Pangenome profiling of novel drug target against vancomycin-resistant Enterococcus faecium.

Enterococcus faecium is a frequent causative agent of nosocomial infection mainly acquired from outgoing hospital patients (Hospital Acquired Infection-HAIs). They are largely involved in the outbreaks of bacteremia, UTI, and endocarditis with a high transmissibility rate. The recent emergence of VRE strain (i.e. vancomycin resistant enterococcus) turned it into high priority pathogen for which new drug research is of dire need. Therefore, in current study, pangenome and resistome analyses were performed for available antibiotic-resistant genomes (n = 216) of E. faecium. It resulted in the prediction of around 5,059 genes as an accessory gene, 1,076 genes as core and 1,558 genes made up a unique genome fraction. Core genes common to all strains were further used for the identification of potent drug targets by applying subtractive genomics approach. Moreover, the COG functional analysis showed that these genomes are highly enriched in metabolic pathways such as in translational, ribosomal, proteins, carbohydrates and nucleotide transport metabolism. Through subtractive genomics it was observed that 431 proteins were non-homologous to the human proteome, 166 identified as essential for pathogen survival while 26 as potential and unique therapeutic targets. Finally, 3-dehydroquinate dehydrogenase was proposed as a potent drug target for further therapeutic candidate identification. Moreover, the molecular docking and dynamic simulation technique were applied to performed a virtual screening of natural product libraries (i.e., TCM and Ayurvedic compounds) along with 3-amino-4,5-dihydroxy-cyclohex-1-enecarboxylate (DHS) as a standard compound to validate the study. Consequently, Argeloside I, Apigenin-7-O-gentiobioside (from Ayurvedic library), ZINC85571062, and ZINC85570908 (TCM library) compounds were identified as potential inhibitors of 3-dehydroquinate dehydrogenase. The study proposed new compounds as novel therapeutics, however, further experimental validation is needed as a follow-up.Communicated by Ramaswamy H. Sarma.

Total Bioactive Contents, Metabolic Profiling, Docking Studies, Antioxidant and Enzyme Inhibition Activities of Convolvulus Arvensis L. and Multivariate Analysis to Unravel a Potential Herb as Natural Resource for Pharmaceutical Industry.

Convolvulus arvensis L. is an evergreen herb growing in various regions of Pakistan. Despite of several medicinal properties associated to this herb, it was not investigated scientifically for its bioactive compounds and detailed pharmaceutical properties. Therefore, its methanolic extract was divided into hexane (CA-H), chloroform (CA-C), ethyl acetate (CA-E) and butanol (CA-B) soluble fractions. CA-H and CA-C were found rich in phenolics (30.73±0.63 and 20.15±0.59 mg GAE/g of the extract, respectively), and the same fractions exhibited significant antioxidant activities (DPPH: 5.23±0.11 & 12.34±0.17 mg TE/g extract, respectively; ABTS: 36.82±0.04 & 56.74±0.61 mg TE/g extract, respectively). Also in CUPRAC activity assay, CA-H and CA-C exhibited highest activities as 87.30±0.46 and 56.74±0.61 mg TE/g extract, respectively, while CA-C was most active in FRAP activity assay with value of 40.21±2.19 mg TE/g extract. Total antioxidant capacity (1.23±0.033 mmol TE/g extract) was also found higher for CA-C, while CA-H activity was also comparable, however, CA-H showed higher metal chelating activity (22.74±0.001 mg EDTAE/g extract) than that of CA-C (17.55±0.22 mg EDTAE/g extract). These activities clearly revealed a direct relation between antioxidant potential and phenolic contents of CA-H and CA-C. In AChE and BChE inhibitory assay, CA-H and CA-E showed better inhibition (AChE: 8.24±0.77 & 4.46±0.007 mg GALAE/g extract; BChE: 5.40±0.02 & 1.92±0.24 mg GALAE/g extract) as compared to other fractions, whereas, against tyrosinase, CA-B was most active (37.35±0.53 mg KAE/g extract). CA-H and CA-C also showed higher inhibitory potential (0.98±0.08 & 0.58±0.01 mmol ACAE/g extract) against α-Amylase; while against α-Glucosidase, CA-E was the most active fraction. UHPLC/MS analysis of the methanolic extract of C. arvensis disclosed the presence of 62 compounds as sterols, triterpenes, flavonoids, fatty acids, alkaloids and coumarins. In Multivariate Analysis, the total phenolic contents were correlated strongly with all antioxidant assays except FRAP and DPPH. Regarding enzyme inhibitory properties, only AChE, BChE and α-amylase were correlated with the total phenolic contents in the extracts. Docking analyses confirmed these findings, as identified compounds had high binding free energy and inhibition constants with the enzymes studied. It was finally concluded that C. arvensis is a potential industrial crop, which can be a component of nutraceuticals and functional foods, if evaluated for its toxicity.

A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study.

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1-3 were characterized by various spectroscopic techniques. The IC50 values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1-3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.

Identification of glucosyl-3-phosphoglycerate phosphatase as a novel drug target against resistant strain of Mycobacterium tuberculosis (XDR1219) by using comparative metabolic pathway approach.

Tuberculosis (TB) is a major global health challenge. It has been afflicting human for thousands of years and is still severely affecting a huge population. The etiological agent of the disease is Mycobacterium tuberculosis (MTB) that survives in the human host in latent, dormant, and non-replicative state by evading the immune system. It is one of the leading causes of infection related death worldwide. The situation is exacerbated by the massive increase in the resistant strains such as multi-drug resistant TB (MDR-TB) and extensive drug-resistant TB (XDR-TB). The resistance is as severe that it resulted in failure of the current chemotherapy regimens (i.e. anti-tubercular drugs). It is therefore imperative to discover the new anti-tuberculosis drug targets and their potential inhibitors. Current study has made the use of in silico approaches to perform the comparative metabolic pathway analysis of the MTBXDR1219 with the host i.e. H. sapiens. We identified several metabolic pathways which are unique to pathogen only. By performing subtractive genomic analysis 05 proteins as potential drug target are retrieved. This study suggested that the identified proteins are essential for the bacterial survival and non-homolog to the host proteins. Furthermore, we selected glucosyl-3-phosoglycerate phosphatase (GpgP, EC 5.4.2.1) out of the 05 proteins for molecular docking analysis and virtual screening. The protein is involved in the biosynthesis of methylglucose lipopolysaccharides (MGLPs) which regulate the biosynthesis of mycolic acid. Mycolic acid is the building block of the unique cell wall of the MTB which is responsible for the resistance and pathogenicity. A relatively larger library consisting of 10,431 compounds was screened using AutoDock Vina to predict the binding modes and to rank the potential inhibitors. No potent inhibitor against MTB GpgP has been reported yet, therefore ranking of compounds is performed by making a comparison with the substrate i.e. glucosyl-3-phosphoglycerate. The obtained results provide the understanding of underlying mechanism of interactions of ligands with protein. Follow up study will include the study of the Protein-Protein Interactions (PPIs), and to propose the potential inhibitors against them.

Evaluation of Antioxidant, Free Radical Scavenging, and Antimicrobial Activity of Quercus incana Roxb.

Considering the indigenous utilization of Quercus incana Roxb., the present study deals with the investigation of antioxidant, free radical scavenging activity, total phenolic content, and antimicrobial activity of Q. incana Roxb. In vitro antioxidant activity of the plant fractions were determined by 1,1-diphenyl-2-picrylhydrazyl and nitric oxide scavenging method. Total phenolic contents were determined by gallic acid equivalent and antimicrobial activities were determined by agar well diffusion method. It was observed that Q. incana Roxb. showed significant antibacterial activity against Gram-positive and Gram-negative bacteria. n-Butanol fraction showed maximum activity against Micrococcus leuteus with 19 mm zone of inhibition. n-Butanol fraction of Q. incana Roxb. showed immense antifungal activity against Aspergillus niger (32 mm ± 0.55) and A. flavus (28 mm ± 0.45). Similarly n-butanol fraction showed relatively good antioxidant activity with IC50 value of 55.4 ± 0.21 µg/mL. The NO scavenging activity of ethyl acetate fraction (IC50 = 23.21 ± 0.31 µg/mL) was fairly good compared to other fractions. The current study of Q. incana Roxb. suggests the presences of synergetic action of some biological active compounds that may be present in the leaves of medicinal plant. Further studies are needed to better characterize the important active constituents responsible for the antimicrobial, antioxidant and free radical scavenging activity.