Suresh Kumar Chitta

Comprehensive structural and functional characterization of Mycobacterium tuberculosis UDP-NAG enolpyruvyl transferase (Mtb-MurA) and prediction of its accurate binding affinities with inhibitors

Tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death in the world. One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), the etiologic agent of TB. The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. In this work, 3D structure model of Mtb-MurA enzyme has been developed for the first time by homology modeling and molecular dynamics simulation techniques. Multiple sequence alignment and 3D structure model provided the putative substrate binding pocket of Mtb-MurA with respect to E. coli MurA. This analysis was helpful in identifying the binding sites and molecular function of the MurA homologue. Molecular docking study was performed on this 3D structure model, using different classes of inhibitors like fosfomycin, cyclic disulfide analog RWJ-3981, pyrazolopyrimidine analog RWJ-110192, purine analog RWJ-140998, 5-sulfonoxy-anthranilic acid derivatives T6361, T6362 and the results showed that the 5-sulfonoxyanthranilic acid derivatives showed the best interaction compared to other inhibitors. We also designed new efficient analogs of T6361 and T6362 which showed even better interaction with Mtb-MurA than the parental 5-sulfonoxy-anthranilic acid derivatives. Further the comparative molecular electrostatic potential and cavity depth analysis of Mtb-MurA suggested several important differences in its substrate and inhibitor binding pocket. Such differences could be exploited in the future for designing a more specific inhibitor for Mtb-MurA enzyme.

Molecular Modeling and Docking Studies of O-Succinylbenzoate Synthase of M. tuberculosis—a Potential Target for Antituberculosis Drug Design

Menaquinone is a lipid-soluble naphthoquinone that is essential for various pivotal functions of bacteria. Naphthoquinone is synthesized from chorismate of the shikimate pathway in microorganisms. Due to its absence in humans and animals, menaquinone biosynthesis has been an attractive target for development of antibiotics against a number of important microbial pathogens, such as Mycobacterium tuberculosis (Mtb). In shikimate pathway, O-succinylbenzoate synthase (OSBS) plays a major role and is one of the major potential drug targets. For Mtb-OSBS, a systematic study was conducted to get an insight about Mtb-OSBS enzyme and the corresponding inhibitors using in silico methods. The 3-D model of Mtb-OSBS was built using structure coordinates of Thermobifida fusca. O-succinylbenzoate synthase, the model, was further refined. The active site amino acids have been identified by comparing the template sequence with the Mtb-OSBS sequence. We identified that Lys108, Asn140, Asp138, Lys110, Glu189, Ser236, Asp188, Arg27, Tyr52, and Ser237 are highly conserved, and these may play a vital role as active residues, similar to that in template protein. As per the competitive binding of substrate (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC)), we screened the SHCHC through AutoDock 4.0. The SHCHC molecule was further modified structurally and optimized through PRODRG server. Docking of the 12 lead molecules for best interactions with Mtb-OSBS has given an insight that all the lead molecules have shown interactions with active site amino acids of Mtb-OSBS. MD simulation analysis report has shown the stable conformation annotations of Mtb-OSBS. These hypothetical studies create another way to develop more potential drugs against the deadly mycobacterium.

Multi-spectroscopic and molecular docking studies on the interaction of new phthalimides with calf-thymus DNA: In vitro free radical scavenging activities

ABSTRACT In this study, we report the synthesis and biological evaluation of novel phthalimide based Schiff base derivatives as promising antioxidant and DNA-binding agents. The structural investigation of the synthesized compounds was determined by spectral and elemental analysis. In vitro DNA-binding studies of title compounds were carried out by UV–Vis, fluorescence, circular dichroism spectroscopic techniques, cyclic voltammetry, thermal denaturation studies, and hydrodynamic measurements to investigate their potential as DNA-binding agents. The DNA binding constant (Kb) of target compounds was obtained from absorption studies between 1.2 × 105 M−1 and 1.27 × 105 M−1, respectively, suggesting that the test compounds have shown good affinity toward calf-thymus DNA. The experimental results of DNA-binding studies reveal a non-intercalative mode of binding between DNA and the synthesized compounds, most probably groove binding. In addition, molecular docking techniques were performed to rationalize the observed binding affinities with the target DNA. Furthermore, antioxidant and free radical scavenging activities of the synthesized compounds were carried out to find out their pharmacological potential. The results indicate that the title compounds displayed good antioxidant activity against DPPH (IC50: 0.727 and 0.656 mg/mL) and H2O2 radicals (IC50: 1.072 and 0.911 mg/mL) comparable to standard ascorbic acid.

A transcriptional repressive role for epithelial-specific ETS factor ELF3 on estrogen receptor-alpha in breast cancer cells

Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERαs DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα and ELF3 could inhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy.

Design and evaluation of new chemotherapeutics of aloe-emodin (AE) against the deadly cancer disease: an in silico study

The Bcl-2 family proteins include pro- and antiapoptotic factors acting as critical arbiters of apoptotic cell death decisions in most circumstances. Evasion of apoptosis is one of the hallmarks of cancer, relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins was observed in many cancers. Since Bax-mediated induction of apoptosis is a crucial mechanism in cancerous cells, we aimed at conducting in silico analysis on Bax in order to predict the possible interactions for anticancer agents. The present report depicts the binding mode of aloe-emodin and its structurally modified derivatives onto Bax. The structural information about the binding site of Bax for docked compounds obtained from this study could aid in screening and designing new anticancer agents or selective inhibitors for chemotherapy against Bax.

Experimental and molecular docking investigation on DNA interaction of N-substituted phthalimides: antibacterial, antioxidant and hemolytic activities.

A series of Schiff base molecules derived from a phthalimide scaffold was investigated as efficient antibacterial, antioxidant and DNA-interacting agents. The spectroscopic characterization of these derivatives was studied in detail using elemental analysis and spectroscopic techniques. The DNA-binding profile of title molecules against Ct-DNA (calf thymus) was investigated by absorbance, fluorescence, hydrodynamics and thermal denaturation investigations. The bacterial inhibition potential of these molecules was investigated against Escherichia coli and Staphylococcus aureus. Molecule 3c emerged as the most active against S. aureus (IC50 : 14.8 μg/mL), whereas compounds 3a and 3b displayed potential antibacterial activities against E. coli (IC50 : 49.7 and 67.6 μg/mL). Molecular docking studies of these compounds against GlcN-6-P synthase were carried out to rationalize antibacterial efficiency of these molecules. These newly synthesized molecules were screened for their scavenging capacity against 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and H2 O2 free radicals and the results were compared with ascorbic acid as synthetic antioxidant. The title molecules 3a, 3b and 3e showed less than 20% hemolysis, which indicated their significant non-toxic behavior.

Oestrogen receptor negativity in breast cancer: a cause or consequence?

Endocrine resistance, which occurs either by de novo or acquired route, is posing a major challenge in treating hormone-dependent breast cancers by endocrine therapies. The loss of oestrogen receptor α (ERα) expression is the vital cause of establishing endocrine resistance in this subtype. Understanding the mechanisms that determine the causes of this phenomenon are therefore essential to reduce the disease efficacy. But how we negate oestrogen receptor (ER) negativity and endocrine resistance in breast cancer is questionable. To answer that, two important approaches are considered: (1) understanding the cellular origin of heterogeneity and ER negativity in breast cancers and (2) characterization of molecular regulators of endocrine resistance. Breast tumours are heterogeneous in nature, having distinct molecular, cellular, histological and clinical behaviour. Recent advancements in perception of the heterogeneity of breast cancer revealed that the origin of a particular mammary tumour phenotype depends on the interactions between the cell of origin and driver genetic hits. On the other hand, histone deacetylases (HDACs), DNA methyltransferases (DNMTs), miRNAs and ubiquitin ligases emerged as vital molecular regulators of ER negativity in breast cancers. Restoring response to endocrine therapy through re-expression of ERα by modulating the expression of these molecular regulators is therefore considered as a relevant concept that can be implemented in treating ER-negative breast cancers. In this review, we will thoroughly discuss the underlying mechanisms for the loss of ERα expression and provide the future prospects for implementing the strategies to negate ER negativity in breast cancers.

Inhibition of Mycobacterium-RmlA by Molecular Modeling, Dynamics Simulation, and Docking

The increasing resistance to anti-tb drugs has enforced strategies for finding new drug targets against Mycobacterium tuberculosis (Mtb). In recent years enzymes associated with the rhamnose pathway in Mtb have attracted attention as drug targets. The present work is on α-D-glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme involved in the biosynthesis of L-rhamnose, of Mtb cell wall. This study aims to derive a 3D structure of RmlA by using a comparative modeling approach. Structural refinement and energy minimization of the built model have been done with molecular dynamics. The reliability assessment of the built model was carried out with various protein checking tools such as Procheck, Whatif, ProsA, Errat, and Verify 3D. The obtained model investigates the relation between the structure and function. Molecular docking interactions of Mtb-RmlA with modified EMB (ethambutol) ligands and natural substrate have revealed specific key residues Arg13, Lys23, Asn109, and Thr223 which play an important role in ligand binding and selection. Compared to all EMB ligands, EMB-1 has shown better interaction with Mtb-RmlA model. The information thus discussed above will be useful for the rational design of safe and effective inhibitors specific to RmlA enzyme pertaining to the treatment of tuberculosis.

Synthesis and Biological Activity of New Resveratrol Derivative and Molecular Docking: Dynamics Studies on NFkB

Resveratrol (RVS) is a naturally occurring antioxidant, able to display an array of biological activities. In the present investigation, a new derivative of RVS, RVS(a), was synthesized, and its biological activity was determined on U937 cells. It was observed that RVS(a) showed pronounced activity on U937 cells than RVS. RVS(a) is able to induce apoptosis in tumor cell lines through subsequent DNA fragmentation. From the EMSA results, it was evident that RVS(a) was able to suppress the activity of NFkB by interfering its DNA binding ability. Furthermore, the molecular interaction analysis (docking and dynamics) stated that RVS(a) has strong association with the IkB-alpha site of NFkB compared with RVS; this binding nature of RVS(a) might be prevent the NFkB binding ability with DNA. The present findings represent the potential activity of propynyl RVS on U937 cells and signifying it as a one of putative chemotherapeutic drugs against cancer.

Insights from Streptococcus pneumoniae glucose kinase structural model.

Streptococcus pneumonia is the common cause of sepsis and meningitis. Emergence of multiple antibiotic resistant strains in the community‐acquired bacterium is catastrophic. Glucose kinase (GLK) is a regulatory enzyme capable of adding phosphate group to glucose in the first step of streptomycin biosynthesis. The activity of glucose kinase was regulated by the Carbon Catabolite Repression (CCR) system. Therefore, it is important to establish the structure‐function relation of GLK in S. pneumoniae. However, a solved structure for S. pneumoniae GLK is not available at the protein data bank (PDB). Therefore, we created a model of GLK from S. pnemoniae using the X‐ray structure of Glk from E. faecalis as template with MODELLER (a comparative modeling program). The model was validated using protein structure checking tools such as PROCHECK, WHAT IF and ProSA for reliability. The active site amino acid Asp114 in the template is retained in S. pneumoniae GLK model (Asp115). Solvent accessible surface area (ASA) analysis of the GLK model showed that known key residues playing important role in active site for ligand binding and metal ion binding are buried and hence not accessible to solvent. The information thus discussed provides insight to the molecular understanding of glucose kinase in S. pneumoniae.