1,720,976 research outputs found
Understanding the determinants for substrate recognition, regulation of enzymatic activity and the development of broad-spectrum inhibitors of coronavirus 3-chymotrypsin-like proteases
Full text linkCoronaviruses include lethal human pathogens like severe acute respiratory syndrome coronavirus (SARS-CoV) and the recently emerged Middle-east respiratory coronavirus (MERS-CoV). Coronavirus also impact global economy by infecting farm animals like pigs (porcine epidemic diarrhea virus, PEDV), cows (bovine coronavirus, BCoV) and poultry (avian infectious bronchitis virus, IBV). Moreover, the global distribution of bats that naturally harbor one or more coronavirus strains heightens the likelihood of emergence of a novel highly pathogenic coronavirus in the near future. To combat infections of existing and emerging coronaviruses, it is important to identify coronavirus drug targets that can be utilized for the development of broad-spectrum anti-coronaviral therapeutics. Viral encoded 3-Chymotrypsin-like protease (3CLpro) is essential for viral polyprotein processing to release non-structural proteins that form the replicase complex machinery for viral genome replication. Due to its indispensable role in coronaviral replication, 3CLpro is an attractive drug target. Moreover, high sequence conservation in the vicinity of active site among 3CLpro proteases from different coronavirus subclasses make them an excellent target for the development of broad-spectrum therapeutics for coronavirus infections. The overarching goal of this project is to investigate enzymatic and structural properties of multiple 3CLpro enzymes encompassing different coronavirus subclasses. Understanding the determinants of structural and functional disparity between different 3CLpro enzymes and the factors regulating these properties will aid in the design of broad-spectrum inhibitors of 3CLpro enzymes. (Abstract shortened by ProQuest.
Crystallographic, kinetic and cellular studies on natural product inspired inhibitors of quinone reductase 2
No full textQuinone reductase 2 (QR2) is a cytosolic enzyme capable for activating compounds and rendering them capable of causing DNA damage (i.e. DNA alkylation). QR2 has recently been implicated in neurodegenerative diseases such as Parkinson\u27s Disease and schizophrenia, where patients show significant elevation in cellular levels of QR2. Also, the increased levels of reactive oxygen species (ROS) in these patients has been correlated with increased QR2 levels, as its electron transfer mechanisms activate quinones to generate ROS. Here, we report the identification of novel QR2 inhibitors that can potentially be used as new therapeutics for the treatment or prevention of neurodegenerative diseases or to decrease overall toxicity levels of xenobiotic compounds due to QR2. Furthermore, we utilized non-toxic natural products as starting scaffolds to identify inhibitors of QR2. First, the chemical extraction on blueberries, grapes and red wine was performed to isolate small molecules capable of binding to and inhibiting QR2 activity. Through the use of X-ray crystallographic assisted dereplication (CrystAssist), quercetin and quercetin-sugar derivatives were identified that bind to QR2. The binding of these molecules was verified using HPLC and UF-LC/MS (Ultra filtration), in conjunction with enzyme kinetic studies. The extracts were also tested for their ability to inhibit QR2, and the IC50 of the blueberry extract was approximately 10 µg/mL. Finally, a series of synthetic analogs was evaluated for their ability to inhibit QR2 activity, and a structure-activity relationship was developed that incorporated kinetic data, e.g. IC50 values and X-ray crystallographic data on QR2-inhibitor complexes. To further identify QR2 compounds with the potential to decrease oxidative stress (because of QR2 inhibition), we evaluated a series of synthetic resveratrol analogs. Similar to the quercetin analogs, we correlated their potency (IC 50 values) with their structures in complex with QR2, utilizing X-ray crystallography. In this series, over 80 resveratrol analogs were evaluated for inhibition towards QR2, of which measurable potency was observed in 30 compounds. The range in affinity of the 30 compounds was over 2000-fold (from 84 nM to 175 µM), the largest range in affinity as well as the largest number of compounds evaluated of one series as QR2 inhibitors. The third natural product scaffold explored was a metabolite, ammosamide, generated from the isolate of deep-sea marine extracts that showed potent QR2 inhibitory activity. A series of synthetic analogs of ammoasamide was found to display exceptionally high affinity towards QR2, with several compounds demonstrating IC50 values in the low nanomolar range. Using X-ray crystallography, the binding orientations of these analogs in complex with QR2 was determined and were found to be unique. Subtle substitutions in the ammosamide ring structures resulted in drastically different orientations within the QR2 active site. In general, the highest affinity compounds preferred a particular orientation, however, the third most potent compound in the series (PVN-4-28) flips over the y-axis and rotates + 90° relative to the two most potent inhibitors (PVN-5-59 and PVN-4-28). These correlations indicate that binding orientation is not entirely driving by potency of the compound. Finally, it was demonstrated that the novel QR2 inhibitors (from the ammosamide series) are capable of reversing cellular toxicity induced upon increasing menadione concentrations. Menadione is a quinone-containing compound that is metabolized by QR2. It has been hypothesized that menadione reduction results in radical formation, that with the reaction with molecular oxygen, superoxide is formed and is cytotoxic. Here, the addition of the ammosamide analogs was found to counteract the toxicity of increasing menadione concentrations. (Abstract shortened by UMI.
Structure-Based Drug Design of Selective BACE1 and BACE2 Inhibitors for the Treatments of Alzheimer\u27s Disease and Type II Diabetes
No full textThe BACE (Beta-site Amyloid precursor protein Cleaving Enzyme) protein family contains two members, BACE1 and BACE2. BACE1 has been demonstrated to be the major β-secretase involved in the production of amyloid beta (Aβ) peptide. The Aβ cascade hypothesis suggests that the accumulation of Aβ peptides in the brain would lead to the formation of plaques and tangles, which are the neuropathological hallmarks of Alzheimer disease (AD). Based upon this, inhibition of the enzymatic activity of BACE1 to prevent the production of Aβ peptides has been proposed as a novel therapeutic approach for the treatment of AD. Recently, the other member of this family, BACE2, has been identified as a potential target for the treatment of Type II diabetes (TIID). In 2011, BACE2 was shown to be a major sheddase of TMEM27, which is a transmembrane protein located on the pancreatic β cells. The full-length TMEM27 on pancreatic β cells would promote the proliferation of pancreatic β cells as well as stimulate the secretion of insulin. However, it loses its mitogenic activity once it is cleaved by BACE2. Therefore, the inhibition of the enzymatic activity of BACE2 would likely maintain the function of TMEM27 on pancreatic β cells and thus benefit TIID patients. This hypothesis has been tested using a BACE2 inactive mouse model and a diabetes mouse model treated with a BACE2 inhibitor. Increased insulin level and improved control of glucose homeostasis have been observed in both models, supporting BACE2 as a promising target for the treatment of TIID. Although both BACE1 and BACE2 have been proposed as promising therapeutic targets for the treatments of AD and TIID, selective inhibitors against either BACE1 or BACE2 are needed. From an animal study, it has been shown that BACE1/2 double knockout mice exhibit a higher mortality rate and a smaller body size compared to wild type mice. Therefore, the design of selective inhibitors is needed to prevent any undesired side effects. However, it is challenging to develop selective inhibitors against only BACE1 or BACE2 since they share high sequence similarity (68%). In this dissertation, the ultimate goal is to develop selective BACE1/2 inhibitors by structure-based drug design approaches. Both BACE1 and BACE2 are expressed in the insoluble inclusion body fractions when using E.coli as the expression system. Although there are a few reported protocols for production and purification of both BACE1 and BACE2 from E.coli in the literature, the ones that have reported methods are either time-consuming or costly. Here, the protein production and purification protocols have been optimized for both BACE1 and BACE2 from E.coli inclusion bodies. BACE1 and BACE2 can both be simply refolded in nanopure water without adding any refolding adjuvants, and it takes only 4 to 5 days to refold each enzyme to reach the maximum activity. After refolding, milligram quantities of BACE1 and BACE2 can be purified via chromatography. The purity of the resulting BACE1 and BACE2 purified proteins is greater than 95 % as judged by SDS-PAGE and their resulting specific activities are 126 and 330 µM/min/mg. The steady-state kinetic parameters of BACE1 and BACE2 were determined using a fluorogenic FRET (fluorescence resonance energy transfer)-based peptide substrate derived from the β-secretase cleavage site of the Swedish APP mutation. The purified BACE1 enzyme has a catalytic efficiency (k cat /Km) of 0.58 min –1 µM–1 (k cat = 8.0 ± 0.4 min–1, K m = 18.3 ± 1.6 µM ). The catalytic efficiency ( kcat / Km) of purified BACE2 is 0.23 min–1 µM–1 ( kcat = 5.7 ± 0.5 min–1, Km = 24.8 ± 4.7 µM). The crystallization conditions for both enzymes were also optimized for structural characterization and drug design. In collaboration with Prof. Arun Ghosh’s lab in the chemistry department at Purdue University, structure activity relationship (SAR) studies were performed to identify potent and selective inhibitors against either BACE1 or BACE2. Since the inhibitory constant (Ki) values ranged from subnanomolar to micromolar for each individual compound tested, different kinetic models were applied to determine the Ki values of compounds For the classical inhibitors, the Michaelis - Menten model was used to fit the kinetic data to obtain individual K i value of each compound. The Morrison equation was utilized for potent inhibitors since the assays were performed under the tight binding condition ([E] ≤ [I]). The detailed steps of applying the Morrison equation for curve fitting are also reported here. First and foremost, an accurate active enzyme concentration has to be determined using a titration exper- iment in order to obtain accurate Ki values with the Morrison equation. (Abstract shortened by ProQuest.
Structure-based design, synthesis and biological evaluation of novel β-secretase inhibitors containing a pyrazole or thiazole moiety as the P3 ligand
No full textWe describe structure-based design, synthesis, and biological evaluation of a series of novel inhibitors bearing a pyrazole (compounds 3a-h) or a thiazole moiety (compounds 4a-e) as the P3 ligand. We have also explored Boc-β-amino-l-alanine as a novel P2 ligand. A number of inhibitors have displayed β-secretase inhibitory potency. Inhibitor 4c has shown potent BACE1 inhibitory activity, Ki = 0.25 nM, cellular EC50 of 194 nM, and displayed good selectivity over BACE2. A model of 4c was created based upon the X-ray structure of 2-bound β-secretase which revealed critical interactions in the active site
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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