<p></p><p>Recently, the emergence and dissemination of SARS-CoV-2 has caused high mortality and enormous economic loss. In the fight against COVID-19, the rapid development of new drug molecules is the need of hour. However, the conventional approaches of drug development is time consuming and expensive in nature. In this study, we have adopted an alternative approach to identify lead molecules from natural sources using high throughput virtual screening approach. Ligands from natural compounds library from Selleck Inc (L1400) have been screened to evaluate their ability to bind and inhibit the main protease (M^pro or 3CL^pro) of SARS-CoV-2, which is a potential drug target. We found that Kaempferol, Quercetin, and Rutin were able to bind at the substrate binding pocket of 3CL^pro with high affinity (10⁵-10⁶ M^-1) and interact with the active site residues such as His41 and Cys145 through hydrogen bonding and hydrophobic interactions. In fact, the binding affinity of Rutin was much higher than Chloroquine (1000 times) and Hydroxychloroquine (100 times) and was comparable to that of the reference drug Remdesivir, which is in clinical trials to treat COVID-19 patients. The results suggest that natural compounds such as flavonoids have the potential to be developed as novel inhibitors of SARS-CoV-2 with a comparable potency as that of Remdesivir. However, their clinical usage on COVID-19 patients is a subject of further investigations and clinical trials.</p><br><p></p>

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrateenzyme interaction during the dsRNA cleavage by HPR.

The worldwide dissemination of CTX-M type b-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other b-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5a transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional b-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC 50 value (6 nM), high affinity (K i = 0.017 mM) and better acylation efficiency (k +2 /K9 = 0.44 mM 21 s 21 ). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s 21 ). Since increasing resistance has been reported against conventional b-lactam antibiotic-inhibitor combinations, we aspire to design a non-b-lactam core containing b-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-b-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it's IC 50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 mM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-b-lactam containing b-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.

The fluoroquinolone antibiotic drug namely ciprofloxacin hydrochloride (CFH) is widely prescribed for the treatment of different bacterial infections. The interaction of CFH with a synthetic polymer, polyvinyl pyrrolidone (PVP), and biopolymer, bovine serum albumin (BSA) was studied by UVvisible and fluorescence spectroscopic methods at different temperatures. The binding constant (K b ) for the CFH-PVP complex was determined from the Benesi-Hildebrand plot. PVP of different molecular weights (MW) (such as 24,000, 40,000, 360,000, and 700,000 gm. mole -1 ) were used for the interaction between CFH and PVP. There was a gradual increase in K b value and the complexation reaction was found to be much enhanced with the augmentation of the MW of PVP. The values of K b were also found to be increased with increasing temperatures as well as with the increase of electrolyte/acetic acid concentration. The Gibbs free energy of binding (∆G 0 ) values of the interaction process was negative which indicates the complex formation is thermodynamically spontaneous. The positive values of enthalpy (∆H 0 ) and entropy (∆S 0 ) of binding connote that the binding force for CFH-PVP complexation is hydrophobic in nature and the complexation is entropy controlled. The negative intrinsic enthalpy (∆H *,0 ) values indicate the high stability of CFH-PVP complexes. Molecular docking calculation discloses the existence of similar binding forces between CFH and PVP obtained by the analysis of experimental data from UV-visible spectroscopic method. The binding constant between CFH and BSA (K b ), quenching constant (K sv ), the number of binding sites (n), and the quenching rate constant (K q ) for the CFH-BSA system were also calculated. The values of K sv , K q , and n for the CFH-BSA system are lower in 0.05 mol kg -1 urea solution and higher in PVP solutions compared to those of aqueous medium.

Alzheimer's disease (AD) is a neurodegenerative disease and the most prevalent form of dementia. The generation of oxygen free radicals and oxidative damage is believed to be involved in the pathogenesis of AD. It has been suggested that date palm, a plant rich in phenolic compounds and flavonoids, can provide an alternative treatment to fight memory loss and cognitive dysfunction due to its potent antioxidant activity. Thus, we studied the effect of flavonoids present in date palm on Aβ1−40 amyloid formation using molecular docking and molecular dynamics simulation. AutoDock. Myricetin was used as a positive control drug. The flavonoids Diosmetin, Luteolin, and Rutin were found to be potent inhibitors of aggregation (docking energies ≤ −8.05 kcal mol−1) targeting Aβ1−40 fibrils (both 2LMO and 6TI5), simultaneously. Further screening by physicochemical properties and drug-likeness analysis suggested that all flavonoids except Rutin followed Lipinski's rule of five. Rutin was, thus, taken as a negative control (due to its violation of Lipinski's rule) to compare its dynamics with Diosmetin. Diosmetin exhibited the highest positive scores for drug likeness. Since Luteolin exhibited moderate drug-likeness and better absorption properties, it was also included in molecular dynamics simulation. Molecular dynamics of shortlisted compounds (Rutin, Diosmetin, and Luteolin) were performed for 200 ns, and the results were analyzed by monitoring root mean square deviations (RMSD), root mean square fluctuation (RMSF) analysis, the radius of gyration (Rg), and solvent accessible surface area (SASA). The results proved the formation of a stable protein-compound complex. Based on binding energies and non-bonded interactions, Rutin and Luteolin emerged as better lead molecules than Diosmetin. However, high MW (610.5), lowest absorption rate (16.04%), and more than one violation of Lipinski's rule make Rutin a less likely candidate as an anti-amyloidogenic agent. Moreover, among non-violators of Lipinski's rule, Diosmetin exhibited a greater absorption rate than Luteolin as well as the highest positive scores for drug-likeness. Thus, we can conclude that Diosmetin and Luteolin may serve as a scaffold for the design of better inhibitors with higher affinities toward the target proteins. However, these results warrant in-vitro and in-vivo validation before practical use.