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StatementThe authors have withdrawn their manuscript owing to the authors decision of withdrawing of the manuscript until all experiments are completed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

IntracellularSalmonellaresides and multiplies in cholesterol-rich specialized compartment calledSalmonella-containing vacuoles (SCVs) and avoids fusion with acidic lysosomes. Given, lysosomes are primary organelle that redistributes LDL derived cholesterol to other organelles; we questioned how lysosomal cholesterol can be transported to SCV. We demonstrate here that peroxisomes are recruited to SCVs in human primary macrophages, epithelial cells and functions as pro-bacterial organelles. Further, this interaction is assisted by SseI, aSalmonellaeffector protein containing mammalian peroxisome targeting sequence. SseI localizes to peroxisome, interacts and activates a host Ras GTPase, ARF-1 on the peroxisome membrane. Activation of ARF-1 leads to recruitment of phosphatidylinsolitol-5- phosphate-4 kinase to generate phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. Accordingly, theΔsseIstrain showed reduced virulence in cell lines and during mice infection. Taken together, our work identified a fascinating mechanism by which a pathogen targets host organelles via its secretory effectors and exploits host metabolic intermediates for its intracellular proliferation.

Recent reports suggest that persistent Epstein-Barr virus (EBV) infection and its recurrent reactivation could instigate the formation of proteinaceous plaques in the brain: a hallmark of Alzheimer's disease (AD). Interestingly, a major genetic risk factor of AD, the apolipoprotein E (ApoE), could also influence the outcome of EBV infection in an individual. The ApoE is believed to influence the proteinaceous plaque clearance from the brain, and its defective functioning could result in the aggregate deposition. The persistent presence of EBV infection in a genetically predisposed individual could create a perfect recipe for severe neurodegenerative consequences. Therefore, in the present study, we investigated the possible interactions between ApoE and various EBV proteins using computational tools. Our results showed possibly stable de-novo interactions between the C-terminal domain of ApoE3 and EBV proteins: EBV nuclear antigen-1 (EBNA1) and BamHI Z fragment leftward open reading frame-1 (BZLF1). The EBNA1 protein of EBV plays a crucial role in establishing latency and replication of the virus. Whereas BZLF1 is involved in the lytic replication cycle. The proposed interaction of EBV proteins at the ligand-binding site of ApoE3 on CTD could interfere with- its capability to sequester amyloid fragments and, hence their clearance from the brain giving rise to AD pathology. This study provides a new outlook on EBV's underexplored role in AD development and paves the way for novel avenues of investigation which could further our understanding of AD pathogenesis.Communicated by Ramaswamy H. Sarma[Figure: see text].

The molecular hybridization concept led us to design a series of galloyl conjugates of flavanones that have potent osteoblast differentiation ability in vitro and promote bone formation in vivo. An array of in vitro studies, especially gene expression of osteogenic markers, evinced compound 5e as the most potent bone anabolic agent, found to be active at 1 pM, which was then further assessed for its osteogenic potential in vivo. From in vivo studies on rat calvaria and a fracture defect model, we inferred that compound 5e, at an oral dose of 5 mg/(kg day), increased the expression of osteogenic genes (RUNX2, BMP-2, Col1, and OCN) and the bone formation rate and significantly promoted bone regeneration at the fracture site, as evidenced by the increased bone volume/tissue fraction compared with vehicle-treated rats. Furthermore, structure-activity relationship studies and pharmacokinetic studies suggest 5e as a potential bone anabolic lead for future osteoporosis drug development.

Neurological disorders affect various aspects of life. Finding drugs for the central nervous system is a very challenging and complex task due to the involvement of the blood–brain barrier, P-glycoprotein, and the drug’s high attrition rates. The availability of big data present in online databases and resources has enabled the emergence of artificial intelligence techniques including machine learning to analyze, process the data, and predict the unknown data with high efficiency. The use of these modern techniques has revolutionized the whole drug development paradigm, with an unprecedented acceleration in the central nervous system drug discovery programs. Also, the new deep learning architectures proposed in many recent works have given a better understanding of how artificial intelligence can tackle big complex problems that arose due to central nervous system disorders. Therefore, the present review provides comprehensive and up-to-date information on machine learning/artificial intelligence-triggered effort in the brain care domain. In addition, a brief overview is presented on machine learning algorithms and their uses in structure-based drug design, ligand-based drug design, ADMET prediction, de novo drug design, and drug repurposing. Lastly, we conclude by discussing the major challenges and limitations posed and how they can be tackled in the future by using these modern machine learning/artificial intelligence approaches.

Nicotinamide N-methyltransferase (NNMT) is a protein coding gene, which methylates the nicotinamide (NA) (vitamin B3) to produce 1-methylnicotinamide (MNA). Several studies have suggested that the overexpression of NNMT is associated with different metabolic disorders like obesity and type-2 diabetes thereby making it an important therapeutic target for development of anti-diabetic agents. Here we describe a workflow for identification of new inhibitors of NNMT from a library of small molecules. In this study, we have hypothesized a four-point pharmacophore model based on the pharmacophoric features of reported NNMT inhibitors in the literature. The statistically significant pharmacophore hypothesis was used to explore the Maybridge compound library that resulted in mapping of 1330 hit compounds on the proposed hypothesis. Subsequently, a total of eight high scoring compounds, showing good protein-ligand interactions in the molecular docking study, were selected for biological evaluation of NNMT activity. Eventually, four compounds were found to show significant inhibitory activity for NNMT and can be further explored to design new derivatives around the identified scaffolds with improved activities as NNMT inhibitors.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged first around December 2019 in the city of Wuhan, China. Since then, several variants of the virus have emerged with different biological properties. This pandemic has so far led to widespread infection cycles with millions of fatalities and infections globally. In the recent cycle, a new variant omicron and its three sub-variants BA.1, BA.2 and BA.3 have emerged which seems to evade host immune defences and have brisk infection rate. Particularly, BA.2 variant has shown high transmission rate over BA.1 strain in different countries including India. In the present study, we have evaluated a set of eighty drugs/compounds using in silico docking calculations in omicron and its variants. These molecules were reported previously against SARS-CoV-2. Our docking and simulation analyses suggest differences in affinity of these compounds in omicron and BA.2 compared to SARS-CoV-2. These studies show that neohesperidin, a natural flavonoid found in Citrus aurantium makes a stable interaction with spike receptor domain of omicron and BA.2 compared to other variants. Free energy binding analyses further validates that neohesperidin forms a stable complex with spike RBD in omicron and BA.2 with a binding energy of -237.9 ± 18.7 kJ/mol and -164.1 ± 17.5 kJ/mol respectively. Key residual differences in the RBD interface of these variants form the basis for differential interaction affinities with neohesperidin as drug binding site overlaps with RBD-human ACE2 interface. These data might be useful for the design and development of novel scaffolds and pharmacophores to develop specific therapeutic strategies against these novel variants.

Acetyl Coenzyme A Carboxylase (AccD6) is a homodimeric protein which is involved in the carboxylation of acetyl coenzyme A to produce malonyl coenzyme A, which plays an important role in the biosyn...

NAD synthetase (NadE) catalyzes the last step in NAD biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction with co-substrates ATP and amide donor ammonia. In this study, we report the crystal structure of Staphylococcus aureus NAD synthetase enzyme (saNadE) at 2.3 A resolution. We used this structure to perform molecular dynamics simulations of apo-enzyme, enzyme-substrate (NadE with ATP and NaAD) and enzyme-intermediate complexes (NadE with NaAD-AMP) to investigate key binding interactions and explore the conformational transitions and flexibility of the binding pocket. Our results show large shift of N-terminal region in substrate bound form which is important for ATP binding. Substrates drive the correlated movement of loop regions surrounding it as well as some regions distal to the active site and stabilize them at complex state. Principal component analysis of atomic projections distinguish feasible trajectories to delineate distinct motions in enzyme-substrate to enzyme-intermediate states. Our results suggest mixed binding involving dominant induced fit and conformational selection. MD simulation extracted ensembles of NadE could potentially be utilized for in silico screening and structure based design of more effective Methicillin Resistant Staphylococcus aureus (MRSA) inhibitors.

Human DNA ligase (hLigI) plays an important role in the process of DNA replication and repair, making it an important target for cancer. In our tireless efforts for finding selective inhibitors against hLigI, this article reports the design, synthesis and biological activity of a series of 71 curcumin-monocarbonyl-dithiocarbamate hybrid molecules. Many of the synthetic compounds tested in this study show a significant inhibitory effect on the human DNA ligase 1 (hLig1) activity (> 90% inhibition) and demonstrated significant anti-proliferative activity against colon cancer cells (DLD-1) in a concentration dependent manner. In addition, at a concentration of 10 µM, certain compounds showed selective activity towards DLD-1 cells as compared to the normal HEK-293 cells. Overall, our studies demonstrate that two compounds in this series (27 and 49) have significantly better DNA ligase 1 inhibition and anti-proliferative activities than the previously reported monocarbonyl-curcumin hybrid (compound 23*). This study brings us closer towards our quest to explore the molecular space for novel hLig1 inhibitors and to find inhibitors that will be suitable for clinical trials in cancer patients.

New chemical scaffolds with novel mechanism of action are urgently needed for the treatment of drug resistant tuberculosis. The oxidative phosphorylation pathway of Mycobacterium tuberculosis consists of multiple clinically validated drug targets. This pathway can function through any one of the two terminal oxidases-the proton pumping cytochrome bc

Insulin-signalling is an important pathway in multiple cellular functions and organismal ageing across the taxa. A strong association of insulin-signalling with Parkinson’s disease (PD) has been proposed but the exact nature of molecular events and genetic associations are yet to be understood. We employed transgenic C. elegans strain harboring human α-synuclein::YFP transgene, towards studying the aggregation pattern of α-synuclein, a PD-associated endpoint, under human insulin (Huminsulin®) treatment and DAF-16/DAF-2 knockdown conditions, independently and in combination. The aggregation was increased when DAF-16 was knocked-down independently or alongwith a co-treatment of Human insulin (HumINS) and decreased when DAF-2 was knocked-down independently or alongwith a co-treatment of HumINS; whereas HumINS treatment per se, reduced the aggregation. Our results depicted that HumINS decreases α-synuclein aggregation via DAF-2/DAF-16 pathway by acting as an antagonist for DAF-2 receptor. Knockdown of reported DAF-2 agonist (INS-6) and antagonists (INS-17 and INS-18) also resulted in a similar effect on α-synuclein aggregation. Further by utilizing bioinformatics tools, we compared the differences between the binding sites of probable agonists and antagonists on DAF-2 including HumINS. Our results suggest that HumINS treatment and DAF-16 expression play a protective role against α-synuclein aggregation and its associated effects.

Trypanothione reductase of Leishmania donovani is a flavin adenine dinucleotide containing homodimeric protein essential for parasite survival. The flavoenzyme utilizes nicotinamide adenine dinucleotide phosphate in the reaction to convert oxidized trypanothione to reduced trypanothione which is further used up by tryparedoxin/tryparedoxin peroxidase system to neutralize the reactive oxygen species generated by the macrophages. Some of the drugs previously reported against the disease include sodium stibogluconate, miltefosine and amphotericin B. However, due to the resistance and toxicity problem associated with these molecules, there is an urgent need to develop new drugs against L. donovani. Trypanothione reductase of L. donovani is one such essential target whose inhibition could lead to a decline in parasite growth. In this work, we have performed a computational studies using Maybridge library of chemical compounds to identify potential inhibitors of Trypanothione reductase of L. donovani. Structure-based virtual screening method in combination with molecular docking was employed to identify and prioritize 30 compounds which were further subjected to molecular dynamics simulation. Ten compounds which showed stable ligand root-mean-square deviation plot, c-alpha backbone and root-mean-square fluctuation were considered for trypanothione reductase inhibition assay and subsequent inhibition studies of parasite growth. Enzyme inhibition assay resulted in shortlisting of four compounds that were found to inhibit Trypanothione reductase of L. donovani. Subsequently, the anti-leishmanial screening highlighted one compound as the potential anti-leishmanial agent, with IC50 value of 15.2 µM, that can be further optimised with medicinal chemistry efforts to improve its activity. Communicated by Ramaswamy H. Sarma

Matrix Metalloproteinase-13 (MMP-13) is a collagenase that regulates the homeostasis of the extracellular matrix (ECM) and basement membrane, as well as the breakdown of type II collagen. Recent research studies on the molecular and cellular mechanisms of cartilage degradation suggest that MMP-13 overexpression triggers osteoarthritis and is considered a promising target for osteoarthritis treatment. The present work employs machine learning-based virtual screening and structure-based rational drug design approaches to identify potential inhibitors of MMP-13 with diverse chemical scaffolds. The twelve top-scoring screened compounds were subjected to biological evaluation to validate the robustness and predictive modeling of ML-based Virtual Screening. It was observed that eight compounds exhibited approximately 44%–60% inhibition at 0.1 µM concentration, and the IC50 lies in the range of 1.9–2.3 µM against MMP-13. Interestingly, two of the compounds, DP01473 and RH01617, showed potent dose-dependent inhibitory activity. Compound DP01473 inhibited MMP-13 by 44%, 50%, and 70%, while compound RH01617 inhibited MMP-13 by 54%, 55%, and 57% at 0.1 μM, 1 μM, and 10 μM concentrations, respectively, and can be further optimized for the design and development of more potent MMP-13 inhibitors. Communicated by Ramaswamy H. Sarma

Over the last few decades, anticancer peptides (ACPs) have turned into potential warheads against cancer. Apart from small molecules and monoclonal antibodies, ACPs have been proven to be effective against cancer cells. ACPs are small cationic peptides that selectively bind to the negatively charged cancer cell membrane and kill them by various mechanisms. In the present study, we prepared a random scrambled library of 1200 peptides from the 100 known ACPs and virtually screened them for their anticancer properties. From in silico-predicted ACPs, 27 peptides were prioritized based on their support vector machine (SVM) score. Based on the SVM score and properties such as hydrophobicity, size, overall net charge, secondary structure, and synthetic feasibility, finally, four peptides were synthesized and screened for their biological activities. Cancer cell membrane-deforming potential of two most active peptides, peptide1 and peptide2 was assessed with molecular dynamics simulation. We found that peptide1 remains adsorbed to the membrane surface, while peptide2 has membrane penetration capability. The present study will be helpful in the computational design of ACPs and understanding their interaction with the cancerous cell's membrane.

Previously, we established adiponectin receptors (AdipoRs) as osteoanabolic target. To discover small molecule agonists of AdipoRs, we studied apigenin and apigenin-6C-glucopyranose (isovitexin) that induced osteoblast differentiation. In-silico, in vitro and omics-based studies were performed. Molecular docking using the crystal structures of AdipoRs showed different interaction profiles of isovitexin and apigenin. In osteoblasts, isovitexin but not apigenin rapidly phosphorylated AMP-activated protein kinase (pAMPK) which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated expression of AdipoRs. Blocking AMPK abolished the osteogenic effect of isovitexin and its effect on AdipoR expression. Isovitexin upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the mitochondrial biogenesis factor in osteoblasts, and the effect was blocked by AMPK inhibition. Upregulation of PGC-1α by isovitexin was accompanied by increased mitochondrial membrane proteins and mitochondrial DNA (mtDNA). Isovitexin via AdipoRs and PGC-1α induced oxidative phosphorylation (OxPhos) and ATP synthesis that resulted in osteoblast differentiation. Isovitexin had no agonistic/antagonistic activity and stimulatory/inhibitory effect in screening platforms for G protein-coupled receptors and kinases, respectively. In vivo, isovitexin upregulated AdipoRs and osteogenic genes, and increased mtDNA in rat calvarium. We conclude that isovitexin selectively via AdipoRs induced osteoblast differentiation that was fuelled by mitochondrial respiration.

The malaria parasite carries two organelles, the apicoplast and mitochondrion, whose DNA genomes must be maintained for optimal function and parasite survival under genotoxic stress. DNA repair mechanism(s) operative within these organelles were explored by mining the Plasmodium falciparum nuclear genome for sequences encoding proteins of major DNA repair pathways with predicted targeting to either organelle. Of the panel of enzymes identified for base excision repair (BER), we characterized the apurinic/apyrimidinic (AP) endonuclease PfApn1-an EndoIV whose homolog is not known in humans. PfApn1 targeted to the mitochondrion and functioned as an AP endonuclease requiring both Zn2+ and Mn2+ ions for maximal activity. Mutation of the critical third metal-binding site residue H542 resulted in the loss of Mn2+ (but not Zn2+ ) binding indicating that Mn2+ bound PfApn1 at this site; this was further supported by molecular dynamic simulation. CD spectra analysis further showed requirement of both metal ions for the attainment of PfApn1 β-strand-rich optimal conformation. PfApn1 also functioned as a 3'-phosphatase that would enable removal of 3'-blocks for DNA polymerase activity during BER. Interestingly, unlike Escherichia coli and yeast EndoIV homologs, PfApn1 lacked 3'-5' exonuclease activity and also did not cleave damaged bases by nucleotide incision repair (NIR). Uncoupling of endonuclease/phosphatase and exonuclease/NIR in PfApn1 suggests that amino acid residues distinct from those critical for endonuclease function are required for exonuclease activity and NIR. Characterization of a critical mitochondrion-targeted AP endonuclease provides evidence for a functional BER pathway in the parasite organelle.

Cervical cancer is a leading cause of cancer-related deaths among women in developing countries. Therefore, development of new chemotherapeutic agents is required. Unlike normal cells, cancer cells contain elevated copper levels which play an integral role in angiogenesis. Thus, targeting copper via copper-specific chelators in cancer cells can serve as effective anticancer strategy. In this work, a copper chelator pregnenolone acetate nucleus-based tetrazole derivative (ligand-L) was synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. DNA binding ability of ligand-L was studied using UV–Vis and fluorescence spectroscopy. Fluorescence spectroscopy studies reveal that quenching constant of ligand- l -DNA and ligand-L-Cu(II) were found to be 7.4 × 103 M−1 and 8.8 × 103 M−1, respectively. In vitro toxicity of ligand-L was studied on human cervical cancer C33A cancer cells. Results showed that ligand-L exhibit significant cytotoxic activity against cervical cancer C33A cells with IC50 value 5.0 ± 1.8 µM. Further, it was found that ligand-L cytotoxicity is due to redox cycling of copper to generate ROS which leads to DNA damage and apoptosis. In conclusion, this is the report where we synthesized pregnenolone acetate-based tetrazole derivative against C33A cells that targets cellular copper to induce pro-oxidant death in cancer cells. These findings will provide significant insights into the development of new chemical molecules with better copper chelating and pro-oxidant properties against cancer cells.

Allosteric modulators of G-protein-coupled receptors have lately gained significant traction in drug discovery. Recent studies have shown that allosteric modulation of serotonin 2C receptor (5-HT2C) as a viable strategy for the treatment of various central nervous system (CNS) disorders. Considering the critical role of 5-HT2C in the modulation of appetite, a selective positive allosteric modulator (PAM) of 5-HT2C offers a new opportunity for anti-obesity therapeutic development. In this study, phenyl cyclopropyl-linked N-heterocycles were synthesized and evaluated at 5-HT2C for agonist and PAM activity. Our study shows that imidazole linked phenyl cyclopropyl methanones has PAM activity on both 5-HT2C and serotonin 2B receptor (5-HT2B). Interestingly, piperazine linked phenyl cyclopropyl methanones (58) was active as PAM of 5-HT2C (increased the Emax of 5-HT to 139%), and as negative allosteric modulator (NAM) of 5-HT2B (decreases EC50 of 5-HT 10 times without affecting Emax). Similar effect of compound 58 was observed with synthetic orthosteric agonist lorcaserin on 5-HT2B. Molecular docking study revealed that all active compounds were binding to the predicted allosteric site on 5-HT2C and shared a common interacting residues. Finally, compound 58 suppressed food intake in Sprague Dawley (SD) rats similar to lorcaserin after i.c.v. administration. Therefore, these results suggest that piperazine moiety is essential for dual activity (PAM & NAM) of compounds 58, and supports the hypothesis of 5-HT2C PAM for the treatment of obesity similar to the full agonist.

Neurological disorders affect various aspects of life. Finding drugs for the central nervous system is a very challenging and complex task due to the involvement of the blood-brain barrier, P-glycoprotein, and the drug's high attrition rates. The availability of big data present in online databases and resources has enabled the emergence of artificial intelligence techniques including machine learning to analyze, process the data, and predict the unknown data with high efficiency. The use of these modern techniques has revolutionized the whole drug development paradigm, with an unprecedented acceleration in the central nervous system drug discovery programs. Also, the new deep learning architectures proposed in many recent works have given a better understanding of how artificial intelligence can tackle big complex problems that arose due to central nervous system disorders. Therefore, the present review provides comprehensive and up-to-date information on machine learning/artificial intelligence-triggered effort in the brain care domain. In addition, a brief overview is presented on machine learning algorithms and their uses in structure-based drug design, ligand-based drug design, ADMET prediction, de novo drug design, and drug repurposing. Lastly, we conclude by discussing the major challenges and limitations posed and how they can be tackled in the future by using these modern machine learning/artificial intelligence approaches.

The synthesis of 5-formyl-6-aryl-6H-indolo[3,2,1-de][1,5] naphthyridine-2-carboxylates by reaction between 1-formyl-9H-β-carbolines and cinnamaldehydes in the presence of pyrrolidine in water with microwave irradiation is described. Pharmacophoric modification of the formyl group offered several new fused β-carboline derivatives, which were investigated for their κ-opioid receptor (KOR) agonistic activity. Two compounds 4 a and 4 c produced appreciable agonist activity on KOR with EC50 values of 46±19 and 134±9 nM, respectively. Moreover, compound-induced KOR signaling studies suggested both compounds to be extremely G-protein-biased agonists. The analgesic effect of 4 a was validated by the increase in tail flick latency in mice in a time-dependent manner, which was completely blocked by the KOR-selective antagonist norBNI. Moreover, unlike U50488, an unbiased full KOR agonist, 4 a did not induce sedation. The docking of 4 a with the human KOR was studied to rationalize the result.

Poly (ADP-ribose) polymerase-1 (PARP1) inhibition strategy for cancer treatment is gaining advantage particularly in patients having a mutation in BRCA1/BRCA2 gene. To date, four drugs have obtained FDA approval and some inhibitors are in clinical trials. To identify more potent PARP1 inhibitors extensive research is going on to enrich the library of PARP1 inhibitors with compounds belonging to different classes. We employed an integrated virtual screening approach to identify potential PARP1 inhibitors. The sequential support vector machine (SVM) and pharmacophore model based virtual screening was carried out on the Maybridge library. The obtained hits were docked in the binding site of the PARP1 catalytic domain and nine drug-like compounds showing good ADME properties and form critical molecular interactions with the binding site residues were considered for the in vitro PARP1 inhibition assay. MD simulations were performed to decipher the stability of the PARP1-ligand complexes. Hydrogen bond interactions were also probed for their stability during MD simulations. We have identified three compounds (BTB02767, GK01172, and KM09200) showing 50% inhibition of PARP1 enzyme activity at 25 μM. BTB02767 and KM09200 have phthalazinone scaffold, while GK01172 bears a thiophene carboxamide scaffold, which could be a new chemotype of PARP1 inhibitors. In conclusion, GK01172 may serve as an important compound for further development of PARP1 inhibitors containing thiophene carboxamide scaffold. Communicated by Ramaswamy H. Sarma

Renin enzyme plays an essential role in the Renin-Angiotensin System (RAS), and it is involved in the pathogenesis of hypertension and several other cardiovascular diseases (CVDs). Inhibition of renin is an effective way to intervene with the pathogenesis of these diseases. Docking-based virtual screening, 3D-Quantitative Structure-Activity Relationship (3D-QSAR), and structure-based drug design are the most frequently used strategies towards discovering novel inhibitors targeting renin. In this study, we have developed a 2D fingerprint-based Deep Neural Network (DNN) classifier for virtual screening and a DNN-QSAR model for biological activity prediction. The resulting hits from the DNN-QSAR model were then subjected to the molecular docking to identify further top hits. Molecular Dynamics (MD) simulation was conducted to get a better insight into the binding mode of identified hits. We have discovered six compounds from the Maybridge chemical database with the predicted IC

Background Defects in DNA repair pathway can lead to double-strand breaks leading to genomic instability. Earlier we have shown that S.pombe Drp1, a Rint1/Tip1 family protein is required for the recovery from DNA damage. Methods Various truncations of Drp1 protein were constructed and their role in DNA damage response and interaction with Rad50 protein has been studied by co-immunoprecipitation and pull-down assays. Results The structural and functional analysis of Drp1 protein revealed that the N-terminus region of Drp1 is indispensable for the survival. The C-terminus truncation mutants, drp1C1Δ and drp1C2Δ exhibit temperature sensitive phenotype and are hypersensitive against DNA damaging agents with elevated level of Rad52-YFP foci at non-permissive temperature indicating the impairment for DNA damage repair pathway. The essential N-terminus region of Drp1 interacts with the C-terminus region of Rad50 and might be involved in influencing the MRN/X function. Small-angle X-ray (SAXS) analysis revealed three-domain like shapes in Drp1 protein while the C-terminus region of Rad50 exhibit unusual bulges. Computational docking studies revealed the amino acid residues at the C-terminus region of Rad50 that are involved in the interaction with the residues present at the N-terminal region of Drp1 indicating the importance of the N-terminal region of Drp1 protein. Conclusions We have identified the region of Drp1 and Rad50 proteins that are involved in the interaction and their role in the DNA damage response pathway has been analyzed. General significance The functional and structural aspects of fission yeast Drp1 protein and its interaction with Rad50 have been elucidated.

Chronic alcoholism (CA) decreases bone mass and increases the risk of hip fracture. Alcohol and its main metabolite, acetaldehyde impairs osteoblastogenesis by increasing oxidative stress. Aldehyde dehydrogenase (ALDH) is the rate-limiting enzyme in clearing acetaldehyde from the body. The clinical relevance of ALDH in skeletal function has been established by the discovery of single nucleotide polymorphism, SNP (rs671) in the ALDH2 gene giving rise to an inactive form of the enzyme (ALDH2*2) that causes increased serum acetaldehyde and osteoporosis in the affected individuals. Subsequent mouse genetics studies have replicated human phenotype in mice and confirmed the non-redundant role of ALDH2 in bone homeostasis. The activity of ALDH2 is amenable to pharmacological modulation. ALDH2 inhibition by disulfiram (DSF) and activation by alda-1 cause reduction and induction of bone formation, respectively. DSF also inhibits peak bone mass accrual in growing rats. On the other hand, DSF showed an anti-osteoclastogenic effect and protected mice from alcohol-induced osteopenia by inhibiting ALDH1a1 in bone marrow monocytes. Besides DSF, there are several classes of ALDH inhibitors with disparate skeletal effects. Alda-1, the ALDH2 activator induced osteoblast differentiation by increasing bone morphogenic protein 2 (BMP2) expression via ALDH2 activation. Alda-1 also restored ovariectomy-induced bone loss. The scope of structure-activity based studies with ALDH2 and the alda-1-like molecule could lead to the discovery of novel osteoanabolic molecules. This review will critically discuss the molecular mechanism of the ethanol and its principal metabolite, acetaldehyde in the context of ALDH2 in bone cells, and skeletal homeostasis.

Mammalian Lst8 interacts with the kinase domain of mTOR and stabilizes its interaction with Raptor regulating cell growth through the mTOR-S6K1 signalling pathway. Fission yeast Wat1, an ortholog of mammalian Lst8 is also an essential component of TOR complex 1 (TORC1) and TOR Complex 2 (TORC2) that control protein kinases essential for metabolic pathways. Here, we show that in response to osmotic stress, the Wat1 protein undergoes hyper-phosphorylation at S116 position. Wat1 interacts with the C-terminal region of Tor1 that also contain kinase domain. Co-immunoprecipitation and molecular modelling studies suggest that Wat1-Tor1 interaction is stabilized by FATC domain of Tor1 protein present at the C-terminal region. We have also demonstrated a physical interaction of Wat1 with Gad8, an AGC family protein kinase that is dependent on phosphorylation of Wat1 at S116 residue. Wat1 phosphorylation is required for the maintenance of vacuolar integrity and sexual differentiation. Collectively, our study reveals Wat1 phosphorylation regulates Gad8 function in a manner dependent on Tor1 interaction.

Unique intrinsic properties of peptides like low toxicity, high biological activity, and specificity make them attractive therapeutic agents. PDZ-binding peptide inhibitors have been demonstrated for curing of Alzheimer, Parkinson, Dementia, and other central nervous system ailments. In this article, we report the successful use of an integrated computational protocol to analyze the structural basis of how peptides bind to the shallow groove of the third PDZ domain (PDZ-3) from the postsynaptic density (PSD-95) protein. This protocol employs careful and precise computational techniques for design of new strategy for predicting novel and potent peptides against PDZ protein. We attempted to generate a pharmacophore model using crystal structure of peptide inhibitor bound to the PDZ-3. A highly specific and sensitive generated pharmacophore model was used for screening virtual database generated using different combination of amino acid substitutions as well as decoy peptide database for its sensitivity and specificity. Identified hit peptides were further analyzed by docking studies, and their stability analyzed using solvated molecular dynamics. Quantum Mechanics/Molecular Mechanics (QM/MM) interaction energy and GMX-PBSA scoring schemes were used for ranking of stable peptides. Computational approach applied here generated encouraging results for identifying peptides against PDZ interaction model. The workflow can be further exercised as a virtual screening technique for reducing the search space for candidate target peptides against PDZ domains.

A concise, one pot and regioselective access to (E)-distyrylbenzenes (DSBs) from arylhalide and secondary phenylenediethanol, a stable precursor for in situ generation of divinylbenzene (DVB) to avoid its polymerization, is described for construction of double C C bond formation via tandem double-dehydrative-double-Heck (D-D-D-H) reaction using Palladium and ionic liquid [hmim]Br as a cooperative catalyst. It is noteworthy that this pot-economy approach also provides direct synthesis of hydroxylated distyrylbenzenes without requirement of protection-deprotection strategy. Importantly, the synthesized DSBs are tested for their protective activity against β amyloid reduction, acetylcholine esterase inhibition, lipid lowering and reactive oxygen species (ROS) reduction properties in transgenic Caenorhabditis elegans model wherein 1,3-bis((E)-4-(trifluoromethyl)styryl)benzene (5c) is found to be active across all above factors thus presenting lead molecule within multi-target-directed ligands (MTDLs) approach. Molecular docking studies were also performed to understand the interactions of potent DSBs with receptors.

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like candidiasis are major global health burden in female reproductive health. In this context a novel non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was found to be more active than the standard drug Metronidazole (MTZ). Compounds were trichomonicidal in the MIC ranges of 4.77–294.1 μM and 32.46–735.20 μM against MTZ-susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at least two out of ten fungal strains tested at MIC of 7.50–240.38 μM. The most active compound (20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and preliminary pharmacokinetic study has shown good distribution and systemic clearance.

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4β7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Molecular hybridization of different pharmacophores to tackle both tumor growth and metastasis by a single molecular entity can be very effective and unique if the hybrid product shows drug-like properties. Here, we report synthesis and discovery of a novel small-molecule inhibitor of PP2A–β-catenin signaling that limits both in vivo tumor growth and metastasis. Our molecular hybridization approach resulted in cancer cell selectivity and improved drug-like properties of the molecule. Inhibiting PP2A and β-catenin interaction by selectively engaging PR55α-binding site, our most potent small-molecule inhibitor diminished the expression of active β-catenin and its target proteins c-Myc and Cyclin D1. Furthermore, it promotes robust E-cadherin upregulation on the cell surface and increases β-catenin–E-Cadherin association, which may prevent dissemination of metastatic cells. Altogether, we report synthesis and mechanistic insight of a novel drug-like molecule to differentially target β-catenin functionality via interacting with a particular subunit of PP2A. Mol Cancer Ther; 16(9); 1791–805. ©2017 AACR.

DNA methylation is an epigenetic change that results in the addition of a methyl group at the carbon-5 position of cytosine residues. DNA methyltransferase (DNMT) inhibitors can suppress tumour growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. Additionally, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects. In the present study, we have designed a workflow by integrating various ligand-based and structure-based approaches to discover new agents active against DNMT1. We have derived a pharmacophore model with the help of available DNMT1 inhibitors. Utilising this model, we performed the virtual screening of Maybridge chemical library and the identified hits were then subsequently filtered based on the Naïve Bayesian classification model. The molecules that have returned from this classification model were subjected to ensemble based docking. We have selected 10 molecules for the biological assay by inspecting the interactions portrayed by these molecules. Three out of the ten tested compounds have shown DNMT1 inhibitory activity. These compounds were also found to demonstrate potential inhibition of cellular proliferation in human breast cancer MDA-MB-231 cells. In the present study, we have utilized a multi-step virtual screening protocol to identify inhibitors of DNMT1, which offers a starting point to develop more potent DNMT1 inhibitors as anti-cancer agents.

Dipeptidyl peptidase 4 (DPP4) is a well-known target for the antidiabetic drugs. However, currently available DPP4 inhibitor screening assays are costly and labor-intensive. It is important to create a robust in silico method to predict the activity of DPP4 inhibitor for the new lead finding. Here, we introduce an R-based Web application SVMDLF (SVM-based DPP4 Lead Finder) to predict the inhibitor of DPP4, based on support vector machine (SVM) model, predictions of which are confirmed by in vitro biological evaluation. The best model generated by MACCS structure fingerprint gave the Matthews correlation coefficient of 0.87 for the test set and 0.883 for the external test set. We screened Maybridge database consisting approximately 53,000 compounds. For further bioactivity assay, six compounds were shortlisted, and of six hits, three compounds showed significant DPP4 inhibitory activities with IC50 values ranging from 8.01 to 10.73 μm. This application is an OpenCPU server app which is a novel single-page R-based Web application for the DPP4 inhibitor prediction. The SVMDLF is freely available and open to all users at http://svmdlf.net/ocpu/library/dlfsvm/www/ and http://www.cdri.res.in/svmdlf/.

SUMMARYDespite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of solubleLeishmania donovaniantigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy againstL. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treatedLeishmania-infected hamsters and peripheral blood mononuclear cells ofLeishmaniapatients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treatedLeishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses inLeishmaniapatients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 A and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

A series of seventeen piperazine derivatives have been synthesized and biologically evaluated for the management of andropause-associated prostatic disorders and depression. Five compounds 16, 19, 20, 21 and 22 significantly inhibited proliferation of androgen-sensitive LNCaP prostatic cell line with EC50 values of 12.4 μM, 15.6 μM, 11.8 μM, 10.4 μM, 12.2 μM respectively and decreased Ca2+ entry through adrenergic-receptor α1A blocking activity. Anti-androgenic behaviour of compound 19 and 22 was evident by decreased luciferase activity. The high EC50 value in AR-negative cells PC3 and DU145 suggested that the cytotoxicity of compounds was due to AR down regulation. Compound 19 reduced the prostate weight of rats by 53.8%. Further, forced-swimming and tail-suspension tests revealed antidepressant-like activity of compound 19, lacking effects on neuromuscular co-ordination. In silico ADMET predictions revealed that the compound 19 had good oral absorption, aqueous solubility, non-hepatotoxic and good affinity for plasma protein binding. Pharmacokinetic and tissue uptake of 19 at 10 mg/kg demonstrated an oral bioavailability of 35.4%. In silico docking studies predicted similar binding pattern of compound 19 on androgen receptor as hydroxyflutamide. Compound 19 appears to be a unique scaffold with promising activities against androgen associated prostatic disorders in males like prostate cancer and BPH and associated depression.

In insulin and leptin signaling pathway, Protein-Tyrosine Phosphatase 1B (PTP1B) plays a crucial controlling role as a negative regulator, which makes it an attractive therapeutic target for both Type-2 Diabetes (T2D) and obesity. In this work, we have generated classification models by using the inhibition data set of known PTP1B inhibitors to identify new inhibitors of PTP1B utilizing multiple machine learning techniques like naïve Bayesian, random forest, support vector machine and k-nearest neighbors, along with structural fingerprints and selected molecular descriptors. Several models from each algorithm have been constructed and optimized, with the different combination of molecular descriptors and structural fingerprints. For the training and test sets, most of the predictive models showed more than 90% of overall prediction accuracies. The best model was obtained with support vector machine approach and has Matthews Correlation Coefficient of 0.82 for the external test set, which was further employed for the virtual screening of Maybridge small compound database. Five compounds were subsequently selected for experimental assay. Out of these two compounds were found to inhibit PTP1B with significant inhibitory activity in in-vitro inhibition assay. The structural fragments which are important for PTP1B inhibition were identified by naïve Bayesian method and can be further exploited to design new molecules around the identified scaffolds. The descriptive and predictive modeling strategy applied in this study is capable of identifying PTP1B inhibitors from the large compound libraries.

Glutamine synthetase is an enzyme that catalyzes the condensation of glutamate and ammonia to form glutamine in the presence of adenosine triphosphate. The wealth of structure-related information about glutamine synthetase across the species is endorsing it as emerging potential drug target. Owing to its well-characterized role in metabolism of Mycobacterium, various high throughput screening studies have been aimed at the identification of inhibitors against MtGS. The present work is focussed on comparative sequence and structural studies of glutamine synthetase and its evaluation as drug target against the infectious diseases. We have done molecular modeling studies of glutamine synthetase of Leishmania and Plasmodium. The structure models and molecular dynamics simulations studies shed light on to the binding modes of substrates viz. adenosine diphosphate, glutamate, ammonia, and metal ions. The comparative studies of MtGS, HsGS, LmGS, and PvGS helped in better understanding of prospects of structure-based inhibitor design. The results suggest that amino acid-binding site is highly conserved, whereas nucleotide-binding site possess subtle variations and thus offers opportunity for specific inhibitor design. Therefore, present study suggests that broad spectrum glutamine synthetase inhibition is feasible and it is potential drug target against infectious diseases.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3–10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% β-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain–phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

Histone Deacetylases (HDACs) play a significant role in the regulation of gene expression by modifying histones and non-histone substrates. Since they are key regulators in the reversible epigenetic mechanism, they are considered as promising drug targets for the treatment of various cancers. In the present study, we have developed a workflow for identification of HDAC1 inhibitors using a multistage virtual screening approach from Maybridge and Chembridge chemical library. Initially, a support vector machine based classification model was generated, followed by generation of a zinc-binding group (ZBG) based pharmacophore model. The hits screened from these models were further subjected to molecular docking. Finally, a set of twenty-three molecules were selected from Maybridge and Chembridge library. The biological evaluation of these hits revealed that three out of the twenty-three tested compounds are showing HDAC1 inhibition along with the moderate anti-proliferative activity. It was found that the identified inhibitors are exerting chromosomal loss effect in growing yeast cells. Further, to extend the activity spectrum of the identified inhibitors, the optimization guidelines were drawn with the hydration site mapping approach by using in silico tool Watermap. Communicated by Ramaswamy H. Sarma

Cathepsin S (CatS) is one of the cysteinyl cathepsins widely studied for its clinical significance and found to be a promising therapeutic target for several diseases; to name a few is arthritis, allergic inflammation, cancer, diabetes, obesity, and cystic fibrosis. Elevated CatS level is a contributing factor for related disorders, and therefore among different strategies to regulate the activity of CatS, one is to design a quality inhibitor. Earlier, we have demonstrated a highly selective CatS inhibitor, RO5444101 interacts primarily with the S2 pocket of the protein which is structurally unique in contrast to other variants of cathepsin. However, the molecular properties of RO5444101 can question its efficacy at the clinical level. In the present study, we have implemented a series of molecular modeling methods to screen the Maybridge library considering the pharmacophoric features of RO5444101 and other relevant inhibitors of CatS. Based on the priority list, eight hits were subjected to biological evaluation. Subsequently, KM07987 was found to be most potent, with the IC50 of

The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC50 < 0.04 μM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c a...