Understanding the determinants for substrate recognition, regulation of enzymatic activity and the development of broad-spectrum inhibitors of coronavirus 3-chymotrypsin-like proteases

Coronaviruses 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

A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis.IMPORTANCEThe 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

Understanding the determinants for substrate recognition, regulation of enzymatic activity and the development of broad-spectrum inhibitors of coronavirus 3-chymotrypsin-like proteases

Coronaviruses 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.

A BAF’ling Approach to Curing HIV

Latency is the primary barrier to the development of a long-sought cure for HIV-1. In this issue of Cell Chemical Biology, Marian et al., (2018) describe the development of novel compounds targeting the BAF chromatin remodeling complex to reverse HIV latency, with the potential to provide a functional cure

InsurTech in insurance: The road ahead for Telematics in India

The Indian insurance sector is characterised by the presence of information asymmetry and moral hazard. Resultantly, the adoption of InsurTech has become a necessity. While telematics, a form of InsurTech, is gaining significant impetus globally, it is still at a nascent stage in India. Against this backdrop, the present study analyses the role of Telematics in reducing information asymmetries in the Indian motor insurance sector, thereby improving the risk assessment process and ensuring that the insurance premium is reflective of the risk taken by the policyholder. The novelty of the study is that it is one of the very few studies that have explored the implementation of Telematics in the Indian context. The study is exploratory and uses a mixed-method approach by analysing the use of telematics in developed countries through document analysis followed by semi-structured interviews with Indian vehicle owners and industry experts. The study highlights that Telematics devices can assist companies in reducing information asymmetries and enable precise ascertainment of insurance premiums based on factors such as driving habits, distance travelled, and driving patterns of the policyholders

Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and <i>in vitro</i> biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and <i>Saccharomyces cerevisiae</i> wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B

Recent Advances in Biomass Pretreatment Technologies for Biohydrogen Production

Hydrogen is an economical source of clean energy that has been utilized by industry for decades. In recent years, demand for hydrogen has risen significantly. Hydrogen sources include water electrolysis, hydrocarbon steam reforming, and fossil fuels, which emit hazardous greenhouse gases and therefore have a negative impact on global warming. The increasing worldwide population has created much pressure on natural fuels, with a growing gap between demand for renewable energy and its insufficient supply. As a result, the environment has suffered from alarming increases in pollution levels. Biohydrogen is a sustainable energy form and a preferable substitute for fossil fuel. Anaerobic fermentation, photo fermentation, microbial and enzymatic photolysis or combinations of such techniques are new approaches for producing biohydrogen. For cost-effective biohydrogen production, the substrate should be cheap and renewable. Substrates including algal biomass, agriculture residue, and wastewaters are readily available. Moreover, substrates rich in starch and cellulose such as plant stalks or agricultural waste, or food industry waste such as cheese whey are reported to support dark- and photo-fermentation. However, their direct utilization as a substrate is not recommended due to their complex nature. Therefore, they must be pretreated before use to release fermentable sugars. Various pretreatment technologies have been established and are still being developed. This article focuses on pretreatment techniques for biohydrogen production and discusses their efficiency and suitability, including hybrid-treatment technology

Recent Advances in Biomass Pretreatment Technologies for Biohydrogen Production

Hydrogen is an economical source of clean energy that has been utilized by industry for decades. In recent years, demand for hydrogen has risen significantly. Hydrogen sources include water electrolysis, hydrocarbon steam reforming, and fossil fuels, which emit hazardous greenhouse gases and therefore have a negative impact on global warming. The increasing worldwide population has created much pressure on natural fuels, with a growing gap between demand for renewable energy and its insufficient supply. As a result, the environment has suffered from alarming increases in pollution levels. Biohydrogen is a sustainable energy form and a preferable substitute for fossil fuel. Anaerobic fermentation, photo fermentation, microbial and enzymatic photolysis or combinations of such techniques are new approaches for producing biohydrogen. For cost-effective biohydrogen production, the substrate should be cheap and renewable. Substrates including algal biomass, agriculture residue, and wastewaters are readily available. Moreover, substrates rich in starch and cellulose such as plant stalks or agricultural waste, or food industry waste such as cheese whey are reported to support dark- and photo-fermentation. However, their direct utilization as a substrate is not recommended due to their complex nature. Therefore, they must be pretreated before use to release fermentable sugars. Various pretreatment technologies have been established and are still being developed. This article focuses on pretreatment techniques for biohydrogen production and discusses their efficiency and suitability, including hybrid-treatment technology