Rudraraju Srilakshmi Reshma

Engineering another class of anti-tubercular lead: Hit to lead optimization of an intriguing class of gyrase ATPase inhibitors.

A structure based medium throughput virtual screening campaign of BITS-Pilani in house chemical library to identify novel binders of Mycobacterium tuberculosis gyrase ATPase domain led to the discovery of a quinoline scaffold. Further medicinal chemistry explorations on the right hand core of the early hit, engendered a potent lead demonstrating superior efficacy both in the enzyme and whole cell screening assay. The binding affinity shown at the enzyme level was further corroborated by biophysical characterization techniques. Early pharmacokinetic evaluation of the optimized analogue was encouraging and provides interesting potential for further optimization.

Inhibitors of the Cysteine Synthase CysM with Antibacterial Potency against Dormant Mycobacterium tuberculosis

Cysteine is an important amino acid in the redox defense of Mycobacterium tuberculosis, primarily as a building block of mycothiol. Genetic studies have implicated de novo cysteine biosynthesis in pathogen survival in infected macrophages, in particular for persistent M. tuberculosis. Here, we report on the identification and characterization of potent inhibitors of CysM, a critical enzyme in cysteine biosynthesis during dormancy. A screening campaign of 17 312 compounds identified ligands that bind to the active site with micromolar affinity. These were characterized in terms of their inhibitory potencies and structure-activity relationships through hit expansion guided by three-dimensional structures of enzyme-inhibitor complexes. The top compound binds to CysM with 300 nM affinity and displays selectivity over the mycobacterial homologues CysK1 and CysK2. Notably, two inhibitors show significant potency in a nutrient-starvation model of dormancy of Mycobacterium tuberculosis, with little or no cytotoxicity toward mammalian cells.

Mycobacterium tuberculosis lysine-ɛ-aminotransferase a potential target in dormancy: Benzothiazole based inhibitors

MTB lysine-ɛ-aminotransferase (LAT) was found to play a crucial role in persistence and antibiotic tolerance. LAT serves as a potential target in the management of latent tuberculosis. In present work we attempted to derivatize the benzothiazole lead identified through high throughput virtual screening of Birla Institute of Technology and Science in house database. For Structure activity relationship purpose 22 derivatives were synthesized and characterized. Among synthesized compounds, eight compounds were found to be more efficacious in terms of LAT inhibition when compared to lead compound (IC50 10.38±1.21µM). Compound 22 exhibits bactericidal action against nutrient starved Mycobacterium tuberculosis (MTB). It also exhibits significant activity in nutrient starvation model (2.9log folds) and biofilm model (2.3log folds).

Profiling of in vitro activities of urea-based inhibitors against cysteine synthases from Mycobacterium tuberculosis

CysK1 and CysK2 are two members of the cysteine/S-sulfocysteine synthase family in Mycobacterium tuberculosis, responsible for the de novo biosynthesis of l-cysteine, which is subsequently used as a building block for mycothiol. This metabolite is the first line defense of this pathogen against reactive oxygen and nitrogen species released by host macrophages after phagocytosis. In a previous medicinal chemistry campaign we had developed urea-based inhibitors of the cysteine synthase CysM with bactericidal activity against dormant M. tuberculosis. In this study we extended these efforts by examination of the in vitro activities of a library consisting of 71 urea compounds against CysK1 and CysK2. Binding was established by fluorescence spectroscopy and inhibition by enzyme assays. Several of the compounds inhibited these two cysteine synthases, with the most potent inhibitor displaying an IC50 value of 2.5µM for CysK1 and 6.6µM for CysK2, respectively. Four of the identified molecules targeting CysK1 and CysK2 were also among the top ten inhibitors of CysM, suggesting that potent compounds could be developed with activity against all three enzymes.

A robust synthesis of functionalized 2H-indazoles via solid state melt reaction (SSMR) and their anti-tubercular activity

A facile and convenient approach has been developed for the synthesis of functionalized indazoles via solid state melt reaction using easily accessible starting materials under catalyst-free conditions. This transformation involves electrocyclization via a conjugated nitrene intermediate obtained under thermal conditions. Further anti-tubercular activity screening of the molecules was undertaken, among the compounds 3a-3x screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, compound 3u (MIC: 4.20μM) was found to be most active and are superior over existing standard drugs ciprofloxacin and ethambutol. Compounds 3c and 3x were found to equally potent as ethambutol. Among most potent compounds in the series, four compounds (3n, 3o, 3p and 3u) showed lower cytotoxicity which could be promising drug candidates for further development.

Structure‐Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand‐ and structure‐based pharmacophore modeling was used to identify structurally diverse small‐molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE ). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)‐5‐(5‐(2‐(1H‐benzo[d]imidazol‐2‐yl)‐2‐cyanovinyl)furan‐2‐yl)isophthalic acid; IC50=4.6±0.1 μm], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)‐3‐(5‐(2‐cyano‐2‐(5‐methyl‐1H‐benzo[d]imidazol‐2‐yl)vinyl)thiophen‐2‐yl)benzoic acid; IC50=0.3±0.2 μm], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H37Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.

Design and development of new class of Mycobacterium tuberculosisl-alanine dehydrogenase inhibitors

Mycobacterium tuberculosisl-alanine dehydrogenase (MTB l-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB l-AlaDH bound with cofactor NAD(+) as a structural framework for virtual screening of our in-house database to identified new classes of l-AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as one of the potent inhibitor with IC50 of 9.22±0.72μM. Further lead optimization by synthesis leads to compound 1-(isonicotinamido)-N(2),N(4)-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with l-AlaDH IC50 of 3.83±0.12μM, 2.0log reduction in nutrient starved dormant MTB model and MIC of 11.81μM in actively replicative MTB.

Design and Identification of Mycobacterium tuberculosis Glutamate Racemase (MurI) Inhibitors

Objective: We determined the correlation of atypical lymphocyte (AL) in dengue illness and accuracy in predicting dengue infection. Statement of the Problem: Diagnosis of dengue during febrile stage has been challenging. There are several existing diagnostics however most are costly and not available in many tropical countries. Atypical lymphocytes (AL) or reactive lymphocytes are activated non-malignant lymphocyte seen in the peripheral blood smear. There were studies mentioning atypical lymphocytes as an adjunct tool in the diagnosis of dengue infection and could be used as marker of disease severity. Methodology: This is a retrospective case control study of randomly selected pediatric patient admitted in a tertiary institution with confirmed dengue fever cases and other febrile illness (OFI). There were 296 who were able to meet the criteria. CBC results were reviewed on the day of admission and day 1 afebrile. Presence and absence of atypical lymphocytes was noted on each patient. Findings: Significantly more proportion of subjects with dengue illness has atypical lymphocyte than those with Other febrile illness (p<0.0001). Of the 155 confirmed cases of dengue, a total of 137 (88.4%) of patients have atypical lymphocyte and 18 (11.6%) found negative. The positive and negative predictive values of atypical lymphocytes were 86.2% and 86.9%, respectively. However no difference was noted when proportion of atypical lymphocyte was compared across dengue severity. Finally, atypical lymphocytes are a significant predictor of dengue fever as derived from logistic regression analysis. The results showed that the risk of a patient with atypical lymphocyte was 41.16 times higher for dengue than those without atypical lymphocyte. Conclusion & Significance: This study shows that the presence of atypical lymphocyte is highly associated with dengue illness. Atypical lymphocyte can be useful in predicting dengue illness. However additional study on the actual quantity of AL is required before the information can be used in usual clinical settings. Research Article Citation: Avegail Cardinal, Vincent Joseph Alba. Atypical Lymphocytes as a Predictor of Dengue Illness among Pediatric Patients Admitted In a Tertiary Institution. Microbiol Infect Dis. 2017; 1(1): 1-6.Background The oral cavity harbours a large number of bacterial species as normal flora existing as biofilm. Dental disease such as dental caries results when there is a shift in the balance of bacteria towards pathogenic species within these biofilms. Objective The objective of this study was to isolation, identification and characterisation of oral bacterial species of patients with dental caries and caries-free healthy control subjects. Materials and Methods A standard bacteriological procedures were followed in the isolation of bacteria. The identification of bacteria was carried out using matrix-associated laser desorption ionisation–time of flight–mass spectrometry (MALDI–TOF–MS) (Bruker MALDI Biotyper system). The characterisation of bacteria involved in the determination of biofilm forming potential and assessment of synergistic antimicrobial action of manuka honey and gentamicin against the oral species. Results A total of 13 bacterial species were isolated from 35 orals samples (10 from patients with dental caries); of which seven bacterial species have been isolated for the first time in Saudi Arabia. The Streptococcus spp. exhibited varied biofilm-forming potential and response to synergistic antimicrobial activity of manuka honey and gentamicin. Conclusion The isolation of seven bacterial species for the first time from dental caries and caries-free subjects in Saudi Arabia warrants a larger prevalence study involving molecular and phenotypic tests to assess their role in health and disease in Saudi population.E the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important feature in the design of universal influenza vaccines. In past decades, investigation of the ADCC epitopes on the highly immunogenic influenza hemagglutinin (HA) head region has been largely absent. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes (E1 and E2) on the HA-head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. Our data demonstrated that sera from the E1-vaccinated mice could induce high ADCC activities. While induction of ADCC response modestly decreased viral load in the lungs of H1N1-infected mice, the elevated ADCC significantly increased mouse alveolar damage and mortality than that of the PBS-vaccinated group. This phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an up-regulated release of cytotoxic granules were observed in the lung tissue of E1vaccinated mice after H1N1 influenza virus challenge. Our data suggested that ADCC elicited by certain domains of HA headregion might have a detrimental rather than protective effect during influenza virus infection. Meanwhile, we have successfully cloned the E1-specific monoclonal antibody and determined the human germline V(D)J combinations on this antibody. The resultant monoclonal antibodies expressed could be visualized on SDS-PAGE and detected in ELISA. With a series of optimization, the platform for cloning ADCC antibodies has been established.

Design and development of novel inhibitors for the treatment of latent tuberculosis

Objective/background: “The captain of all these men of death”, is the apt sobriquet for the age-old disease tuberculosis (TB). Despite the availability of many drugs, cases of increasing resistance in the forms of multi-drug and extensively drug-resistant TB and persistence [characteristic of Mycobacterium tuberculosis (MTB)] make the eradication of TB a nightmare. Approval of bedaquiline by the Food and Drug Administration focused attention on quinoline scaffolds for development of new anti-TB agents. Lysine ɛ-aminotransferase (LAT) in MTB plays a pivotal role in regulating amino acid synthesis, which in turn affects mycobacterial persistence. Here, developed quinoline inhibitors that targeted LAT with an objective to eliminate dormant forms of mycobacterium. Methods: Using e-pharmacophore approaches, quinolone (PBD: 2CJD) leads were found to inhibit lysine binding to LAT. To investigate structural activity relationships, 21 analogues were synthesized and characterized based on the identified lead molecules. Results: Among the derivatives, N-(pyridin-2-yl methyl)-2-(4-(quinolin-4-yl) piperazin-1-yl) acetamide was identified as a potent molecule, with an IC50 for LAT of 1.04 μM. In nutrient-starved and zebra fish models, this molecule exhibited logarithmic reductions of 2.1- and 2.2-fold, respectively, at a concentration of 10 μg/mL. The compound also exhibited good activity against persistent forms of mycobacteria (biofilm model), showing logarithmic reduction of 2.8-fold. Additionally, the hit molecule showed concentration-dependent kill kinetics against dormant forms of mycobacteria, and were devoid of cytotoxicity against RAW cell lines 264.7 at concentrations of 50 μM. Conclusion: Our results indicated that the hit molecule showed activity against both active and persistent forms of infection, which is ideal for new anti-TB agents. This molecule requires further pharmacokinetic and dynamic screening for development as new drug candidate.