed the interaction between Ld-cTXN and Ld-cTXNPx proteins. In addition, for the first time, we demonstrated at the translational level that Ld-cTXN protein is upregulated in Amp B resistant isolates accompanied by enhanced peroxidase activity, as compared to sensitive strains. Thus, our results show that Ld-cTXN and Ld-cTXNPx proteins acts in concert by physical interaction to form a strong peroxide stress detoxification system in Leishmania and their upregulation in Amp B resistant isolates imparts better stress tolerance, and hence fitter pathogens, as compared to sensitive strains.

Analysis of sequence, structure of GAPDH of Leishmania donovani and its interactions

Drug resistance acquired by Leishmania donovani (Ldv) is a major problem in the treatment and control of visceral leishmaniasis (VL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme has been targeted as is found in other protozoan which cause diseases like sleeping sickness. GAPDH gene of Ldv (AG83 strain) was amplified, sequenced, and modeled on the basis of crystal structure of Leishmania mexicana. The model of the Ldv GAPDH exhibited NAD-binding domain with Rossmann folding. Virtual screening of different experimentally proved compounds with the crystal and the modeled structures of GAPDH of Leishmania strains revealed diverse binding affinities of different compounds. Comparison of binding affinities (based on different programs) of compounds revealed that discovery studio v2.5 (Ligandfit) was able to predict the most hit compounds, the best hit compounds against GAPDH of Leishmania strains are hydrazine, vetrazine, and benzyl carbazate. It is predicted that patients suffering from both VL and cardiac disorders (atrial fibrillation) may benefit if they are treated with warfarin in conjunction with first-line antileishmanial therapies such as miltefosine and AmBisome.

Glucose-6-phosphate dehydrogenase and Trypanothione reductase interaction protects Leishmania donovani from metalloid mediated oxidative stress

Abstract Exploration of metabolons as viable drug target is rare in kinetoplastid biology. Here we present a novel protein‐protein interaction among Glucose‐6‐phosphate dehydrogenase (LdG6PDH) and Trypanothione reductase (LdTryR) of Leishmania donovani displaying interconnection between central glucose metabolism and thiol metabolism of this parasite. Digitonin fractionation patterns observed through immunoblotting indicated localisation of both LdG6PDH and LdTryR in cytosol. In‐silico and in‐vitro interaction observed by size exclusion chromatography, co‐purification, pull‐down assay and spectrofluorimetric analysis revealed LdG6PDH and LdTryR physically interact with each other in a NADPH dependent manner. Coupled enzymatic assay displayed that NADPH generation was severely impaired by addition of SbIII, AsIII and TeIV extraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co‐purification patterns and pull‐down assays also depicted that metalloids (SbIII, AsIII and TeIV) hinder the in‐vitro interaction of these two enzymes. Surprisingly, metalloids at sub‐lethal concentrations induced the in‐vivo interaction of LdG6PDH and LdTryR, as analyzed by pull‐down assays and fluorescence microscopy signifying protection against metalloid mediated ROS. Inhibition of LdTryR by thioridazine in LdG6PDH‐/‐ parasites resulted in metalloid induced apoptotic death of the parasites due to abrupt fall in reduced thiol content, disrupted NADPH/NADP+ homeostasis and lethal oxidative stress. Interestingly, clinical isolates of L.donovani resistant to SAG exhibited enhanced interaction between LdG6PDH and LdTryR and showed cross resistivity towards AsIII and TeIV. Thus, our findings propose the metabolon of LdG6PDH and LdTryR as an alternate therapeutic target and provide mechanistic insight about metalloid resistance in Visceral Leishmaniasis. Graphical abstract Figure. No Caption available. HighlightsMetabolon analysis of Leishmania is rare.G6PDH and TryR of L.donovani interact physically.Sb, As and Te affects this interaction in vitro and in vivo.Inhibition of LdTryR in G6PDH‐/‐ parasites leads to metalloid induced apoptosis.Interaction of LdG6PDH and LdTryR modulates LdMRPA, affecting metalloid resistance.

Structure, evolution and virtual screening of NDM-1 strain from Kolkata

β-lactam antibiotics are utilised to treat bacterial infection. β-lactamase enzymes (EC 3.5.2.6) are produced by several bacteria and are responsible for their resistance to β-lactam antibiotics like penicillin, cephamycins and carbapenems. New Delhi Metallo-β-lactamase (NDM-1) is a gene that makes bacteria resistant to β-lactam antibiotics. Preparing a compound against NDM-1 will require additional investment and development by drug manufacturers as the current antibiotics will not treat patients with NDM-1 resistance. NDM-1 of Kolkata showed convergent-type evolution with other NDM-1 producing strains. The modelled structure exhibited α-β-α barrel-type domain along with Zn metallo-β-lactamase N-terminal domain. Compounds belonging to cephalosporins (relatively resistant to β-lactamase) and other antibiotics ceftaroline, ceftobiprole, piperacillin, penamecillin, azidocillin, cefonicid, tigecycline and colistin have exhibited better binding affinity with the modelled NDM-1.