Rani Mansuri

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.

Inhibitor designing, virtual screening, and docking studies for methyltransferase: A potential target against dengue virus.

Aim: Aim of this work was to design and identify some S-adenosyl-L-homocysteine (SAH) analogs as inhibitors of S-adenosyl-L-methionine-dependent methyltransferase (MTase) protein using computational approaches. Introduction: According to the current scenario the dengue has been a global burden. The people are being killed by dengue virus in an abundant number. Despite of lot of research being going on dengue worldwide, there is no single drug which can kill its virus. This creates an urge for new drug target identification and designing. MTase has been reported as an effective target against dengue virus as it catalyzes an essential step in methylation and capping of viral RNA for viral replication. Materials and Methods: The crystal structure of MTase in complex with SAH was used for designing new analogs of SAH. SAH analogs designed were analyzed on the basis of docking, ADMET, and toxicity analysis done using Discovery Studio 3.5. Results: Seventeen analogs found noncarcinogenic, nonmutagenic, as well as good ADMET properties and good drug-like profile. Conclusion: These SAH analogs, inhibitors of MTase may act as drugs against dengue virus. Further synthesis and biological testing against dengue virus is under observation.

Vaccine potential of HLA A2 epitopes from Leishmania Cysteine Protease Type III (CPC)

Although the precise host‐defence mechanisms are not completely understood, T‐cell‐mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA‐A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA‐A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8+ T‐cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN‐γ production, which indicates its pivotal role in protective immune response. The IFN‐γ production was mainly in a CD8+ T‐cells‐dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I‐dependent manner. Moreover, no role of the CD3+ T cell was observed in the IL‐10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.

In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinskis rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL.

Computational Elucidation of Structural Basis for Ligand Binding with Mycobacterium tuberculosis Glucose-1-Phosphate Thymidylyltransferase (RmlA)

Glucose-1-Phosphate Thymidylyltransferase (RmlA) is one of the enzymes in rhamnose biosynthesis pathway, where rhamnose acts as linker of peptidoglycan and arabinogalacton in the cell wall, therefore RmlA is a potential enzyme for the survival of Mycobacterium tuberculosis (Mtb). To go into the depth of the structure for exploring binding regions, homology model of RmlA was built in Prime, Schrodinger v9.2. The model with lowest Discrete Optimized Potential Energy (DOPE) score of -35524.17 kcal/mol and RMSD of 0.1 Å with the template (1H5R_B) was subjected to Molecular Dynamics Simulation (MDS) for 5 ns to achieve its stable folding state. The tertiary structure of the proposed model is composed of α/β/α sandwich type protein with quasi-Rossmann type folding pattern. The substrate, deoxy Thymidine tri phosphate (dTTP) comprises of triphosphate (R1) and methyl (R2) side chains where, R1 is highly essential for the survival of Mtb. Therefore, nineteen side chain analogues of dTTP were designed by substituting R1 and R2 chain of dTTP using Combi Glide, Schrodinger v9.2 and docked with the target RmlA protein. Out of which two analogues such as, 6-[(2R,3S,5R)-5-[5-(2- aminoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]-3-hydroxyoxolan-2 yl] hexanoic acid (COMP- 11) and 4-(2-{1-[(1S,3S,4S)-3-(5-carboxypentyl)-4-hydroxy-2-methylidenecyclopentyl]-2,4-dioxo- 1,2,3,4-tetrahydropyrimidin-5-yl}ethyl)morpholin-4-ium (COMP-12) showed the highest GLIDE score (-12.55 Kcal/mol and -11.58 Kcal/mol respectively) than that of substrate (-9.725 Kcal/mol). During simulations, hydrogen bonding profile between the two top hits and protein ranges up to 5 strong polar contacts which were much stronger than that of substrate. Similarly, the computational binding free energy of both the analogues was found to be less than -70 Kcal/mol which is much lower than that of substrate (-52.84 Kcal/mol). All these results suggest that these two compounds have more stable interaction than that of substrate inside the solvent condition and can be used as competitive inhibitors.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut

BackgroundIn vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment.MethodsIn silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays.ResultsWe identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1.ConclusionOur results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Computational elucidation, mutational and hot spot-based designing of potential inhibitors against human acid-sensing ion channels (hASIC-1a) to treat various physiological conditions

Acid-sensing ion channels are ligand/proton-gated ion channels belonging to the family of the degenerin/epithelial Na+ channel (DEG/ENaC). They function as a sodium-selective pore for Ca2+ entry into neuronal cells during pathological conditions. The blocking of this channel has therapeutic importance, because at basal physiological pH (7.2), it is in a closed state and under a more acidic condition, and the ASIC1a ion channel is activated. To investigate the different states of the hASIC1a channel based on mutational analysis, structure-based virtual screening and molecular dynamics simulation studies. The system showed stability after 30 ns (after 1500 frame), and it was stabilized to an average value around 2.2Å. During the simulation, the ion channel residues in persistent contact with toxin PcTx1 were D237, E238, D347, D351, E219 and E355. These residues are important physiologically for the activation of the channel. From in silico alanine scanning, the significant hotspots obtained in hASIC1 are E344, P347, F352, D351, E355 and E219. From the sitemap analysis, it was evident that the sitemap found one of the active sites at the PcTx1 binding site with a site score of 1.086 and a D-score of 1.035 for hASIC1. We obtained a few promising hits and final potential hits from the virtual screening in hASIC1 that made interactions with the residues in the acidic pocket (E344, P347, F352, D351, E355 and E219). Based on these studies, the hits and scaffolds of potential therapeutic interest against various pathological conditions are associated with hASIC1a for future studies.