As new medications are used to treat COVID-19, many studies have reported that proteins such as spike, polymerase and proteases are prone to high levels of mutation that can create resistance to therapy over time [1]. Thus, it becomes necessary to, not only target other viral proteins such as the non-structural proteins (nsp’s), but to also target the most conserved residues of these proteins. A synergistic combination of bioinformatics, computer-aided drug-design and in vitro studies can feed into better understanding of SARS-CoV-2 (SC-2) and therefore help in the development of small molecule inhibitors against the nsp’s. As part of our initial anti-viral work, a pharmacophore study on nsp15 found a hit molecule (INS316) that made interactions with Ser293, Lys344 and Leu345 residues [2] which are highly conserved across SC-2.

We have performed multiple sequence alignment studies on different datasets i.e., ~200,000, ~1 million and ~11 million sequences of SC-2 Orf1ab sequences to identify the most conserved residues. These residues were then visualized on 3D protein X-ray structures using MOE software. We found that there were known and novel binding pocket residues that were 100% conserved in our datasets. Our results indicate that these highly conserved pockets can be targeted for developing promising SC-2 inhibitors. We have also performed mutational analysis and have found different mutational hotspots across the nsp’s. Our group has recently been selected to enter two international challenges organized by the CACHE consortium to discover inhibitors of the RNA binding tunnel of SC-2 nsp13 and the Mac1 domain of SC-2 nsp3. We have used a tiered screening workflow which included the use of volume/shape information of the binding pockets (fastROCS), use of in-house pharmacophore generation software (MoPBS [3]/MOE) and performed docking in the binding pocket (FRED) to rank hits for subsequent clustering and to identify compounds that bind to these conserved pockets. Our results on nsp3 will be presented here.

References

1. Pachetti, M., Marini, B., Benedetti, F., Giudici, F., Mauro, E., Storici, P., Masciovecchio, C., Angeletti, S., Ciccozzi, M., Gallo, R. C., Zella, D., & Ippodrino, R. (2020). Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. Transl. Med., 18(1), 179. https://doi.org/10.1186/s12967-020-02344-6
2. Kandwal, S., & Fayne, D. (2022). Repurposing drugs for treatment of SARS-CoV-2 infection: computational design insights into mechanisms of action. Biomol. Struct. Dyn., 40(3), 1316–1330. https://doi.org/10.1080/07391102.2020.1825232
3. Braun, J., & Fayne, D. (2022). Mapping of Protein Binding Sites using clustering algorithms - Development of a pharmacophore based drug discovery tool. Mol. Graph. Model., 115, 108228. https://doi.org/10.1016/j.jmgm.2022.108228

G-quadruplexes (G4) are DNA secondary structures that play important roles in the regulation of gene expression in human cells and have been proposed as therapeutic targets in cancer. At the same time, putative G-quadruplex forming sequences have also been found on the genome of parasites T. brucei, L. major and P. falciparum suggesting they could also be explored as therapeutic targets.

We have designed and prepared thio sugars as vectors for naphthalene diimides, a classical G-quadruplex ligand, aiming to improve binding to G4 and biostability. Recent results on their efficacy against cancer cells and parasitic infections will be presented.

Silver nanoparticles (AgNPs) with broad-spectrum antimicrobial properties are gaining increasing interest in fighting multidrug-resistant bacteria. Herein we describe the synthesis of AgNPs stabilized by polyvinyl alcohol (PVA) with high purity and homogeneous sizes using radiolysis. Solvated electrons and reducing radicals are induced from solvent radiolysis and no other chemical reducing agents are needed to reduce the metal ions. Another advantage of this method is that it leads to sterile colloidal suspensions, which can be directly used for medical applications. We systematically investigated the effect of the silver salt precursor on the optical properties, particle size and morphology of the resulting colloidal AgNPs. With Ag₂SO₄ precursor, the AgNPs displayed a narrow size distribution (20 ± 2 nm). In contrast, AgNO₃ and AgClO₄ precursors lead to inhomogeneous AgNPs of various shapes. Moreover, the optimized AgNPs synthesized from Ag₂SO₄ were stable upon storage in water and phosphate-buffered saline (PBS) and were effective in inhibiting the growth of Staphylococcus. aureus (S. aureus) at a concentration of 0.6 μg·mL⁻¹ and completely eradicated it at a concentration of 5.6 μg·mL⁻¹. When compared with various conventional methods and other different strategies, the remarkable bactericidal ability against S. aureus of the AgNPs produced here opens up new avenues for further applications in medicine and other domains.

Abstract:

MXene are a group of two-dimensional (2D) transition metal carbides and carbonitrides. MXene have a wide range of uses in biotechnology, drug delivery, and bioimaging. Due to their biocompatibility and excellent antimicrobial activities, these two-dimensional (2D) nanomaterials have gained significant attention. MXene are one of these nanomaterials that demonstrated excellent bactericidal, fungicidal activity, and antimicrobial characteristics. Luliconazole drug are a highly effective antifungal drug in vitro against dermatophytes. However, luliconazole drugs suffers from less stability, poor solubility, low permeability, rapid metabolism, and rapid elimination from the topical area of the skin. For the improvement of drug stability, skin permeability, and efficacy, these drugs were loaded onto MXene nanocarriers, and for adhesion to the skin, drug-loaded MXene was coated with a mucoadhesive polymer. Sulphur-containing amino acids present in epithelial cells formed strong Di-sulphide bonds with thiolate pullulan polymer and adhered strongly. The poor bioavailable luliconazole-loaded nanocarrier showed sub-therapeutic effects because a major portion of the dose never reached the targeted sites. Thus, the purpose of our study was to improve the antimicrobial activity of the particular drug while lowering the dose, dose frequency, ensuring high stability, and delivering it to the targeted site. Many studies had been conducted to increase the bioavailability of badly absorbed antimicrobial drugs to enhance their clinical efficacies. Drug-loaded MXene nanocarriers were synthesized by incubating the drug with the MXene dispersion method. Drug-loaded MXene nanocarriers were characterized by different techniques such as ATR-FTIR, DSC, TGA, XRD, HR-TEM, NMR, and DLS. In this research work, thiolate polymer-coated drug-loaded MXene was synthesized and evaluated for its application as a carrier for the delivery of antimicrobial drugs.

The environmental levels of paroxetine (PAR), a selective serotonin reuptake inhibitor antidepressant, have risen due to increased consumption combined with the inefficiency of wastewater treatment removal. The functional conservation of important SSRI targets in vertebrates raises concerns about the potential adverse effects of exposure to pharmaceutically active molecules, such as PAR, on non-target organisms. This study addressed the effects of PAR chronic exposure (21-days) on adult zebrafish, assessing the effects of a low (40 µg/L - environmentally relevant concentration) and a high (400 µg/L – worst-case scenario) concentration in fish swimming behaviour and stress response. PAR-exposed fish displayed reduced swimming activity, stress response impairment and disruption of thigmotactic behaviour. These PAR-induced behavioural modifications may directly influence animal fitness, which, in turn, may lead to population and community disruption.