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.

The synthesis of new heterocyclic structures is a crucial issue in medicinal chemistry, which is constantly seeking for new active molecules. In the past 20 years, our research group has focused on the synthesis of DYRK1A inhibitors containing a thiazole ring fusionned with a quinazolin-4-one, a heterocyclic system present in many natural or synthetic molecules of biological interest. Two highly affine compounds, EHT1610 and FC162 were then identified and particularly studied. Docking studies highlighted the role of the V-shape of our compounds in their ability to inhibit DYRK1A. Recently, we described a synthetic method for access to 2-cyanobenzothiazoles from N-Arylcyanothioformamides via Pd-Catalyzed/Cu-Assisted C-H Functionalization/Intramolecular C-S bond formation. This regiospecific cyclization incited us to develop novel synthetic routes to obtain regioisomers of EHT1610 and FC162, our reference compounds. After an optimization step, this methodology allowed the synthesis of linear thiazolo[4,5-g] and [5,4-g] quinazoline regioisomers and chemical analogs of the two V-shaped leads. Preliminary results of kinase inhibition and cytotoxic evaluation of the target compounds are presented in this communication.

Introduction: According to the Diagnostic And Statistical Manual Of Mental Disorders–5 (DSM-5), attention-deficit/hyperactivity disorder (ADHD) is “a persistent pattern of inattention and/or hyperactivity-impulsivity interfering in functioning or development, as characterized by inattention and/or hyperactivity and impulsivity” [1], globally affecting 5%–7.2% of youth and 2.5%–6.7% of adults [2]. The treatment mostly focus on symptoms. Methylphenidate (MPH) and amphetamine (AMPH), are well-known psychostimulants for their ability to promote monoamine levels increase [3] used in ADHD [4]. Herein, we evaluated AMPH and MPH potential to promote neurite outgrowth and synaptogenesis in undifferentiated SH-SY5Y neuronal cells.

Material and Methods: Unifferentiated SH-SY5Y neuronal human cells were exposed to clinically relevant concentrations of either MPH or AMPH (0.001, 0.01, 0.1, 1, and 10μM) for 96 hours to evaluate their cytotoxicity through MTT reduction assay. Thereafter, neurite outgrowth and synaptogenesis, were evaluated through Western blotting, using the higher cellular density (25000 cm²) and microphotographs analysis to count neurites using NeuronJ software at the lower cellular density (6250 cells/cm²). To validate the experimental protocol, we evaluated the neurite outgrowth of SH-SY5Y after 96 hours of exposure to b-NGF (25 or 50 ng/mL) as a positive control. Statistical analysis was conducted using GraphPad Prism. When the distribution was normal, a parametric analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test. When data did not follow a normal distribution, statistical analysis was performed using the Kruskal–Wallis test, followed by Dunn's post-hoc test when a significant p was reached. Results were considered significantly different when p < 0.05.

Results: Cytotoxic evaluation revealed that AMPH 0.1 μM induced cytotoxicity at the lower cellular density, while the lower concentration worked as cytotoxic thought the MTT assay at the higher cellular density after 96 hours of exposure. Importantly, both concentrations of b-NGF reduced cytotoxicity at both cellular densities, as expected. The expression of neuronal proteins synaptophysin, PSD95 and GAP43 were not affected by the drugs, as well as by the positive control b-NGF. Finally, no changes in neurite outgrowth was evoked by AMPH or MPH or even b-NGF (50 ng/mL).

Conclusions: As far as we know, we are conducting the first study using clinically relevant concentrations of MPH and AMPH in a paradigm of acute exposure to evaluate the effects of neurogenesis in undifferentiated SH-SY5Y cells. More studies are needed for shorter periods of exposure attempting to reveal new data and experimental paradigms to assess the neuroprotection potential on these amphetamines.

Acknowledgements:

This work was supported by national funds from Fundação para a Ciência e a Tecnologia (FCT), I.P., in the scope of the project “EXPL/MED-FAR/0203/2021”. V.M.C acknowledges FCT for her grant (SFRH/BPD/110001/2015) that was funded by national funds through FCT under the Norma Transitória – DL57/2016/CP1334/CT0006.

References:

1. Bloch M, Clark J, Harris J, King B, Leckman J, Margolis A, Paul D, Pliszka S, Rice M, Wetherby A. Section II - Diagnostic Criteria and Codes. In: Diagnostic and Statistical Manual of Mental Disorders,5th ed. G B. American Psychiatric Association: American Psychiatric Association, 2013 pp. 69-77.
2. Abdelnour E, Jansen MO, Gold JA. ADHD Diagnostic Trends: Increased Recognition or Overdiagnosis? Mo Med 2022, 119467-473
3. Faraone SV. The pharmacology of amphetamine and methylphenidate: Relevance to the neurobiology of attention-deficit/hyperactivity disorder and other psychiatric comorbidities. Neurosci Biobehav Rev 2018, 87255-270 10.1016/j.neubiorev.2018.02.001.
4. Castells X, Blanco-Silvente L, Cunill R. Amphetamines for attention deficit hyperactivity disorder (ADHD) in adults. Cochrane Database Syst Rev 2018, 8Cd007813 10.1002/14651858.CD007813.pub3.

Background: We present and discuss our recent findings on the chemistry of Astragalus galactites. More than 20 of potentially bioactive constituents, including saponins, polyphenolic acids, polysaccharides, alkaloids, and flavonoids, were putatively identified in extracts of A.galactites with the aid of GC-QTOF-MS, and UPC2-QTOF-MS. This article provides a first-time such effort for the untargeted metabolite profiling of A.galactites.

Objective: This study aimed to shotgun analyze biological active substances in A.galactites, a plant used in Mongolian traditional medicine.

Material and Methods: The plant sample was collected in the Tahiltayn Mountain area (47º 91’ 44” N, 106º 70’ 63” E), Songino Khairhan district, Mongolia, (May 2021). Untargeted metabolite profiling analyses were performed in various plant organs (aerial parts, leaves, roots, and flowers) and previously isolated fractions (XYZ) of chloroform extract using GC-QTOF-MS and UPC2-QTOF-MS instruments.

Results: Twenty-three compounds, eight phenolic acid derivatives, seven flavonoids, two diterpenoid derivatives, two monoterpenoids, one sesquiterpenoid, two fatty acids, and one lignan were identified by UPC2-QTOF-MS analysis in the chloroform fraction of A.galactites. Further 16 compounds, of which 14 are polysaccharide derivatives and two alkaloids were also identified putatively by GC-MS analysis in the extract of leaves, flowers, roots, and aerial parts of A.galactites.

Conclusion: A shotgun metabolite profiling showed the presence of numerous polyphenolic compounds, polysaccharides, and saponins to be containing in A.galactites samples, a medicinal plant for its anti-inflammatory properties. The presence of the above-mentioned compounds supports the pharmacological effects and radical scavenging activities we reported (XYZ) previously.

Oncological diseases are one of the most common public health problems and second leading cause of death after cardiovascular disease. Natural products or synthetic compounds inspired from natural products continue to be excellent sources for new drug candidates. Isoxazole and oxazole rings containing nitrogen and oxygen atoms are considered as one of prime scaffolds for the drug discovery. Their structural features make it possible for multiple weak non-covalent interactions such as hydrogen bonds, coordination bonds, π-π stacking, hydrophilic interactions and so on. Such in medicinal chemistry isoxazole and oxazole compounds could readily bind with a variety of enzymes and receptors in biological systems and show broad biological activities like antibacterial, antifungal, antiviral, antitubercular, anticancer, anti-inflammatory and so on. Series of heterocyclic compounds containing pyrrolo[3,4-d]isoxazole framework have been studied for their antiproliferative activity against human cervical carcinoma (HeLa), murine fibroblast (3T3) and SV-40 transformed murine fibroblast (3T3-SV40) cell lines. It was found using confocal microscopy that granular actin was distributed diffusely in the cytoplasm in up to 71% of treated cells after their treatment with tested compounds while actin filaments were disappeared. Number of cells with filopodium-like membrane protrusions was significantly reduced after treatment with some of tested compounds (from 92 % in control cells up to 18% after treatment). The obtained results support the antitumor effect of the studied compounds and encourage the extension of the study in order to improve the anticancer activity and reduce the toxicological risks of obtained compounds.

The delivery of nucleic acids into muscle tissue is a pressing issue that needs to be resolved to advance the field of gene therapy for neuromuscular diseases. The transfection of muscle tissue is highly challenging primarily due to the absence of mitotic divisions in myofibrils, making it difficult for plasmid DNA to enter the nucleus. We have developed non-viral carriers based on arginine-histidine-rich peptides, and have shown their ability to deliver DNA into the muscle cells. Anionic peptide coating was developed to increase the stability of the polyplexes. The main task of current study was to reduce polyplexes package volume in order to increase transfection efficiency in vivo. Arginine-histidine-rich and glutamic acid-containing peptides were synthesized and polyplexes were formed with plasmids carrying the