BackgroundThe Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India.MethodsA total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations.ResultsThe sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding.ConclusionsHence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Aim: This study was conducted to find the best suited freely available software for modelling of proteins by taking a few sample proteins. The proteins used were small to big in size with available crystal structures for the purpose of benchmarking. Key players like Phyre2, Swiss-Model, CPHmodels-3.0, Homer, (PS)2, (PS)2-V2, Modweb were used for the comparison and model generation. Results: Benchmarking process was done for four proteins, Icl, InhA, and KatG of Mycobacterium tuberculosis and RpoB of Thermus Thermophilus to get the most suited software. Parameters compared during analysis gave relatively better values for Phyre2 and Swiss-Model. Conclusion: This comparative study gave the information that Phyre2 and Swiss-Model make good models of small and large proteins as compared to other screened software. Other software was also good but is often not very efficient in providing full-length and properly folded structure.

Quinoline-based compounds are one of the most important classes of N-heterocyclics exhibiting a wide spectrum of biological activities. There is a constant demand for the synthesis of new quinoline-based molecules suitable for therapeutic applications. Here a new strategy is developed to synthesize different quinoline-based quinazolinones catalytically using InCl 3 through a cyclocondensation reaction of isatoic anhydride and aniline with 2-substituted-quinoline-3-carbaldehyde to produce 2-(2-phenoxyquinolin-3-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one derivatives (> 90 % yields). In contrast, the reduced electrophilicity at the metal center allows the hydrazide ligand to bind In(III) and serendipitously forms benzoic(2-phenoxyquinoline-3-carbonyl) carbamic anhydride (∼ 70 % yields). The mechanistic aspects of these reactions were rationally explained. The structure and purity of all the isolated derivatives were assessed by spectroscopic and analytical data. In view of a strong correlation existing between inflammation and cancer progression, the ex-vivo anti-inflammatory effect of the ligands was established. The anti-cancer property of the proposed ligand was delineated with a specific SIRT protein family. Further, the computational docking studies on the binding abilities of 22 synthesized compounds with the Sirt1 protein were discussed in detail.

Diabetes mellitus (DM) has spread across the globe, increasing the risk of obesity, cardiovascular disease, and other comorbidities. Despite substantial research into the development of diabetic treatments that are effective in lowering blood glucose levels, their efficiency is short‐lived due to unpleasant side effects such as weight gain and hypoglycemia. The discovery of secondary metabolites in the prevention and treatment of diabetes and its complications has an incentive to take interest in plant‐based medications, and enzyme inhibitors have the potential to aid in the treatment and management of DM. This study aims to isolate, characterize, and analyse the influence of berberine‐like alkaloids from alcoholic Cardiospermum halicacabum extract in vitro and in silico, as a possible inhibitor of Dipeptidyl peptidase‐IV (DPP‐IV) and α‐amylase, two essential enzymes involved in diabetes. The alkaloid from C. halicacabum was identified as berberine, with an m/z of 336.1263. Purified berberine inhibits DPP‐IV with an IC50 of 16.328 ± 1.344 μM and inhibits α‐amylase by 72% at 10 μg/mL. In‐silico studies demonstrated that berberine was found to bind to the active site of both DPP‐IV and α‐amylase. The precise mechanism underlying the observation has to be researched further in order to investigate C. halicacabum's anti‐diabetic effects and argue for its possible application as alternative medicine.

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Background: Enterococcus faecalis (Ef) infections are becoming dreadfully common in hospital environments. Infections caused by Ef are difficult to treat because of its acquired resistance to different class of antibiotics, making it a multidrug resistant bacteria. Key pathogenic factor of Ef includes its ability to form biofilm on the surface of diagnostic and other medical devices. Sortase A (SrtA) is a cysteine transpeptidase which plays a pivotal role in the formation of biofilm in Ef, hence, it is considered as an important enzyme for the pathogenesis of Ef. Thus, inhibition of Sortase A will affect biofilm formation, which will reduce its virulence and eventually Ef infection will be abridged. Objective: To find potential inhibitors of Enterococcus faecalis Sortase A (EfSrtA) through in-silico and in-vitro methods. Methods: Gene coding for EfSrtA was cloned, expressed and purified. Three-dimensional model of EfSrtA was created using Swiss-Model workspace. In-silico docking studies using Autodock vina and molecular dynamics simulations of the modelled structures using Gromacs platform were performed to explore potential lead compounds against EfSrtA. In-vitro binding experiments using spectrofluorometric technique was carried out to confirm and validate the study. Results: In-silico docking and in-vitro binding experiments revealed that curcumin, berberine and myricetin bound to EfSrtA at nanomolar concentrations with high affinity. Conclusion: This is a first structural report of EfSrtA with curcumin, berberine and myricetin. Taking in account the herbal nature of these compounds, the use of these compounds as inhibitors will be advantageous. This study validated curcumin, berberine and myricetin as potential inhibitors of EfSrtA.

Antiretroviral therapy is the single existing therapy for patients infected with HIV; however, it has drawbacks in terms of toxicity and resistance. Thus, there is a continuous need to explore safe and efficacious anti-retroviral agents. C-Phycocyanin (C-PC) is a phycobiliprotein, which has been known for various biological properties; however, its effect on HIV-1 replication needs revelation. This study aimed to identify the inhibitory effects of C-PC on HIV-1 using in vitro and in silico approaches and to assess its role in the generation of mitochondrial reactive oxygen species (ROS) during HIV-1 infection. In vitro anti-HIV-1 activity of C-PC was assessed on TZM-bl cells through luciferase gene assay against four different clades of HIV-1 strains in a dose-dependent manner. Results were confirmed in PBMCs, using the HIV-1 p24 antigen assay. Strong associations between C-PC and HIV-1 proteins were observed through in silico molecular simulation-based interactions, and the in vitro mechanistic study confirmed its target by inhibition of reverse transcriptase and protease enzymes. Additionally, the generation of mitochondrial ROS was detected by the MitoSOX and DCF-DA probe through confocal microscopy. Furthermore, our results confirmed that C-PC treatment notably subdued the fluorescence in the presence of the virus, thus reduction of ROS and the activation of caspase-3/7 in HIV-1-infected cells. Overall, our study suggests C-PC as a potent and broad in vitro antiviral and antioxidant agent against HIV-1 infection.

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