Insight into the screening of potential beta-lactamase inhibitors as anti-bacterial chemical agents through pharmacoinformatics study.

Drug resistance is an unsolved and major concern in the bacterial infection. Continuous development of drug-resistance to the antibiotics exponentially rises the danger of bacterial infections. Chemical components from the plants are becoming a major resource of potentially effective therapeutic chemical agents for the wide range of diseases including bacterial infections. In the current study, pharmacoinformatics methodologies were implemented on more than two hundred known phytochemicals to find promising beta-lactamase inhibitors for therapeutically effective anti-bacterial agents. Initially, the molecular docking-based score was used to reduce the chemical space of the selected dataset. Fourteen molecules were found to have more affinity towards the beta-lactamase in compared to the well-known anti-bacterial agent, Avibactam. Binding interactions analysis revealed the strong binding interactions between phytochemicals and catalytic amino residues. For further analysis, molecular dynamics (MD) simulations, density functional theory (DFT) and in silico pharmacokinetics studies were performed. Parameters from MD simulations studies suggested that selected molecules are strong enough to retain in the active site in different orientations of the beta-lactamase. The orbital energies obtained from the DFT study was undoubtedly explained the potentiality of the selected compounds for being effective beta-lactamase inhibitors. The drug-likeness and acceptable pharmacokinetics parameters were observed using in silico ADME analysis. Therefore, observations from the multiple pharmacoinformatics approach explained without any doubt that selected molecules are potential enough being promising anti-bacterial compounds. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Nature's therapy for COVID-19: Targeting the vital non-structural proteins (NSP) from SARS-CoV-2 with phytochemicals from Indian medicinal plants.

Background: Containing COVID-19 is still a global challenge. It has affected the "normal" world by targeting its economy and health sector. The effect is shifting of focus of research from life threatening diseases like cancer. Thus, we need to develop a medical solution at the earliest. The purpose of this present work was to understand the efficacy of 22 rationally screened phytochemicals from Indian medicinal plants obtained from our previous work, following drug-likeness properties, against 6 non-structural-proteins (NSP) from SARS-CoV-2. Methods: 100 ns molecular dynamics simulations were performed, and relative binding free energies were computed by MM/PBSA. Further, principal component analysis, dynamic cross correlation and hydrogen bond occupancy were analyzed to characterize protein-ligand interactions. Biological pathway enrichment analysis was also carried out to elucidate the therapeutic targets of the phytochemicals in comparison to SARS-CoV-2. Results: The potential binding modes and favourable molecular interaction profile of 9 phytochemicals, majorly from Withania somnifera with lowest free binding energies, against the SARS-CoV-2 NSP targets were identified. It was understood that phytochemicals and 2 repurposed drugs with steroidal moieties in their chemical structures formed stable interactions with the NSPs. Additionally, human target pathway analysis for SARS-CoV-2 and phytochemicals showed that cytokine mediated pathway and phosphorylation pathways were with the most significant p-value. Conclusions: To summarize this work, we suggest a global approach of targeting multiple proteins of SARS-CoV-2 with phytochemicals as a natural alternative therapy for COVID-19. We also suggest that these phytochemicals need to be tested experimentally to confirm their efficacy.

The natural way forward: Molecular dynamics simulation analysis of phytochemicals from Indian medicinal plants as potential inhibitors of SARS-CoV-2 targets.

The pandemic COVID-19 has become a global panic-forcing life towards a compromised "new normal." Antiviral therapy against SARS-CoV-2 is still lacking. Thus, development of natural inhibitors as a prophylactic measure is an attractive strategy. In this context, this work explored phytochemicals as potential inhibitors for SARS-CoV-2 by performing all atom molecular dynamics simulations using high performance computing for 8 rationally screened phytochemicals from Withania somnifera and Azadirachta indica and two repurposed drugs docked with the spike glycoprotein and the main protease of SARS-CoV-2. These phytochemicals were rationally screened from 55 Indian medicinal plants in our previous work. MM/PBSA, principal component analysis (PCA), dynamic cross correlation matrix (DCCM) plots and biological pathway enrichment analysis were performed to reveal the therapeutic efficacy of these phytochemicals. The results revealed that Withanolide R (-141.96 KJ/mol) and 2,3-Dihydrowithaferin A (-87.60 KJ/mol) were with the lowest relative free energy of binding for main protease and the spike proteins respectively. It was also observed that the phytochemicals exhibit a remarkable multipotency with the ability to modulate various human biological pathways especially pathways in cancer. Conclusively we suggest that these compounds need further detailed in vivo experimental evaluation and clinical validation to implement them as potent therapeutic agents for combating SARS-CoV-2.

In vitro and in silico study of Aloe vera leaf extract against human breast cancer.

Aloe vera leaf contains some bioactive compounds that have a strong binding affinity toward estrogen receptor as compared to standard drug tamoxifen. In this study, we have found that the IC50 of Aloe vera leaf extract against breast cancer cell line (MCF-7) is 23 µg/mL which is much lower than the IC50 (332 µg/mL) of Aloe vera leaf extract against non-cancerous cell line (NIH-3T3). We have also calculated the total concentration of phenolic acid (385.662 µg/mL), flavonoids (160.402 µg/mL) and alkaloids (276.754 µg/mL) in Aloe vera leaf extract. The free radical scavenging activity of Aloe vera leaf extract is 67% to 89% (at 50 to 300 µg/ml). Our virtual molecular docking study suggests that bioactive compounds like Aloe-emodin (-8.8 Kcal/mol), 7-hydroxy-2,5 dimethylchromone (-7.5 Kcal/mol), Beta-sitosterol (-7.3 Kcal/mol) etc. have a greater binding affinity toward estrogen alpha receptor as compared to standard drug Tamoxifen (-6.4 Kcal/mol). [Formula: see text].

Lactate dehydrogenase A regulates autophagy and tamoxifen resistance in breast cancer.

Estrogen receptor (ER) antagonist, tamoxifen has been universally used for the treatment of the ER-positive breast cancer; however, the inevitable emergence of resistance to tamoxifen obstructs the successful treatment of this cancer. So, there is an immediate requirement for the search of a novel therapeutic target for treatment of this cancer. Acquired tamoxifen-resistant breast cancer cell lines MCF-7 (MCF-7/TAM-R) and T47D (T47D/TAM-R) showed higher apoptotic resistance accompanied by induction of pro-survival autophagy compared to their parental cells. Besides, tamoxifen resistance was associated with reduced production of ATP and with overexpression of glycolytic pathways, leading to induced autophagy to meet the energy demand. Further, our study revealed that LDHA; one of the key molecules of glycolysis in association with Beclin-1 induced pro-survival autophagy in tamoxifen-resistant breast cancer. Mechanistically, pharmacological and genetic inhibition of LDHA reduced the pro-survival autophagy, with the restoration of apoptosis and reverting back the EMT like phenomena noticed in tamoxifen-resistant breast cancer. In total, targeting LDHA opened a novel strategy to interrupt autophagy and tamoxifen resistance in breast cancer.

Cutaneotrichosporon (Trichosporon) debeurmannianum: A Rare Yeast Isolated from Blood and Urine Samples.

Cutaneotrichosporon (Trichosporon) debeurmannianum is a rarely isolated yeast from clinical samples. Nine isolates of this yeast were identified from clinical samples within a period of 3 years from June 2012 to May 2015. These isolates were from blood and urine samples sent to a clinical mycology laboratory of a tertiary care hospital in Assam, North East India. Clinically, the patients were diagnosed as septicemia and urinary tract infection. The age of the patients ranged from 2 to 50 years. Identification was made by sequencing the ITS region of ribosomal RNA gene. Antifungal susceptibility test by disk diffusion method (CLSI, M44-A) showed all the isolates to be sensitive to fluconazole and voriconazole. Vitek 2 compact commercial yeast identification system misidentified this yeast as Cryptococcus laurentii and low discrimination Cryptococcus laurentii/Trichosporon mucoides. This species was originally named as Trichosporon debeurmannianum. In 2015, this yeast has been included into new genera Cutaneotrichosporon based on an integrated phylogenetic classification of the Tremellomycetes. To the best of our knowledge, this is the first report of identification of this species from blood and urine samples of clinically suspected cases. We are reporting these isolates because of their rarity in clinical samples. The pathogenic potential and epidemiological relevance of this yeast remains to be seen.

Novel Insights into the Molecular Interaction of a Panduratin A Derivative with the Non Structural Protein (NS3) of Dengue Serotypes: A Molecular Dynamics Study.

BACKGROUND: The ligand PKP10 having substitution of Cl- at R2 and R3 positions of ring A of Panduratin A i.e., ((1R,2S,5S)-5-(2,3-dichlorophenyl)-3-methyl-2-(3-methylbut-2-nyl)cyclohex-3- enyl)(2,6-dihydroxy-4-methylphenyl)methanone hydrate) has been observed to block the Nuclear Receptor Binding Protein binding site of Non Structural protein 3 in all dengue serotypes. In continuation with our earlier study, we have reported sixty novel Panduratin A derivatives compounds where substitution was done in positions 2 and 3 position of the benzyl ring A of Panduratin A with various substituents. METHODS: We selected ((1R,2S,5S)-5-(2,3-dichlorophenyl)-3-methyl-2-(3-methylbut-2-nyl)cyclohex-3- nyl) (2,6-dihydroxy-4-methylphenyl) methanone hydrate) (PKP10) for molecular dynamics (MD) simulations as it constantly produced lowest CDocker interaction energy of among all the sixty five derivatives. The CDocker interaction energy was predicted to be -140.804, -79.807, -78.217 and -84.073 Kcalmol-1 respectively against NS3 protein of dengue serotypes (DENV1-4). To understand the dynamics of the PKP10 with NS3 protein, each complex was subjected to molecular dynamics simulations of 50 ns in aqueous solution. MD (Molecular Dynamics) simulation study revealed that the binding of ligand PKP10 at the active site of NS3 induces a conformational change in all serotypes which was well supported by principal component analysis. RESULT: To the best of our knowledge, this is first ever study which provided atomistic insights into the interaction of PKP10 with NS3 protein of dengue serotypes. CONCLUSION: The result from our study along with in vitro studies is expected to open up better avenues to develop inhibitors for dengue virus in the near future.

Molecular Modeling and Dynamics Simulation Analysis of KATNAL1 for Identification of Novel Inhibitor of Sperm Maturation.

BACKGROUND: Hormone based birth control often causes various side effects. A recent study revealed that temporary infertility without changing hormone levels can be attained by inhibiting Katanin p60 ATPase-containing subunit A-like 1 protein (KATNAL1) which is critical for sperm maturation in the testes. OBJECTIVE: This study aimed at attaining the most energetically stable three dimensional (3D) structure of KATNAL1 protein using comparative modeling followed by screening of a ligand library of known natural spermicidal compounds for their binding affinity with KATNAL1. This in turn may inhibit the development of mature sperm in the seminiferous epithelium. METHOD: A series of computational techniques were used for building the 3D structure of KATNAL1 which was further optimized by molecular dynamics (MD) simulation. For revealing the ATP binding mode of KATNAL1, docking study was carried out using the optimized model obtained from the MD simulation. The docking study was also employed to test the binding efficiency of the ligand library. RESULTS: Molecular docking study confirmed the ATP binding of KATNAL1 with various hydrophobic and hydrogen bond interactions. Binding efficiency of the ligand library suggested that calotropin, a cardenolide of Calotropis procera showed the highest binding efficiency against the target protein without toxicity. MD simulation of the docked complex validated the results of the docking study. CONCLUSION: This study revealed the ATP binding mode of KATNAL1 and identified calotropin as a potential lead molecule against it showing high binding efficiency with good bioavailability and no mutagenicity. Further in vitro and in vivo bioassay of calotropin could facilitate the development of novel non-hormonal male-specific contraceptive in near future.

Clinically relevant yeast species identified by sequencing the internal transcribed spacer region of r-RNA gene and Vitek 2 compact (YST card) commercial identification system: Experience in a Tertiary Care Hospital in Assam, Northeast India.

In this retrospective study from 2012 to 2015, 333 clinical isolates of yeasts were identified using Vitek 2 Compact System YST ID card (Biomerieux, France) and internal transcribed spacer (ITS) sequencing. Eighteen species were identified by ITS sequencing. Candida albicans was the most common species (46.5%), followed by Candida tropicalis (27%). The total species supported by Vitek System was 11 (61.11%). The sensitivity of the system in identifying these 11 species was 66.66%-100%; specificity 98.37%-100%; positive predictive value 70%-100%, negative predictive value 96.05%-100%, and diagnostic accuracy 96.99%-100%. Diagnostic accuracy of ITS1 and ITS2 sequences individually was 98.49% and 100% using NCBI Genbank database.

Physiological Proteins in Therapeutics: A Current Review on Interferons.

Interferons are produced in vivo and are one of the prime components of natural defense system of animals. They are released by the viral infected cells and provide protection to the neighboring cells against viral infection. The cyto-protective property of the proteins ignited the thought of their pharmaceutical adaptation for therapeutic use against viral diseases in individuals in whom the interferons released naturally are not sufficient to combat the situation. Interferon supplements have been found to complement various antiviral drugs. Considering the efficacy of interferons in regulating angiogenesis and immunomodulation, they can be adapted for therapy of the killer diseases like cancer and AIDS. We have come ahead more than twenty five years after the approval of clinical use of interferon as drugs and are today really in a position to promise a disease free life to our present and next generation. Interferon therapy will be contributing a big share to the upcoming remedies for the new diseases and we are thus armed to fight back the deadly viral threats. Interferons have been modified [pegylated etc.] and have already been adapted to some extent in certain diseases and are in regular use in some. Thus interferons if modified as per need and used in combination with either antiviral drugs, antibiotics, antioxidants may strengthen our defense system effectively to bring about a strong protection against wide range of diseases.

Drug Discovery and Development of Type 2 Diabetes Mellitus: Modern-Integrative Medicinal Approach.

Type 2 diabetes is a disorder of ages, which has become deadlier because of life style modification and adaptation in the modern world. Extensive sudy of the pathophysiology of diabetes has opened up various mysteries about the disease and has helped us to know and understand diabetes in a better manner. Presently, we know many minute details about the pathophysiology of diabetes mellitus and are thus well weaponed to fight against it. Treatment regime has been evolving daily. Besides the conventional anti-diabetic drugs, integrated medicinal approach for treating diabetes type 2 with a compact therapeutic approach consisting of various targeted treatments for individual symptoms associated with the disease are being tried currently. Diabetes associated complications like high blood pressure, hyperglycemia, microalbumuria, dyslipidemia, pro -coagulation, etc. are being targeted and dealt with individually in the integrative medicinal approach. The results are promising and thus ignite hope for a better and more successful handling of diabetes and diabetes related pathophysiological complications in near future.

Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs.

The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest.

Mitogen Activated Protein Kinase (MPK) Interacts With Auxin Influx Carrier (OsAux/LAX1) Involved in Auxin Signaling in Plant.

BACKGROUND: Mitogen activated protein kinases (MPKs) are serine/threonine protein kinases that contain characteristic T-x-Y motif in the activation loop region. MPKs are important signaling molecules involved in diverse signaling cascades that regulate plant growth, development and stress responses by conducting phosphorylation events in their target proteins. MPKs phosphorylate their target proteins at either S-P/T-P (Serine/Proline/Threonine) amino acid. To understand, if MPKs are involved in the auxin signaling cascade, we identified probable target proteins of MPKs involved in auxin signaling or transport processes. RESULTS: A genome-wide search of the rice genome database led us to identification of the OsAux/LAX1 gene as a potential downstream target protein of MPKs. In-silico analysis predicted that MPKs interact with OsAux/LAX1 proteins which were validated by a yeast two-hybrid assay that showed OsMPK3, OsMPK4 and OsMPK6 are physically interact with OsAux/LAX1 protein. CONCLUSION: The yeast two-hybrid interaction showed that MPKs are directly involved in auxin signaling events in plants. This is the first study to report direct involvement of MPKs in the auxin signaling pathway.

Genome-wide identification of Calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events.

BACKGROUND: Calcium ions, the most versatile secondary messenger found in plants, are involved in the regulation of diverse arrays of plant growth and development, as well as biotic and abiotic stress responses. The calcineurin B-like proteins are one of the most important genes that act as calcium sensors. RESULTS: In this study, we identified calcineurin B-like gene family members from 38 different plant species and assigned a unique nomenclature to each of them. Sequence analysis showed that, the CBL proteins contain three calcium binding EF-hand domain that contains several conserved Asp and Glu amino acid residues. The third EF-hand of the CBL protein was found to posses the D/E-x-D calcium binding sensor motif. Phylogenetic analysis showed that, the CBL genes fall into six different groups. Additionally, except group B CBLs, all the CBL proteins were found to contain N-terminal palmitoylation and myristoylation sites. An evolutionary study showed that, CBL genes are evolved from a common ancestor and subsequently diverged during the course of evolution of land plants. Tajima's neutrality test showed that, CBL genes are highly polymorphic and evolved via decreasing population size due to balanced selection. Differential expression analysis with cold and heat stress treatment led to differential modulation of OsCBL genes. CONCLUSIONS: The basic architecture of plant CBL genes is conserved throughout the plant kingdom. Evolutionary analysis showed that, these genes are evolved from a common ancestor of lower eukaryotic plant lineage and led to broadening of the calcium signaling events in higher eukaryotic organisms.

Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants.

BACKGROUND: Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. RESULTS: A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. CONCLUSION: We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.

In silico screening of antifolate based novel inhibitors from Brucea mollis Wall. ex kurz against quadruple mutant drug resistant PfDHFR.

Plasmodium falciparum is the most lethal form of the genus Plasmodium which causes malaria, a 'disease of antiquity'. Globally it affects the health and socio-economic development of a large population especially in Sub-Saharan Africa and Southeast Asia. The Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. Mutations at the active site of PfDHFR have resulted in decrease drug binding affinity of DHFR-inhibitors. In the present study we selected ten compounds of Brucea mollis Wall. Ex kurz and checked for their drug likeness using various computational tools and potential interactions with PfDHFR by molecular docking study. Soulameanone, a quassinoid of Brucea mollis Wall. Ex kurz showed better binding affinity when compared to pyrimethamine for both wild and quadruple mutant drug resistant PfDHFR. In addition, similar isomers of soulameanone were screened for their drug likeness and to study their interactions with PfDHFR. Twenty three compounds showed better binding affinity compared to soulameanone.

Insight into structural organization and protein-protein interaction of non structural 3 (NS3) proteins from dengue serotypes.

Dengue infections produce a distinct character of virus-induced intracellular membrane alterations which are associated with the viral replication machinery. Currently, the NS3 protein is being targeted for antiviral therapy against dengue. NS3 protein of dengue virus interacts with nuclear receptor binding protein (NRBP) of human causing cell trafficking between the Endoplasmic Reticulum (ER) and Golgi, which interacts with Rac3, a member of the Rho-GTPase family. No crystal structure of the NRBP is available for any species, thus limiting the complete understanding of structure- function relationships of this protein. The present study deals with the molecular modeling of the viral protein (NS3 of DENV1-4), the host protein (NRBP) and their interactions through protein-protein docking study. Theoretical threedimensional structures of the NRBP and NS3 were modeled using the Modeller 9v8, and the evaluated models were docked using GRAMM-X to study the mode of protein-protein interaction (NRBP as receptor and NS3 as ligand). The docked docking complexes were further evaluated for interaction analysis by the RosettaDock Server. Suface and interface residues were observed along with hydrogen and hydrophobic interaction. The conserved residues forming hydrogen interaction of NRBP with DENV1-4 serotypes were found to be GLN 305, SER 363 and GLN 379.

Design, virtual screening and docking study of novel NS3 inhibitors by targeting protein-protein interacting sites of dengue virus - a novel approach.

Currently dengue is a serious disease which has become a global burden in the last decade. Unfortunately, there are no effective drugs and vaccines against this disease. DENV non-structural protein (NS) 3, which is viral protease which is a potential target for antiviral therapy. Targeting this we performed homology modeling and protein-protein docking study of NS3 with NRBP (Nuclear Receptor Binding Protein) of human as it has been proved that NS3 of DENV interacts with NRBP which causes cellular trafficking in human cell. To carry out search of novel DENV protease inhibitors by in silico screening panduratin molecule was selected. 65 novel compounds were designed which involved substituting positions 1-5 of the benzyl ring A (4hydroxy-panduratinA) with various substituents. The protein-protein docking showed that the aminoacid residues of NS3 which were interacting with NRBP were found to be Ala 325, Asp 324, Phe 326, Asp 335, Glu 336, Glu 328, Asp 485, Gln 478, Arg 459, Gly 446 and Leu 480. These residues were targeted by the ligands which showed excellent binding affinity as binding energy. The ligand PKP10 showed lowest binding energy. It is also observed that the interface residues participated in the protein-protein interaction are being inhibited by the ligands.

Binding Energy calculation of GSK-3 protein of Human against some anti-diabetic compounds of Momordica charantia linn (Bitter melon).

Diabetes is one of the major life threatening diseases worldwide. It creates major health problems in urban India. Glycogen Synthase Kinase-3 (GSK-3) protein of human is known for phosphorylating and inactivating glycogen synthase which also acts as a negative regulator in the hormonal control of glucose homeostasis. In traditional medicine, Momordica charantia is used as antidiabetic plant because of its hypoglycemic effect. Hence to block the active site of the GSK-3 protein three anti-diabetic compounds namely, charantin, momordenol & momordicilin were taken from Momordica charantia for docking study and calculation of binding energy. The aim of present investigation is to find the binding energy of three major insulin-like active compounds against glycogen synthase kinase-3 (GSK-3), one of the key proteins involved in carbohydrate metabolism, with the help of molecular docking using ExomeTM Horizon suite. The study recorded minimum binding energy by momordicilin in comparison to the others.