Using Synthetic Biology methods to construct a functional estrogen biosensor based on split Nanoluciferase activity

The presence of estrogenic compounds (endocrine-disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time-consuming techniques such as liquid chromatography-mass spectrometry and high-performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses methodology developed in the Wells lab (Elledge et al, 2021) to detect SARS-CoV-2 antibodies. This methodology takes advantage of the split Nanoluciferase (spLUC) protein divided into two functional domains (designated SmBit and LgBit). Based on rational engineering design we express dimerization dependent LgBit and SmBit fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric proteins will dimerize in the presence of estrogen, reconstitute the split luciferase enzyme and reestablish enzyme activity. Cells can be lysed, and luminescence detected to quantify estrogen present in the sample. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual-functional biosensor and its effectiveness for detection of estrogenic compounds in contaminated water. NSF-REU-1852150, REU Site: A multisite REU in Synthetic Biology, 2019.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

Structural insights into SARS-CoV-2 main protease conformational plasticity.

The spread of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants underscores the need for insights into the structural properties of its structural and non-structural proteins. The highly conserved homo-dimeric chymotrypsin-like protease (3CL MPRO ), belonging to the class of cysteine hydrolases, plays an indispensable role in processing viral polyproteins that are involved in viral replication and transcription. Studies have successfully demonstrated the role of MPRO as an attractive drug target for designing antiviral treatments because of its importance in the viral life cycle. Herein, we report the structural dynamics of six experimentally solved structures of MPRO (i.e., 6LU7, 6M03, 6WQF, 6Y2E, 6Y84, and 7BUY including both ligand-free and ligand-bound states) at different resolutions. We have employed a structure-based balanced forcefield, CHARMM36m through state-of-the-art all-atoms molecular dynamics simulations at µ-seconds scale at room temperature (303K) and pH 7.0 to explore their structure-function relationship. The helical domain-III responsible for dimerization mostly contributes to the altered conformational states and destabilization of MPRO . A keen observation of the high degree of flexibility in the P5 binding pocket adjoining domain II-III highlights the reason for observation of conformational heterogeneity among the structural ensembles of MPRO . We also observe a differential dynamics of the catalytic pocket residues His41, Cys145, and Asp187, which may lead to catalytic impairment of the monomeric proteases. Among the highly populated conformational states of the six systems, 6LU7 and 7M03 forms the most stable and compact MPRO conformation with intact catalytic site and structural integrity. Altogether, our findings from this extensive study provides a benchmark to identify physiologically relevant structures of such promising drug targets for structure-based drug design and discovery of potent drug-like compounds having clinical potential.

Disruption of 3CLpro protease self-association by short peptides as a potential route to broad spectrum coronavirus inhibitors.

Coronaviruses have posed a persistent threat to human health over the last two decades. Despite the accumulated knowledge about coronavirus-related pathogens, development of an effective treatment for its new variant COVID-19 is highly challenging. For the highly-conserved and main coronavirus protease 3CLpro, dimerization is known to be essential for its catalytic activity and thereby for virus proliferation. Here, we assess the potential of short peptide segments to disrupt dimerization of the 3CLpro protease as a route to block COVID-19 proliferation. Based on the X-ray structure of the 3CLpro dimer, we identified the SPSGVY126QCAMRP dodecapeptide segment as overlapping the hotspot regions on the 3CLpro dimer interface. Using computational blind docking of the peptide to the 3CLpro monomer, we found that the SPSGVY126QCAMRP peptide has favourable thermodynamic binding (ΔG= -5.93 kcal/mol) to the hotspot regions at the 3CLpro dimer interface. Importantly, the peptide was also found to preferentially bind to the hotspot regions compared to other potential binding sites lying away from the dimer interface (ΔΔG=-1.31 kcal/mol). Docking of peptides corresponding to systematic mutation of the V125 and Y126 residues led to the identification of seven peptides, SPSGHAQCAMRP, SPSGVTQCAMRP, SPSGKPQCAMRP, SPSGATQCAMRP, SPSGWLQCAMRP, SPSGAPQCAMRP and SPSGHPQCAMRP, that outperform the wild-type SPSGVY126QCAMRP peptide in terms of preferential binding to the 3CLpro dimer interface. These peptides have the potential to disrupt 3CLpro dimerization and therefore could provide lead structures for the development of broad spectrum COVID-19 inhibitors.Communicated by Ramaswamy H. Sarma.

Defective ORF8 dimerization in SARS-CoV-2 delta variant leads to a better adaptive immune response due to abrogation of ORF8-MHC1 interaction.

In India, during the second wave of the COVID-19 pandemic, the breakthrough infections were mainly caused by the SARS-COV-2 delta variant (B.1.617.2). It was reported that, among majority of the infections due to the delta variant, only 9.8% percent cases required hospitalization, whereas only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections cases were observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Downregulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant resulted in structural instability of ORF8 dimer caused by disruption of hydrogen bonds and salt bridges as revealed by structural analysis and MD simulation studies. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild-type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 of SARS-CoV-2 delta variant may not be hindering the MHC-I expression thereby resulting in a better immune response against the SARS-CoV-2 delta variant, which partly explains the possible reason for sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India.

Regulation of Retroviral and SARS-CoV-2 Protease Dimerization and Activity through Reversible Oxidation.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

Double chelation of Iron through dimer formation of favipiravir: Density functional theory analysis

This work was performed to examine an idea about full chelation of Iron (Fe) by well-known favipiravir (Fav) as a possible mechanism of action for medication of COVID-19 patients. To this aim, formations of Fe- mediated dimers of Fav were investigated by performing density functional theory (DFT) computations of electronic and structural features for singular and dimer models. The results indicated that the models of dimers were suitable for formation, in which two cis (D1) and trans (D2) models were obtained regarding the configurations of two Fav counterparts towards each other. Energy results indicated that formation of D1 was slightly more favorable than formation of D2. Molecular orbital features affirmed hypothesized interacting sites of Fav for Fe-mediated dimers formations, in which atomic charges and other molecular orbital related representations affirmed such achievements. Moreover, detection of such dimer formation was also possible by monitoring variations of molecular orbitals features. As a consequence, formations of Fe-mediated dimers of Fav could be achievable for possible removal of excess of Fe as a proposed mechanism of action for Fav in medication of COVID-19 patients.

Mutational analysis of ORF-8 of SARS-CoV-2 -a window to immunotherapy

Background: TGF-Beta plays an important role in immune evasion in oncology. Similarly, SARSCov-2, the causal agent of the COVID-19 pandemic, also has an immune evasion function. This is mediated by ORF-8 through its interaction with multiple immune regulatory elements, including TGF-beta. This is a mutational analysis of ORF-8. Methods: We took advantage of the database of millions of SARS-CoV-2 genomes are archived and organized in phylogenetic relationships to show the evolution of ORF-8. Site numbering and genome structure use Wuhan-Hu-1/2019 as reference. The phylogeny is rooted relative to early samples from Wuhan. Temporal resolution assumes a nucleotide substitution rate of 8 × 10-4 subs per site per year. ( https://nextstrain.org/). The epidemiological data provided at https://ourworldindata.org/coronavirus was used to determine the property of the variants using mortality and infectivity data at the site. Results: Scan of ORF-8 revealed a high rate of mutation at aa119 and aa120. More importantly, the mutation at 120 or 119 that resulted in null ORF8 clearly delineates the pre-Delta and Delta SARSCov-2. In fact, all the delta lineages exhibited the null mutation at 119/120. This region is important for the dimerization of ORF-8 and possibly its interaction with host TGF-beta. All other variants, including the alpha variants, are wild type (aa120 = F). Monitoring the mutations over the last several months indicated that the delta variants have now picked up the wild type F at aa120 (Faa120) in Egypt or the L at aa 120 (Laa120) in India. The epidemiology of Egypt and India indicates that the Faa120 is more immune evasive and suggestive that more infectious but not more lethal. Conclusions: This is an opportunity to monitor in real-time the evolution of ORF-8 and how it is interacting with the host immune system. Additionally, since our current clinical trial on TGF-beta inhibitors is in India and Latin America, it is an opportunity to correlate clinical findings to molecular and epidemiological data for these variants. If we are correct, the Faa120 will emerge as the dominant variant in the next wave of COVID-19.

A crystallographic snapshot of SARS-CoV-2 main protease maturation process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N- and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domainthree over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous Nand C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro;also known as 3CL(pro)) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anticoronavirus agents. We solved the crystal structures of the main protease of SARSCoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19.

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

The role of dancing duplexes in biology and disease.

Across species, a common protein assembly arises: proteins containing structured domains separated by long intrinsically disordered regions, and dimerized through a self-association domain or through strong protein interactions. These systems are termed "IDP duplexes." These flexible dimers have roles in diverse pathologies including development of cancer, viral infections, and neurodegenerative disease. Here we discuss the role of disorder in IDP duplexes with similar domain architectures that bind hub protein, LC8. LC8-binding IDP duplexes are categorized into three groups: IDP duplexes that contain a self-association domain that is extended by LC8 binding, IDP duplexes that have no self-association domain and are dimerized through binding several copies of LC8, and multivalent LC8-binders that also have a self-association domain. Additionally, we discuss non-LC8-binding IDP duplexes with similar domain organizations, including the Nucleocapsid protein of SARS-CoV-2. We propose that IDP duplexes have structural features that are essential in many biological processes and that improved understanding of their structure function relationship will provide new therapeutic opportunities.

Asymmetric dynamics of dimeric SARS-CoV-2 and SARS-CoV main proteases in an apo form: Molecular dynamics study on fluctuations of active site, catalytic dyad, and hydration water.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world. It is necessary to examine the viral proteins that play a notorious role in the invasion of our body. The main protease (3CLpro) facilitates the maturation of the coronavirus. It is thought that the dimerization of 3CLpro leads to its catalytic activity; the detailed mechanism has, however, not been suggested. Furthermore, the structural differences between the predecessor SARS-CoV 3CLpro and SARS-CoV-2 3CLpro have not been fully understood. Here, we show the structural and dynamical differences between the two main proteases, and demonstrate the relationship between the dimerization and the activity via atomistic molecular dynamics simulations. Simulating monomeric and dimeric 3CLpro systems for each protease, we show that (i) global dynamics between the two different proteases are not conserved, (ii) the dimerization stabilizes the catalytic dyad and hydration water molecules behind the dyad, and (iii) the substrate-binding site (active site) and hydration water molecules in each protomer fluctuate asymmetrically. We then speculate the roles of hydration water molecules in their catalytic activity.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

1H, 13C, and 15N backbone chemical shift assignments of the C-terminal dimerization domain of SARS-CoV-2 nucleocapsid protein.

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

The Computational Intervention of Macrolide Antibiotics in the Treatment of COVID-19.

BACKGROUND: The spike (S) glycoprotein of SARS corona virus (SARS-CoV-2) and human Angiotensin- converting enzyme 2 (ACE2), are both considered the key factors for the initiation of virus infection. The present work is an effort for computational target to block the spike proteins (S) and ACE2 receptor proteins with Macrolide antibiotics like Azithromycin, (AZM), Clarithromycin (CLAM) and Erythromycin (ERY) along with RNA-dependent RNA polymerase (RdRp). METHODS: Three-dimensional structure of the SARS-CoV-2RdRp was built by the SWISS-MODEL server, the generated structure showed 96.35% identity to the available structure of SARS-Coronavirus NSP12 (6NUR), for model validity, we utilized the SWISS-model server quality parameters and Ramachandran plots. RESULTS: These compounds were able to block the residues (Arg553, Arg555, and Ala558) surrounding the deep grove catalytic site (Val557) of RdRp and thus plays an important role in tight blocking of enzyme active site. Reference drug Remdesivir was used to compare the docking score of antibiotics with RdRp. Docking value exhibited good binding energy (-7.7 up to -8.2 kcal/mol) with RdRp, indicating their potential as a potent RdRp inhibitor. Interaction of CLAM and ERY presented low binding energy (-6.8 and -6.6) with the ACE2 receptor. At the same time, CLAM exhibited a good binding affinity of -6.4 kcal/mol, making it an excellent tool to block the attachment of spike protein to ACE2 receptors. Macrolides not only affected the attachment to ACE2 but also blocked the spike proteins further, consequently inhibiting the internalization in the host cell. Three Alkyl bonds between Arg555, Ala558, and Met542 by CLAM and two Alkyl bonds of Arg624 and Lys621 by ERY plays an important role for RdRp inactivation, that can prevent the rise of newly budded progeny virus. These macrolides interacted with the main protease protein in the pocket responsible for the dimerization and catalytic function of this protein. The interaction occurred with residue Glu166, along with the catalytic residues (Tyr343, and His235) of Endoribonuclease (NSP15) protein. CONCLUSION: The present study gives three-way options either by blocking S proteins or ACE2 receptor proteins or inhibiting RdRp to counter any effect of COVID-19 by macrolide and could be useful in the treatment of COVID-19 till some better option available.

Impact of dimerization and N3 binding on molecular dynamics of SARS-CoV and SARS-CoV-2 main proteases.

SARS-CoV-2 main protease is one of the major targets in drug development efforts against Covid-19. Even though several structures were reported to date, its dynamics is not understood well. In particular, impact of dimerization and ligand binding on the dynamics is an important issue to investigate. In this study, we performed molecular dynamics simulations of SARS-CoV and SARS-CoV-2 main proteases to investigate influence of dimerization on the dynamics by modeling monomeric and dimeric apo and holo forms. The dimerization causes an organization of the interdomain dynamics as well as some local structural changes. Moreover, we investigated impact of a peptide mimetic (N3) on the dynamics of SARS-CoV and SARS-CoV-2 Mpro. The ligand binding to the dimeric forms causes some key local changes at the dimer interface and it causes an allosteric interaction between the active sites of two protomers. Our results support the idea that only one protomer is active on SARS-CoV-2 due to this allosteric interaction. Additionally, we analyzed the molecular dynamics trajectories from graph theoretical perspective and found that the most influential residues - as measured by eigenvector centrality - are a group of residues in active site and dimeric interface of the protease. This study may form a bridge between what we know about the dynamics of SARS-CoV and SARS-CoV-2 Mpro. We think that enlightening allosteric communication of the active sites and the role of dimerization in SARS-CoV-2 Mpro can contribute to development of novel drugs against this global health problem as well as other similar proteases. Communicated by Ramaswamy H. Sarma.

Current concepts in the development of therapeutics against human and animal coronavirus diseases by targeting NP.

The coronavirus (CoV) infects a broad range of hosts including humans as well as a variety of animals. It has gained overwhelming concerns since the emergence of deadly human coronaviruses (HCoVs), severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2015. Very recently, special attention has been paid to the novel coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 due to its high mobility and mortality. As the COVID-19 pandemic continues, despite vast research efforts, the effective pharmaceutical interventions are still not available for clinical uses. Both expanded knowledge on structure insights and the essential function of viral nucleocapsid (N) protein are key basis for the development of novel, and potentially, a broad-spectrum inhibitor against coronavirus diseases. This review aimed to delineate the current research from the perspective of biochemical and structural study in cell-based assays as well as virtual screen approaches to identify N protein antagonists targeting not only HCoVs but also animal CoVs.

Caffeine-Operated Synthetic Modules for Chemogenetic Control of Protein Activities by Life Style.

A genetically encoded caffeine-operated synthetic module (COSMO) is introduced herein as a robust chemically induced dimerization (CID) system. COSMO enables chemogenetic manipulation of biological processes by caffeine and its metabolites, as well as caffeinated beverages, including coffee, tea, soda, and energy drinks. This CID tool, evolved from an anti-caffeine nanobody via cell-based high-throughput screening, permits caffeine-inducible gating of calcium channels, tumor killing via necroptosis, growth factors-independent activation of tyrosine receptor kinase signaling, and enhancement of nanobody-mediated antigen recognition for the severe acute respiratory distress coronavirus 2 (SARS-CoV-2) spike protein. Further rationalized engineering of COSMO leads to 34-217-fold enhancement in caffeine sensitivity (EC50 = 16.9 nanomolar), which makes it among the most potent CID systems like the FK506 binding protein (FKBP)-FKBP rapamycin binding domain (FRB)-rapamycin complex. Furthermore, bivalent COSMO (biCOMSO) connected with a long linker favors intramolecular dimerization and acts as a versatile precision switch when inserted in host proteins to achieve tailored function. Given the modularity and high transferability of COMSO and biCOSMO, these chemical biology tools are anticipated to greatly accelerate the development of therapeutic cells and biologics that can be switched on and off by caffeinated beverages commonly consumed in the daily life.