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).

Anti-tubercular activities of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues endowed with high activity toward non-replicative Mycobacterium tuberculosis.

Thirty three derivatives of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues were synthesized by molecular modification of a reported antimycobacterial molecule (GSK163574A). Compounds were evaluated in vitro against actively replicative and nutrient starved non-replicative Mycobacterium tuberculosis (MTB), enzymatic screening and cytotoxicity against RAW 264.7 cell line. Among the compounds, 2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (5c) was found to be the most active compound against non-replicative MTB with 2.7 log reduction of bacteria at 10µg/mL and was more potent than isoniazid (1.2 log reduction) and rifampicin (2.0 log reduction) at same dose level. Compound 5c also showed activity against MTB alanine dehydrogenase enzyme with IC50 of 1.82±0.42µM and showed 25% cytotoxicity against RAW 264.7 cell line at 50µg/mL.

Design, synthesis and biological evaluation of imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives as Mycobacterium tuberculosis pantothenate synthetase inhibitors.

In the present study, we have designed imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives from earlier reported imidazo[1,2-a]pyridine based Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibitors. We synthesized thirty compounds and they were evaluated for MTB PS inhibition study, in vitro anti-TB activities against replicative and non-replicative MTB, in vivo activity using Mycobacterium marinum infected Zebra fish and cytotoxicity against RAW 264.7 cell line. Among them compound 2-methyl-N'-(4-phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bc) emerged as potent compound active against MTB PS with IC50 of 0.53±0.13 µM, MIC of 3.53 µM, 2.1 log reduction against nutrient starved MTB, with 33% cytotoxicity at 50 µM. It also showed 1.5 log reduction of M. marinum load in Zebra fish at 10mg/kg.

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.

Microgel mechanics in biomaterial design.

The field of polymeric biomaterials has received much attention in recent years due to its potential for enhancing the biocompatibility of systems and devices applied to drug delivery and tissue engineering. Such applications continually push the definition of biocompatibility from relatively straightforward issues such as cytotoxicity to significantly more complex processes such as reducing foreign body responses or even promoting/recapitulating natural body functions. Hydrogels and their colloidal analogues, microgels, have been and continue to be heavily investigated as viable materials for biological applications because they offer numerous, facile avenues in tailoring chemical and physical properties to approach biologically harmonious integration. Mechanical properties in particular are recently coming into focus as an important manner in which biological responses can be altered. In this Account, we trace how mechanical properties of microgels have moved into the spotlight of research efforts with the realization of their potential impact in biologically integrative systems. We discuss early experiments in our lab and in others focused on synthetic modulation of particle structure at a rudimentary level for fundamental drug delivery studies. These experiments elucidated that microgel mechanics are a consequence of polymer network distribution, which can be controlled by chemical composition or particle architecture. The degree of deformability designed into the microgel allows for a defined response to an imposed external force. We have studied deformation in packed colloidal phases and in translocation events through confined pores; in all circumstances, microgels exhibit impressive deformability in response to their environmental constraints. Microgels further translate their mechanical properties when assembled in films to the properties of the bulk material. In particular, microgel films have been a large focus in our lab as building blocks for self-healing materials. We have shown that their ability to heal after damage arises from polymer mobility during hydration. Furthermore, we have shown film mobility dictates cell adhesion and spreading in a manner that is fundamentally different from previous work on mechanotransduction. In total, we hope that this Account presents a broad introduction to microgel research that intersects polymer chemistry, physics, and regenerative medicine. We expect that research intersection will continue to expand as we fill the knowledge gaps associated with soft materials in biological milieu.

Ultrasoft, highly deformable microgels.

Microgels are colloidally stable, hydrogel microparticles that have previously been used in a range of (soft) material applications due to their tunable mechanical and chemical properties. Most commonly, thermo and pH-responsive poly(N-isopropylacrylamide) (pNIPAm) microgels can be fabricated by precipitation polymerization in the presence of the co-monomer acrylic acid (AAc). Traditionally pNIPAm microgels are synthesized in the presence of a crosslinking agent, such as N,N'-methylenebisacrylamide (BIS), however, microgels can also be synthesized under 'crosslinker free' conditions. The resulting particles have extremely low (<0.5%), core-localized crosslinking resulting from rare chain transfer reactions. AFM nanoindentation of these ultralow crosslinked (ULC) particles indicate that they are soft relative to crosslinked microgels, with a Young's modulus of ∼10 kPa. Furthermore, ULC microgels are highly deformable as indicated by a high degree of spreading on glass surfaces and the ability to translocate through nanopores significantly smaller than the hydrodynamic diameter of the particles. The size and charge of ULCs can be easily modulated by altering reaction conditions, such as temperature, monomer, surfactant and initiator concentrations, and through the addition of co-monomers. Microgels based on the widely utilized, biocompatible polymer polyethylene glycol (PEG) can also be synthesized under crosslinker free conditions. Due to their softness and deformability, ULC microgels are a unique base material for a wide variety of biomedical applications including biomaterials for drug delivery and regenerative medicine.

Enabling the (3 + 2) cycloaddition reaction in assembling newer anti-tubercular lead acting through the inhibition of the gyrase ATPase domain: lead optimization and structure activity profiling.

DNA gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis, is absent in humans and is a well validated target for anti-tubercular drug discovery. In this study, a moderately active inhibitor of Mycobacterium tuberculosis GyrB, the pharmaceutically unexploited domain of DNA gyrase, was reengineered using a combination of molecular docking and medicinal chemistry strategies to obtain a lead series displaying considerable in vitro enzyme efficacy and bacterial kill against the Mycobacterium tuberculosis H37Rv strain. Biophysical investigations using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain. Furthermore, the molecules were completely devoid of hERG toxicity up to 30 µM, as evaluated in a zebra fish model with a good selectivity index, and from a pharmaceutical point of view, turned out as potential candidates against TB.

Development of 2-amino-5-phenylthiophene-3-carboxamide derivatives as novel inhibitors of Mycobacterium tuberculosis DNA GyrB domain.

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis (Mtb), unlike other bacteria and its absence in human being makes it a clinically validated target for developing anti-tubercular leads against Mtb. In the present study, our effort was to optimize and synthesize a series of compounds by a combination of molecular docking, and synthetic chemistry approach for better activity. A series of twenty eight substituted 2-amino-5-phenylthiophene-3-carboxamide derivatives were designed based on our earlier reported Mtb GyrB inhibitor lead. Hit expansion of the previously identified lead by chemical synthesis led to improved inhibitor with an IC50 value of 0.86±0.81µM against Mtb DNA gyrase supercoiling and Mycobacterium smegmatis GyrB IC50 of 1.35±0.58µM. Further a biophysical investigation using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain.

Design and synthesis of novel quinoline-aminopiperidine hybrid analogues as Mycobacterium tuberculosis DNA gyraseB inhibitors.

Antibiotics with good therapeutic value and novel mechanism of action are becoming increasingly important in today's battle against bacterial resistance. One of the popular targets being DNA gyrase, is currently becoming well-established and clinically validated for the development of novel antibacterials. In the present work, a series of forty eight quinoline-aminopiperidine based urea and thiourea derivatives were synthesized as pharmacophoric hybrids and evaluated for their biological activity. Compound, 1-(4-chlorophenyl)-3-(1-(6-methoxy-2-methylquinolin-4-yl)piperidin-4-yl)thiourea (45) was found to exhibit promising in vitro Mycobacterium smegmatis GyrB IC50 of 0.95 ± 0.12 µM and a well correlated Mycobacterium tuberculosis (MTB) DNA gyrase supercoiling IC50 of 0.62 ± 0.16 µM. Further, compound 45 also exhibited commendable MTB MIC, safe eukaryotic cytotoxic profile with no signs of cardiotoxicity in zebrafish ether-a-go-go-related gene (zERG).

Microgel film dynamics modulate cell adhesion behavior.

A material's mechanical properties greatly control cell behavior at the cell­substrate interface. In this work, we demonstrate that microgel multilayers have unique elastic and viscoelastic-like properties that can be modulated to produce morphological changes in fibroblasts cultured on the film. Protein adsorption is also examined and the data are contrasted with the number of cells adhered. The dynamic interaction of cell and substrate is only partially explained by conventional understanding of surface­receptor interactions and substrate elasticity. Viscoelasticity, a mechanical property not often considered, plays a significant role at cellular length and time scales for microgel films.

Development of novel tetrahydrothieno[2,3-c]pyridine-3-carboxamide based Mycobacterium tuberculosis pantothenate synthetase inhibitors: molecular hybridization from known antimycobacterial leads.

Twenty six 2,6-disubstituted 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives were designed by molecular hybridization approach using and synthesized from piperidin-4-one by five step synthesis. Compounds were evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among the compounds, 6-(4-nitrophenylsulfonyl)-2-(5-nitrothiophene-2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (11) was found to be the most active compound with IC50 of 5.87 ± 0.12 µM against MTB PS, inhibited MTB with MIC of 9.28 µM and it was non-cytotoxic at 50 µM. The binding affinity of the most potent inhibitor 11 was further confirmed biophysically through differential scanning fluorimetry.

Identification and development of 2-methylimidazo[1,2-a]pyridine-3-carboxamides as Mycobacterium tuberculosis pantothenate synthetase inhibitors.

In the present study, we used crystal structure of mycobacterial pantothenate synthetase (PS) bound with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid inhibitor for virtual screening of antitubercular compound database to identify new scaffolds. One of the identified lead was modified synthetically to obtain thirty novel analogues. These synthesized compounds were evaluated for Mycobacterium tuberculosis (MTB) PS inhibition study, in vitro antimycobacterial activities and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, N'-(1-naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b) was found to be the most active compound with IC50 of 1.90 ± 0.12 µM against MTB PS, MIC of 4.53 µM against MTB with no cytotoxicity at 50 µM. The binding affinity of the most potent inhibitor 5b was further confirmed biophysically through differential scanning fluorimetry.

Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of Mycobacterium tuberculosis InhA.

A series of twenty seven substituted 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives were designed based on our earlier reported Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein reductase (InhA) lead. Compounds were evaluated for MTB InhA inhibition study, in vitro activity against drug-sensitive and -resistant MTB strains, and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30) was found to be the most promising compound with IC50 of 5.12 ± 0.44 µM against MTB InhA, inhibited drug sensitive MTB with MIC 17.11 µM and was non-cytotoxic at 100 µM. The interaction with protein and enhancement of protein stability in complex with compound 30 was further confirmed biophysically by differential scanning fluorimetry.

Salicylanilide diethyl phosphates as potential inhibitors of some mycobacterial enzymes.

Antimycobacterially active salicylanilide diethyl phosphates were evaluated to identify their potential drug target(s) for the inhibition of several mycobacterial enzymes, including isocitrate lyase, L-alanine dehydrogenase (MtAlaDH), lysine ε-aminotransferase, chorismate mutase, and pantothenate synthetase. The enzymes are related to the nongrowing state of Mycobacterium tuberculosis. Salicylanilide diethyl phosphates represent new candidates with significant inhibitory activity especially against L-alanine dehydrogenase. The most active MtAlaDH inhibitor, 5-chloro-2-[(3-chlorophenyl)carbamoyl]phenyl diethyl phosphate, has an IC50 of 4.96 µM and the best docking results. Other mycobacterial enzymes were mostly inhibited by some derivatives but at higher concentrations; isocitrate lyase showed the highest resistance to salicylanilide diethyl phosphates.

Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening.

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure-activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC(50) of 17.7 µM for purified CysK1 and MIC of 7.6 µM for M. tuberculosis, with little or no cytotoxicity (>50 µM).

Homology modeling, docking and structure-based virtual screening for new inhibitor identification of Klebsiella pneumoniae heptosyltransferase-III.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative opportunistic pathogen commonly associated with hospital-acquired infections that are often resistant even to antibiotics. Heptosyltransferase (HEP) belongs to the family of glycosyltransferase-B (GT-B) and plays an important in the synthesis of lipopolysaccharides (LPS) essential for the formation of bacterial cell membrane. HEP-III participates in the transfer of heptose sugar to the outer surface of bacteria to synthesize LPS. LPS truncation increases the bacterial sensitivity to hydrophobic antibiotics and detergents, making the HEP as a novel drug target. In the present study, we report the 3D homology model of K. pneumoniae HEP-III and its structure validation. Active site was identified based on similarities with known structures using Dali server, and structure-based pharmacophore model was developed for the active site substrate ADP. The generated pharmacophore model was used as a 3D search query for virtual screening of the ASINEX database. The hit compounds were further filtered based on fit value, molecular docking, docking scores, molecular dynamics (MD) simulations of HEP-III complexed with hit molecules, followed by binding free energy calculations using Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA). The insights obtained in this work provide the rationale for design of novel inhibitors targeting K. pneumoniae HEP-III and the mechanistic aspects of their binding. Communicated by Ramaswamy H. Sarma.

A Sizeable Solitary Pedunculated Peripheral Osteoma of the Hard Palate: A Case Report.

Osteomas are benign, slow growing, usually sessile osteogenic tumors of unknown etiology. They arise from proliferation of either cancellous or compact bone. They can be central, peripheral or extra-skeletal based on their origin from endosteum, periosteum or extra-skeletal soft tissue respectively. They are commonly found in the skull and facial bones, the most common site in the maxillofacial region being paranasal sinuses. Among the jaw bones the most common site is the mandible. The hard palate is a rare site for such osteomas with a very few cases reported in literature. We report a case of large pedunculated peripheral osteoma of the hard palate in a 38 year old male. The tumor was excised with no recurrence after 3 years followup.

Multiple e-pharmacophore modelling pooled with high-throughput virtual screening, docking and molecular dynamics simulations to discover potential inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH).

Development of new antimalarial drugs continues to be of huge importance because of the resistance of malarial parasite towards currently used drugs. Due to the reliance of parasite on glycolysis for energy generation, glycolytic enzymes have played important role as potential targets for the development of new drugs. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a key enzyme for energy generation of malarial parasites and is considered to be a potential antimalarial target. Presently, there are nearly 15 crystal structures bound with inhibitors and substrate that are available in the protein data bank (PDB). In the present work, we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 1.4 × 102-1.3 × 106 nM efficacy and optimized the pharmacophore based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput virtual screening (HTVS) combined with molecular docking, ADME predictions and molecular dynamics simulation led to the identification of 20 potential compounds which could be further developed as novel inhibitors for PfLDH.

Inhibitor binding studies of Mycobacterium tuberculosis MraY (Rv21 56c): Insights from molecular modeling, docking, and simulation studies.

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M.tb) or tubercule bacillus, and H37Rv is the most studied clinical strain. The recent development of resistance to existing drugs is a global health-care challenge to control and cure TB. Hence, there is a critical need to discover new drug targets in M.tb. The members of peptidoglycan biosynthesis pathway are attractive target proteins for antibacterial drug development. We have performed in silico analysis of M.tb MraY (Rv2156c) integral membrane protein and constructed the three-dimensional (3D) structure model of M.tb MraY based on homology modeling method. The validated model was complexed with antibiotic muraymycin D2 (MD2) and was used to generate structure-based pharmacophore model (e-pharmacophore). High-throughput virtual screening (HTVS) of Asinex database and molecular docking of hits was performed to identify the potential inhibitors based on their mode of interactions with the key residues involved in M.tb MraY-MD2 binding. The validation of these molecules was performed using molecular dynamics (MD) simulations for two best identified hit molecules complexed with M.tb MraY in the lipid bilayer, dipalmitoylphosphatidyl-choline (DPPC) membrane. The results indicated the stability of the complexes formed and retained non-bonding interactions similar to MD2. These findings may help in the design of new inhibitors to M.tb MraY involved in peptidoglycan biosynthesis.

Discovery of novel inhibitors of Mycobacterium tuberculosis MurG: homology modelling, structure based pharmacophore, molecular docking, and molecular dynamics simulations.

MurG (Rv2153c) is a key player in the biosynthesis of the peptidoglycan layer in Mycobacterium tuberculosis (Mtb). This work is an attempt to highlight the structural and functional relationship of Mtb MurG, the three-dimensional (3D) structure of protein was constructed by homology modelling using Discovery Studio 3.5 software. The quality and consistency of generated model was assessed by PROCHECK, ProSA and ERRAT. Later, the model was optimized by molecular dynamics (MD) simulations and the optimized model complex with substrate Uridine-diphosphate-N-acetylglucosamine (UD1) facilitated us to employ structure-based virtual screening approach to obtain new hits from Asinex database using energy-optimized pharmacophore modelling (e-pharmacophore). The pharmacophore model was validated using enrichment calculations, and finally, validated model was employed for high-throughput virtual screening and molecular docking to identify novel Mtb MurG inhibitors. This study led to the identification of 10 potential compounds with good fitness, docking score, which make important interactions with the protein active site. The 25 ns MD simulations of three potential lead compounds with protein confirmed that the structure was stable and make several non-bonding interactions with amino acids, such as Leu290, Met310 and Asn167. Hence, we concluded that the identified compounds may act as new leads for the design of Mtb MurG inhibitors.