Molecular docking based virtual screening of natural compounds as potential BACE1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Beta-site APP cleaving enzyme1 (BACE1) catalyzes the rate determining step in the generation of Aβ peptide and is widely considered as a potential therapeutic drug target for Alzheimer’s disease (AD). Active site of BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein with the help of flap. Currently, there are no marketed drugs available against BACE1 and existing inhibitors are mostly pseudopeptide or synthetic derivatives. There is a need to search for a potent inhibitor with natural scaffold interacting with flap and Asp dyad. This study screens the natural database InterBioScreen, followed by three-dimensional (3D) QSAR pharmacophore modeling, mapping, in silico ADME/T predictions to find the potential BACE1 inhibitors. Further, molecular dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bond throughout the simulation. This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrates have been earlier used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility, and easy permeability through the blood–brain barrier. Our finding will be helpful in identify the potential leads to design novel BACE1 inhibitors for AD therapy.     

Cytochrome P450 reductase: a harbinger of diffusible reduced oxygen species

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1:1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100:1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically 'wasteful' and potentially 'hazardous' owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize "uncoupling" on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.     

Cholinesterase Inhibitors and Hospitalization for Bradycardia: A Population-Based Study

Background

Cholinesterase inhibitors are commonly used to treat dementia. These drugs enhance the effects of acetylcholine, and reports suggest they may precipitate bradycardia in some patients. We aimed to examine the association between use of cholinesterase inhibitors and hospitalization for bradycardia.

Methods and Findings

We examined the health care records of more than 1.4 million older adults using a case-time-control design, allowing each individual to serve as his or her own control. Case patients were residents of Ontario, Canada, aged 67 y or older hospitalized for bradycardia between January 1, 2003 and March 31, 2008. Control patients (3∶1) were not hospitalized for bradycardia, and were matched to the corresponding case on age, sex, and a disease risk index. All patients had received cholinesterase inhibitor therapy in the 9 mo preceding the index hospitalization. We identified 1,009 community-dwelling older persons hospitalized for bradycardia within 9 mo of using a cholinesterase inhibitor. Of these, 161 cases informed the matched analysis of discordant pairs. Of these, 17 (11%) required a pacemaker during hospitalization, and six (4%) died prior to discharge. After adjusting for temporal changes in drug utilization, hospitalization for bradycardia was associated with recent initiation of a cholinesterase inhibitor (adjusted odds ratio [OR] 2.13, 95% confidence interval [CI] 1.29–3.51). The risk was similar among individuals with pre-existing cardiac disease (adjusted OR 2.25, 95% CI 1.18–4.28) and those receiving negative chronotropic drugs (adjusted OR 2.34, 95% CI 1.16–4.71). We found no such association when we replicated the analysis using proton pump inhibitors as a neutral exposure. Despite hospitalization for bradycardia, more than half of the patients (78 of 138 cases [57%]) who survived to discharge subsequently resumed cholinesterase inhibitor therapy.

Conclusions

Among older patients, initiation of cholinesterase inhibitor therapy was associated with a more than doubling of the risk of hospitalization for bradycardia. Resumption of therapy following discharge was common, suggesting that the cardiovascular toxicity of cholinesterase inhibitors is underappreciated by clinicians.Please see later in the article for the Editors'' Summary     

Proteome-wide analysis of single-nucleotide variations in the N-glycosylation sequon of human genes

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can result in the gain of glycosylation. Although the general importance of glycosylation is well known and acknowledged, the effect of variation on the actual glycoproteome of an organism is still mostly unknown. In this study, we focus on a comprehensive analysis of non-synonymous single nucleotide variations (nsSNV) that lead to either loss or gain of the N-glycosylation motif. We find that 1091 proteins have modified N-glycosylation sequons due to nsSNVs in the genome. Based on analysis of proteins that have a solved 3D structure at the site of variation, we find that 48% of the variations that lead to changes in glycosylation sites occur at the loop and bend regions of the proteins. Pathway and function enrichment analysis show that a significant number of proteins that gained or lost the glycosylation motif are involved in kinase activity, immune response, and blood coagulation. A structure-function analysis of a blood coagulation protein, antithrombin III and a protease, cathepsin D, showcases how a comprehensive study followed by structural analysis can help better understand the functional impact of the nsSNVs.     

Multilocus sequence evaluation for differentiating species of the trematode Family Gastrothylacidae,with a note on the utility of mitochondrial COI motifs in species identification

Amphistomiasis, a neglected trematode infectious disease of ruminants, is caused by numerous species of amphistomes belonging to six families under the Superfamily Paramphistomoidea. In the present study, four frequently used DNA markers, viz. nuclear ribosomal 28S (D1–D3 regions), 18S and ITS2 and mitochondrial COI genes, as well as sequence motifs from these genes were evaluated for their utility in species characterization of members of the amphistomes' Family Gastrothylacidae commonly prevailing in Northeast India. In sequence and phylogenetic analyses the COI gene turned out to be the most useful marker in identifying the gastrothylacid species, with the exception of Gastrothylax crumenifer, which showed a high degree of intraspecific variations among its isolates. The sequence analysis data also showed the ITS2 region to be effective for interspecies characterization, though the 28S and 18S genes were found unsuitable for the purpose. On the other hand, sequence motif analysis data revealed the motifs from the COI gene to be highly conserved and specific for their target species which allowed accurate in silico identification of the gastrothylacid species irrespective of their intraspecific differences. We propose the use of COI motifs generated in the study as a potential tool for identification of these species.     

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.     

Gatifloxacin Versus Ofloxacin for the Treatment of Uncomplicated Enteric Fever in Nepal: An Open-Label,Randomized, Controlled Trial

Background

Fluoroquinolones are the most commonly used group of antimicrobials for the treatment of enteric fever, but no direct comparison between two fluoroquinolones has been performed in a large randomised trial. An open-label randomized trial was conducted to investigate whether gatifloxacin is more effective than ofloxacin in the treatment of uncomplicated enteric fever caused by nalidixic acid-resistant Salmonella enterica serovars Typhi and Paratyphi A.

Methodology and Principal Findings

Adults and children clinically diagnosed with uncomplicated enteric fever were enrolled in the study to receive gatifloxacin (10 mg/kg/day) in a single dose or ofloxacin (20 mg/kg/day) in two divided doses for 7 days. Patients were followed for six months. The primary outcome was treatment failure in patients infected with nalidixic acid resistant isolates. 627 patients with a median age of 17 (IQR 9–23) years were randomised. Of the 218 patients with culture confirmed enteric fever, 170 patients were infected with nalidixic acid-resistant isolates. In the ofloxacin group, 6 out of 83 patients had treatment failure compared to 5 out of 87 in the gatifloxacin group (hazard ratio [HR] of time to failure 0.81, 95% CI 0.25 to 2.65, p = 0.73). The median time to fever clearance was 4.70 days (IQR 2.98–5.90) in the ofloxacin group versus 3.31 days (IQR 2.29–4.75) in the gatifloxacin group (HR = 1.59, 95% CI 1.16 to 2.18, p = 0.004). The results in all blood culture-confirmed patients and all randomized patients were comparable.

Conclusion

Gatifloxacin was not superior to ofloxacin in preventing failure, but use of gatifloxacin did result in more prompt fever clearance time compared to ofloxacin. Trial registration: ISRCTN 63006567 (www.controlled-trials.com).     

Baculovirus studies in new,indigenous lepidopteran cell lines

Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.     

Lack of association between Glu298Asp polymorphism and coronary artery disease in North Indians

Nitric Oxide (NO) is an important molecule carrying number of different functions in humans. Published studies suggest that it may inhibit several key steps involved in the pathogenesis of atherosclerosis. Inhibition or reduction of NO due to Glu298Asp polymorphism may accelerate atherosclerosis. The aim of this study was to determine whether Glu298Asp polymorphism is implicated in the pathogenesis of coronary artery disease (CAD) among North Indian population from the state of Uttar Pradesh, India. We selected 253 CAD patients and 174 healthy, normotensive, non-diabetic controls, which were matched for gender and ethnicity. The Glu298Asp (rs1799983) variant was detected by genotyping subjects, using a polymerase chain reaction followed by restriction fragment length polymorphism. There was no significant difference found in the genotypic and allelic frequencies between patients and controls. Our study indicated that Glu298Asp polymorphism does not play any critical role in the pathogenesis of CAD, at least in North Indian population.