SARS-CoV-2 Nsp13 Catalytic Efficiency is Regulated by ATP: Mg2+ Stoichiometry and Functional Cooperativity Among Nsp13 Molecules

Coronavirus disease 19 (COVID-19) is a highly contagious and lethal disease caused by the SARS-CoV-2 positive-strand RNA virus. Nonstructural protein 13 (Nsp13) is the highly conserved ATPase/helicase required for replication of the SARS-CoV-2 genome which allows for the infection and transmission of COVID-19. We biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein expressed using a eukaryotic cell-based system and characterized its catalytic functions, focusing on optimization of its reaction conditions and assessment of functional cooperativity among Nsp13 molecules during unwinding of duplex RNA substrates. These studies allowed us to carefully determine the optimal reaction conditions for binding and unwinding various nucleic acid substrates. Previously, ATP concentration was suggested to be an important factor for optimal helicase activity by recombinant SARS-CoV-1 Nsp13. Apart from a single study conducted using fixed concentrations of ATP, the importance of the essential divalent cation for Nsp13 helicase activity had not been examined. Given the importance of the divalent metal ion cofactor for ATP hydrolysis and helicase activity, we assessed if the molar ratio of ATP to Mg2+ was important for optimal SARS-CoV-2 Nsp13 RNA helicase activity. We determined that Nsp13 RNA helicase activity was dependent on ATP and Mg2+ concentrations with an optimum of 1 mM Mg2+ and 2 mM ATP. Next, we examined Nsp13 helicase activity as a function of equimolar ATP:Mg2+ ratio and determined that helicase activity decreased as the equimolar concentration increased, especially above 5 mM. We determined that Nsp13 catalytic functions are sensitive to Mg2+ concentration suggesting a regulatory mechanism for ATP hydrolysis, duplex unwinding, and protein remodeling, processes that are implicated in SARS-CoV-2 replication and proofreading to ensure RNA synthesis fidelity. Evidence is presented that excess Mg2+ impairs Nsp13 helicase activity by dual mechanisms involving both allostery and ionic strength. In addition, using single-turnover reaction conditions, Nsp13 unwound partial duplex RNA substrates of increasing doublestranded regions (16-30 base pairs) with similar kinetic efficiency, suggesting the enzyme unwinds processively in this range under optimal reaction conditions. Furthermore, we determined that Nsp13 displayed sigmoidal behavior for helicase activity as a function of enzyme concentration, suggesting that functional cooperativity and oligomerization are important for optimal activity. The observed functional cooperativity of Nsp13 protomers suggests the essential coronavirus RNA helicase has roles in RNA processing events beyond its currently understood involvement in the SARS-CoV-2 replication-transcription complex (RTC), in which it was suggested that only one of the two Nsp13 subunits has a catalytic function, whereas the other has only a structural role in complex stability. Altogether, the intimate regulation of Nsp13 RNA helicase by divalent cation and protein oligomerization suggests drug targets for modulation of enzymatic activity that may prove useful for the development of novel anti-coronavirus therapeutic strategies. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Ecofriendly aminochalcogenation of alkenes: a green alternative to obtain compounds with potential anti-SARS-CoV-2 activity

Here, we report a solvent- and metal-free methodology for the aminochalcogenation of alkenes, using molecular iodine as a catalyst, DMSO as a stoichiometric oxidant, and different nucleophiles under microwave irradiation. This ecofriendly approach provided the desired products with good to excellent yields in just 20 minutes. In addition, twenty compounds obtained by this methodology were referred for evaluation of potential antiviral activity against the coronavirus SARS-CoV-2. Two of the evaluated compounds (named 4d and 5b) showed potent antiviral activity, with a low cytotoxic profile resulting in a promising selectivity index.Copyright © 2023 The Royal Society of Chemistry.

Structural elucidation of 1:4:4 stochiometric form of thymine – gallic acid cocrystal hydrate: Hirshfeld surface analysis, 3D energy framework, DFT calculations, and SARS CoV-2 docking studies

Physical and chemical property enhancement of an active pharmaceutical ingredient through a multicomponent form is an integral part of the pharmaceutical research. A novel binary compound of thymine (TY) with gallic acid (GA) has been obtained by a solid-state grinding process and preliminarily characterized by FTIR technique. The single crystals were obtained and the 3D molecular crystal structure was confirmed by X-ray diffraction studies. The novel binary compound was crystallized as a cocrystal hydrate [TY-GA-H2O] in the monoclinic P21/c space group with an 1:4:4 stoichiometry. Crystal structure analysis revealed the presence of both intra and intermolecular hydrogen bond interactions, which resulted in the formation of various supramolecular architectures. Further, the structural analysis explored the presence of lone pair–π and π…π interactions apart from the conventional hydrogen bond interactions. Furthermore, the Hirshfeld surface analysis was carried out to quantify the contribution of hydrogen bond interactions in stabilizing the crystal structure. The individual interaction energies and the total interaction energy between the molecules were computed through energy framework analysis. HOMO-LUMO plots and MEP surface were generated to understand the electronic structure and chemical reactive sites of the molecule respectively. Finally, the molecular docking analysis was carried out to examine the antiviral properties of the novel TY-GA cocrystal hydrate.

Studying Kinetic and Thermodynamic Parameters of the Interaction of Ceftriaxone with Albumin Extracted from Healthy and Covid-19 Plasma

Because it has been well defined, bovine serum albumin (BSA) is highly suited to pharmacological effects, biotransformation, and bio-distribution of medicines initial research. Drug–protein interactions in the blood stream are known to have a significant impact on drug distribution, free concentration, and metabolism. Coronavirus disease 2019 (COVID-19) has spread globally as a severe pandemic. It is a serious threat to healthcare systems, economies, and is devastating to some populations, such as the elderly and those with comorbidities. Unfortunately, there is still no effective cure for COVID-19, especially the critically ill patients. The link between the mortality risk of patients hospitalized for COVID-19 and the function of blood albumin levels has been investigated. Because of albumin biological significance, the study goal conducted to apply spectroscopic methods to explore and comparing the kinetic and thermodynamic aspects of ceftriaxone's interaction with BSA, albumin isolated from healthy and covid-19 plasma. A pooled plasma from healthy and hospitalized covid-19 individuals (Karbala Province / Iraq) was used to purify albumin using HPLC technique. UV-vis spectrophotometric measurements of albumin-ceftriaxone complex formation recorded at different pH (7, 7.2, 7.4, 7.6 & 7.8) in phosphate buffer solution, and at six different temperatures (298, 301, 304, 307, 310 & 313) K. The equilibrium constant and the thermodynamic parameter such as ΔG, ΔH and ΔS were calculated. The drug-albumin (BSA, healthy and Covid-19 albumin) complexes are stable in 60 to 300 minutes, as evidenced by the steady absorbance studies. The reaction is from the first false order for drug-albumin (BSA, healthy and covid-19 plasma) complexes. Our finding suggesting the reaction is from the first false order. In the case of plasma albumin from individuals infected with - Covid-19, the values of (R2) are closer together. This is because the medication dominates the formation of a more stable complex with albumin. The stoichiometric ratio (coordination number) of complex between ceftriaxone and albumin at 298 k and pH=7.4 is 1:1. The Gibbs free energy for albumin-ceftriaxone is negative, indicating that the reaction is spontaneous. The positive enthalpy of contact indicates that the process is endothermic, requiring energy input. Positive enthalpy and entropy change also refer to the hydrophobic association and electrostatic contact that occurs between albumin molecules and ceftriaxone. It is worth noting that the complex formed between bovine albumin and the medication has less absorbency at pH = 7.4. That is, the complex is more stable, and it prefers natural helical shapes. Additionally, as the pH value shifts away from physiological (> 7.4 <), the intensity of complex absorption increases.

Harry Potter Themed Digital Escape Room for Addressing Misconceptions in Stoichiometry

Chemistry is often seen as an and content-heavy subject. Many students struggle with chemistry because they attempt to memorize the content without understanding the concepts. As a result, students often have misconceptions. COVID-19 has driven teaching and learning online, and an escape room teaching method, which is a way to enhance student engagement, has gained popularity among educators in higher education. This study examines the effectiveness of teaching through a digital escape room as compared to a typical online lesson with a collaborative learning method to address misconceptions in stoichiometry. A Harry Potter themed digital escape room is created to spark the students' interest in chemistry and address misconceptions. Thirty-eight students from the Nanyang Polytechnic Foundation Program participated in this study. The students completed a pretest, a post-test, and a survey, in addition to participating in the digital escape room and a typical online lesson. Four topics were covered in this study: balancing chemical equations, calculating empirical formulas, identifying the type of chemical bonding, and interpreting element symbols. Out of these four topics, it was discovered that students tended to have difficulty calculating empirical formulas. It was found that, on average, students showed a 10% improvement in test scores after being taught through the digital escape room. This result is similar to results obtained from a typical online lesson with a collaborative learning method (9% improvement). This implies that a digital escape room is equally as effective as a typical online lesson with a collaborative learning method at addressing misconceptions. Teaching through a digital escape room has shown potential additional benefits of enhancing soft skills, promoting teamwork, the ability to work under time pressure, communication skills, innovation competency, and increasing student motivation. The researcher recommends the use of a digital escape room to complement typical lessons for these additional benefits.

Lung on a Chip Development from Off-Stoichiometry Thiol-Ene Polymer.

Current in vitro models have significant limitations for new respiratory disease research and rapid drug repurposing. Lung on a chip (LOAC) technology offers a potential solution to these problems. However, these devices typically are fabricated from polydimethylsiloxane (PDMS), which has small hydrophobic molecule absorption, which hinders the application of this technology in drug repurposing for respiratory diseases. Off-stoichiometry thiol-ene (OSTE) is a promising alternative material class to PDMS. Therefore, this study aimed to test OSTE as an alternative material for LOAC prototype development and compare it to PDMS. We tested OSTE material for light transmission, small molecule absorption, inhibition of enzymatic reactions, membrane particle, and fluorescent dye absorption. Next, we microfabricated LOAC devices from PDMS and OSTE, functionalized with human umbilical vein endothelial cell (HUVEC) and A549 cell lines, and analyzed them with immunofluorescence. We demonstrated that compared to PDMS, OSTE has similar absorption of membrane particles and effect on enzymatic reactions, significantly lower small molecule absorption, and lower light transmission. Consequently, the immunofluorescence of OSTE LOAC was significantly impaired by OSTE optical properties. In conclusion, OSTE is a promising material for LOAC, but optical issues should be addressed in future LOAC prototypes to benefit from the material properties.

Patient-blood management for COVID19 convalescent plasma therapy: relevance of affinity and donor-recipient differences in concentration of neutralizing antibodies.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) is being extensively investigated as a treatment, with mixed results to date. Overall, there has been a generalized lack of appropriateness in prescriptions, which, in the field of transfusion medicine, is termed patient-blood management. OBJECTIVES: We aimed to separate study design variables that could affect clinical outcome after CCP therapy. We focus here on variables such as pretransfusion antibody testing in recipients, dose adjustments and antibody affinity measurements. SOURCES: We searched PubMed and preprint servers for relevant preclinical and clinical studies discussing each of these variables in the field of CCP therapy. CONTENT: We show evidence that neglecting those variables has affected the outcomes of the vast majority of CCP clinical trials to date. IMPLICATIONS: A better understanding of such variables will improve the design of the next generation of CCP clinical trials. This will likely lead to better clinical outcomes and will minimize risks of immune evasion from subneutralizing doses of neutralizing antibodies.

The Mechanism of SARS-CoV-2 Nucleocapsid Protein Recognition by the Human 14-3-3 Proteins.

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.