Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

Trash to treasure: Sulfonation-assisted transformation of waste masks into high-performance carbon anode for sodium-ion batteries

The global pandemic of COVID-19 poses significant challenge to the recycling of disposable polypropylene (PP)-based waste masks. Herein, a simple but effective sulfonation route has been proposed to transform PP-based waste masks into value-added hard carbon (CM) anode materials for advanced sodium-ion batteries. The sulfonation treatment improves the thermal stability of the PP molecule, preventing their complete decomposition and the release of massive gas molecules during the carbonization process. Meanwhile, the oxygen functional groups introduced during sulfonation effectively facilitates the cross-linking between the PP chains, hindering the rearrangement of carbon microcrystalline structures and enhancing its structural disorder. As a result, the prepared hard carbon anode (CM-180) with a high disorder degree and minimal surface defects realizes a high sodium storage capacity of 327.4 mAh g−1 with excellent cycle and rate capability. In addition, when coupled with O3–NaNi1/3Fe1/3Mn1/3O2 cathode, the fabricated sodium-ion full cell delivers a high energy density of 238 Wh kg−1 and achieves an outstanding rate capability with a retained capacity of 75 mAh g−1 even at an ultrahigh current rate of 50 C. This work offers a novel insight into transforming the waste masks to value-added hard carbons with promising prospects for sodium-ion batteries. © 2023

Biobased polymer SF/PHBV composite nanofiber membranes as filtration and protection materials

With the emergence of the COVID-19, masks and protective clothing have been used in huge quantities. A large number of non-degradable materials have severely damaged the ecological environment. Now, people are increasingly pursuing the use of environmentally friendly materials to replace traditional chemical materials. Silk fibroin (SF) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have received increasing attention because of their unique biodegradability and biocompatibility. In this paper, a series of biodegradable SF/PHBV nanofiber membranes with different PHBV content were fabricated by using electrospinning technology. The morphology of the electrospun SF/PHBV composite nanofiber was observed by scanning electron microscopy (SEM). The average diameters of the pure SF, SF/PHBV (4/1), SF/PHBV (3/1), and SF/PHBV (2/1) nanofibers were 55.16 ± 12.38 nm, 75.93 ± 21.83 nm, 69.35 ± 21.55 nm, and 61.40 ± 12.31 nm, respectively. Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) were used to explore the microstructure of the electrospun SF/PHBV composite nanofiber. The crystallization ability of the composite nanofiber was greatly improved with the addition of PHBV. The results of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicated that the thermal stability of SF was better than PHBV obviously, so SF could improve the thermal stability of the composite materials within a certain range. The mechanical properties of the electrospun nanofiber membranes were evaluated by using a universal testing machine. In general, the elongation of the composite nanofiber membranes decreased, and the breaking strength increased with the addition of PHBV. The small pore size of the nanofiber membranes ensured that they had good application prospects in the field of filtration and protection. When the spinning time was 1 h, the filtration efficiency of SF/PHBV/PLA composite materials remained above 95%. © 2021 The Textile Institute.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Half face mask with regulation thermal stability system for continuous air flow: Stabimask

This article presents the design and prototyping of an electronic mask as personal protective equipment for the virus pandemic known as COVID - 19. Needs were identified such as: tightness and comfort. Requirements for use for long periods of time;this was considered for the design, also it has an adjustable ventilation system. The mask was simulated and validated with Solidworks Flow Simulation software, in addition a PID control model was implemented, thereby, it was shown that enough flow is generated to vary the temperature in a range of 20 to 37.2 ° C inside the mask. The design considers an outlet duct and an inlet duct with filters that prevent the entry of polluting particles, providing adequate protection. The prototype was made by 3D printing, And the thermal stability was achieved with the implementation of the temperature regulation system. The results obtained were validated, and they allow to future research to provide greater efficiency to masks. © 2022 ACM.

Key dimer interface residues impact the catalytic activity of 3CLpro, the main protease of SARS-CoV-2.

3C-like protease (3CLpro) processes and liberates functional viral proteins essential for the maturation and infectivity of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19. It has been suggested that 3CLpro is catalytically active as a dimer, making the dimerization interface a target for antiviral development. Guided by structural analysis, here we introduced single amino acid substitutions at nine residues at three key sites of the dimer interface to assess their impact on dimerization and activity. We show that at site 1, alanine substitution of S1 or E166 increased by twofold or reduced relative activity, respectively. At site 2, alanine substitution of S10 or E14 eliminated activity, whereas K12A exhibited ∼60% relative activity. At site 3, alanine substitution of R4, E290, or Q299 eliminated activity, whereas S139A exhibited 46% relative activity. We further found that the oligomerization states of the dimer interface mutants varied; the inactive mutants R4A, R4Q, S10A/C, E14A/D/Q/S, E290A, and Q299A/E were present as dimers, demonstrating that dimerization is not an indication of catalytically active 3CLpro. In addition, present mostly as monomers, K12A displayed residual activity, which could be attributed to the conspicuous amount of dimer present. Finally, differential scanning calorimetry did not reveal a direct relationship between the thermodynamic stability of mutants with oligomerization or catalytic activity. These results provide insights on two allosteric sites, R4/E290 and S10/E14, that may promote the design of antiviral compounds that target the dimer interface rather than the active site of severe acute respiratory syndrome coronavirus 2 3CLpro.

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS-CoV-2 protease 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.

Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease.

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145-His41 catalytic dyad. We here characterized the catalytic roles of the cysteine-histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine-histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

Higher binding affinity of furin for SARS-CoV-2 spike (S) protein D614G mutant could be associated with higher SARS-CoV-2 infectivity.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has caused an exponential rise in death rates and hospitalizations. The aim of this study was to characterize the D614G substitution in the severe acute respiratory syndome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S protein), which may affect viral infectivity. METHODS: The effect of D614G substitution on the structure and thermodynamic stability of the S protein was analyzed with use of DynaMut and SCooP. HDOCK and PRODIGY were used to model furin protease binding to the S protein RRAR cleavage site and calculate binding affinities. Molecular dynamics simulations were used to predict the S protein apo structure, the S protein-furin complex structure, and the free binding energy of the complex. RESULTS: The D614G substitution in the G clade of SARS-CoV-2 strains introduced structural mobility and decreased the thermal stability of the S protein (ΔΔG = -0.086 kcal mol-1). The substitution resulted in stronger binding affinity (Kd = 1.6 × 10-8) for furin, which may enhance S protein cleavage. The results were corroborated by molecular dynamics simulations demonstrating higher binding energy of furin and the S protein D614G mutant (-61.9 kcal mol-1 compared with -56.78 kcal mol-1 for wild-type S protein). CONCLUSIONS: The D614G substitution in the G clade induced flexibility of the S protein, resulting in increased furin binding, which may enhance S protein cleavage and infiltration of host cells. Therefore, the SARS-CoV-2 D614G substitution may result in a more virulent strain.

Structural analysis of ACE2 variant N720D demonstrates a higher binding affinity to TMPRSS2.

AIMS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel member of the betacoronaviruses family affecting the lower respiratory tract mainly through binding to angiotensin converting enzyme 2 (ACE2) via its S-protein. Genetic analysis of (ACE2) gene revealed several variants that have been suggested to regulate the interaction with S protein. This study investigates the N720D variant, positioned in the collectrin-like domain (CLD) at proximity to type II transmembrane serine protease (TMPRSS2) cleavage site. MAIN METHODS: The effect of N720D variant on ACE2 structure and thermodynamic stability was studied by DynaMut. HDOCK was utilised to model TMPRSS2 protease binding to ACE2 WT and D720 variant cleavage site. PRODIGY was used to calculate binding affinities and MD simulation tools calculated the at 100 ns for ACE2 apo structure and the ACE2-TMPRSS2 complex. KEY FINDINGS: The N720D variant is a more dynamic structure with a free energy change (ΔΔG): -0.470 kcal/mol. As such, introducing a tighter binding affinity of Kd = 3.2 × 10-10 M between TMPRSS2 and N720D variant. RMSD, RMSF calculations showed the N720D variant is less stable, however, RMSF values of the D720-TMPRSS2 complex reflected a slower dynamic motion. SIGNIFICANCE: The hotspot N720D variant in the CLD of ACE2 affected the stability and flexibility of ACE2 by increasing the level of motion in the loop region, resulting in a more favourable site for TMPRSS2 binding and cleavage. Consequently, this would facilitate S-protein binding and can potentially increase viral entry highlighting the importance of variants affecting the ACE2-TMPRSS2 complex.