Targeting SARS-CoV-2 Main Protease: A Successful Story Guided by an In Silico Drug Repurposing Approach.

The SARS-CoV-2 main protease (Mpro) is a crucial enzyme for viral replication and has been considered an attractive drug target for the treatment of COVID-19. In this study, virtual screening techniques and in vitro assays were combined to identify novel Mpro inhibitors starting from around 8000 FDA-approved drugs. The docking analysis highlighted 17 promising best hits, biologically characterized in terms of their Mpro inhibitory activity. Among them, 7 cephalosporins and the oral anticoagulant betrixaban were able to block the enzyme activity in the micromolar range with no cytotoxic effect at the highest concentration tested. After the evaluation of the degree of conservation of Mpro residues involved in the binding with the studied ligands, the ligands' activity on SARS-CoV-2 replication was assessed. The ability of betrixaban to affect SARS-CoV-2 replication associated to its antithrombotic effect could pave the way for its possible use in the treatment of hospitalized COVID-19 patients.

Design, synthesis, biological activity evaluation and structure-activity relationships of new steroidal aromatase inhibitors. The case of C-ring and 7ß substituted steroids.

In this work, new steroidal aromatase inhibitors (AIs) were designed, synthesized, and tested. In one approach, C-ring substituted steroids namely those functionalized at C-11 position with an α or ß hydroxyl group or with a carbonyl group as well as C-9/C-11 steroidal olefins and epoxides were studied. It was found that the carbonyl group at C-11 is more beneficial for aromatase inhibition than the hydroxyl group, and that the C-ring epoxides were more potent than the C-ring olefins, leading to the discovery of a very strong AI, compound 7, with an IC50 of 0.011 µM, better than Exemestane, the steroidal AI in clinical use, which presents an IC50 of 0.050 µM. In another approach, we explored the biological activity of A-ring C-1/C-2 steroidal olefins and epoxides in relation to aromatase inhibition and compared it with the biological activity of C-ring C-9/C-11 steroidal olefins and epoxides. On the contrary to what was observed for the C-ring olefins and epoxides, the A-ring epoxides were less potent than A-ring olefins. Finally, the effect of 7ß-methyl substitution on aromatase inhibition was compared with 7α-methyl substitution, showing that 7ß-methyl is better than 7α-methyl substitution. Molecular modelling studies showed that the 7ß-methyl on C-7 seems to protrude into the opening to the access channel of aromatase in comparison to the 7α-methyl. This comparison led to find the best steroidal AI (12a) of this work with IC50 of 0.0058 µM. Compound 12a showed higher aromatase inhibition capacity than two of the three AIs currently in clinical use.

HSV-1 Glycoprotein D and Its Surface Receptors: Evaluation of Protein-Protein Interaction and Targeting by Triazole-Based Compounds through In Silico Approaches.

Protein-protein interactions (PPI) represent attractive targets for drug design. Thus, aiming at a deeper insight into the HSV-1 envelope glycoprotein D (gD), protein-protein docking and dynamic simulations of gD-HVEM and gD-Nectin-1 complexes were performed. The most stable complexes and the pivotal key residues useful for gD to anchor human receptors were identified and used as starting points for a structure-based virtual screening on a library of both synthetic and designed 1,2,3-triazole-based compounds. Their binding properties versus gD interface with HVEM and Nectin-1 along with their structure-activity relationships (SARs) were evaluated. Four [1,2,3]triazolo[4,5-b]pyridines were identified as potential HSV-1 gD inhibitors, for their good theoretical affinity towards all conformations of HSV-1 gD. Overall, this study suggests promising basis for the design of new antiviral agents targeting gD as a valuable strategy to prevent viral attachment and penetration into the host cell.

Identification of Small Molecular Chaperones Binding P23H Mutant Opsin through an In Silico Structure-Based Approach.

N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.

The Spike Mutants Website: A Worldwide Used Resource against SARS-CoV-2.

A large number of SARS-CoV-2 mutations in a short period of time has driven scientific research related to vaccines, new drugs, and antibodies to combat the new variants of the virus. Herein, we present a web portal containing the structural information, the tridimensional coordinates, and the molecular dynamics trajectories of the SARS-CoV-2 spike protein and its main variants. The Spike Mutants website can serve as a rapid online tool for investigating the impact of novel mutations on virus fitness. Taking into account the high variability of SARS-CoV-2, this application can help the scientific community when prioritizing molecules for experimental assays, thus, accelerating the identification of promising drug candidates for COVID-19 treatment. Below we describe the main features of the platform and illustrate the possible applications for speeding up the drug discovery process and hypothesize new effective strategies to overcome the recurrent mutations in SARS-CoV-2 genome.

Targeting SARS-CoV-2 nsp13 Helicase and Assessment of Druggability Pockets: Identification of Two Potent Inhibitors by a Multi-Site In Silico Drug Repurposing Approach.

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.

Computational Study Reveals the Role of Water Molecules in the Inhibition Mechanism of LAT1 by 1,2,3-Dithiazoles.

The L-type amino acid transporter LAT1, involved in many biological processes including the overexpression of some tumors, is considered a potential pharmacological target. The 1,2,3-Dithiazole scaffold was predicted to inhibit LAT1 by the formation of an intermolecular disulfide bond with the thiolate group of cysteine(s). As a result of the identification of these irreversible covalent inhibitors, we decided to deeply investigate the recognition stage and the covalent interaction, characterizing the chemical structures of the selected ligands. With the aim to provide new insights into the access of the ligands to the binding pocket and to reveal the residues involved in the inhibition, we performed docking, molecular dynamics simulations, and density functional theory-based investigation of three 1,2,3-dithiazoles against LAT1. Our computational analysis further highlighted the crucial role played by water molecules in the inhibition mechanism. The results here presented are consistent with experimental observations and provide insights that can be helpful for the rational design of new-to-come LAT1's inhibitors.

The Se-S Bond Formation in the Covalent Inhibition Mechanism of SARS-CoV-2 Main Protease by Ebselen-like Inhibitors: A Computational Study.

The inhibition mechanism of the main protease (Mpro) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of Mpro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se-S covalent bond mediated by EBS and EBS-OH on Mpro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of Mpro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.

Exploring the Assembly of Resorc[4]arenes for the Construction of Supramolecular Nano-Aggregates.

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the ability of resorc[4]arenes to self-assemble in polar solutions, to form supramolecular aggregates, and to promote water-solubility of an isoflavone endowed with anti-cancer activity, namely Glabrescione B (GlaB). Accordingly, we synthesized several architectures featuring a different pattern of substitution on the upper rim including functional groups able to undergo acid dissociation (i.e., carboxyl and hydroxyl groups). The aggregation phenomenon of the amphiphilic resorc[4]arenes has been investigated in a THF/water solution by UV-visible spectroscopy, at different pH values. Based on their ionization properties, we demonstrated that the supramolecular assembly of resorc[4]arene-based systems can be modulated at given pH values, and thus promoting the solubility of GlaB.

Current Updates on Naturally Occurring Compounds Recognizing SARS-CoV-2 Druggable Targets.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified in China as the etiologic agent of the recent COVID-19 pandemic outbreak. Due to its high transmissibility, this virus quickly spread throughout the world, causing considerable health issues. The scientific community exerted noteworthy efforts to obtain therapeutic solutions for COVID-19, and new scientific networks were constituted. No certified drugs to efficiently inhibit the virus were identified, and the development of de-novo medicines requires approximately ten years of research. Therefore, the repurposing of natural products could be an effective strategy to handle SARS-CoV-2 infection. This review aims to update on current status of the natural occurring compounds recognizing SARS-CoV-2 druggable targets. Among the clinical trials actually recruited, some natural compounds are ongoing to examine their potential role to prevent and to treat the COVID-19 infection. Many natural scaffolds, including alkaloids, terpenes, flavonoids, and benzoquinones, were investigated by in-silico, in-vitro, and in-vivo approaches. Despite the large data set obtained by a computational approach, experimental evidences in most cases are not available. To fill this gap, further efforts to validate these results are required. We believe that an accurate investigation of naturally occurring compounds may provide insights for the potential treatment of COVID-19 patients.

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study.

Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme-substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.

Naturally-Occurring Alkaloids of Plant Origin as Potential Antimicrobials against Antibiotic-Resistant Infections.

Antibiotic resistance is now considered a worldwide problem that puts public health at risk. The onset of bacterial strains resistant to conventional antibiotics and the scarcity of new drugs have prompted scientific research to re-evaluate natural products as molecules with high biological and chemical potential. A class of natural compounds of significant importance is represented by alkaloids derived from higher plants. In this review, we have collected data obtained from various research groups on the antimicrobial activities of these alkaloids against conventional antibiotic-resistant strains. In addition, the structure-function relationship was described and commented on, highlighting the high potential of alkaloids as antimicrobials.

Why hydroxy-proline improves the catalytic power of the peptidoglycan N-deacetylase enzyme: insight from theory.

Nature exploits different strategies for enhancing the catalytic activity of enzymes, often resorting to producing beneficial mutations. The case of post-translational proline hydroxylation mutation in the active site of polysaccharide deacetylase (PDA) Bc1960 from Bacillus cereus is an interesting example of how small chemical modifications can cause significant improvements in enzymatic activity. In the present study the deacetylation mechanism promoted by both OH-proline (2Hyp) and standard proline (Pro) containing PDA is investigated using density functional theory. Although the mechanism presented for the two examined enzymes is in agreement with protease catalysis in metalloenzymes, the analysis along the potential energy surface (PES) reveals that the intermediate and product benefit energetically from the presence of the hydroxyl group on the proline. Our calculations provide evidence that for PDA-2Hyp, the hydrogen bond network established by the -OH group on the Cα of the proline with its closest neighbors stabilizes the transition states and, consequently, the reaction takes advantage of this. These results further contribute towards explaining the different catalytic activity experimentally observed for the polysaccharide deacetylase enzymes.

Pathway involving the N155H mutation in HIV-1 integrase leads to dolutegravir resistance.

Background: Dolutegravir, an integrase strand-transfer inhibitor (STI), shows a high genetic barrier to resistance. Dolutegravir is reported to be effective against viruses resistant to raltegravir and elvitegravir. In this study, we report the case of a patient treated with dolutegravir monotherapy. Failure of dolutegravir treatment was observed concomitant with the appearance of N155H-K211R-E212T mutations in the integrase (IN) gene in addition to the polymorphic K156N mutation that was present at baseline in this patient. Methods: The impact of N155H-K156N-K211R-E212T mutations was studied in cell-free, culture-based assays and by molecular modelling. Results: Cell-free and culture-based assays confirm that selected mutations in the patient, in the context of the polymorphic mutation K156N present at the baseline, lead to high resistance to dolutegravir requiring that the analysis be done at timepoints longer than usual to properly reveal the results. Interestingly, the association of only N155H and K156N is sufficient for significant resistance to dolutegravir. Modelling studies showed that dolutegravir is less stable in IN/DNA complexes with respect to the WT sequence. Conclusions: Our results indicate that the stability of STI IN/DNA complexes is an important parameter that must be taken into account when evaluating dolutegravir resistance. This study confirms that a pathway including N155H can be selected in patients treated with dolutegravir with the help of the polymorphic K156N that acts as a secondary mutation that enhances the resistance to dolutegravir.

Real-life 3D therapy failure: Analysis of NS5A 93H RAS plus 108 K polymorphism in complex with ombitasvir by molecular modeling.

We report a real-life 3D therapy failure in a patient treated with ombitasvir (OMV)/paritaprevir/ritonavir and dasabuvir without ribavirin (3D-R). He had therapy failure at week 12 after the end of treatment. We detected resistance-associated substitutions (RASs) plus polymorphisms on NS3, NS5A, and NS5B target regions by population sequencing (15% cut-off) at baseline, at relapse and during follow-up. About this, NS5A RASs generally persist longer than resistances in the other target genes and may impact treatment outcome. Therefore, to evaluate OMV drug-resistance mechanism, we studied the acquired RAS plus polymorphisms on NS5A phosphoprotein by computational studies. OMV showed a higher affinity towards baseline and 93H/108 K mutant structure (follow-up) with respect to 93H/R108 mutant structure (relapse) on phosphoprotein. By Molecular Dynamics simulations (MDs), structural information about the protein stability in presence of OMV were observed. According to our data, molecular modeling approach has proved to be a powerful method to evaluate the impact of these RASs plus specific amino acid (AA) changes on phosphoprotein.

In Silico and In Vitro Study of Antioxidant Potential of Urolithins.

In this work, quantum chemical calculations based on density functional theory (DFT) were performed to predict the antioxidant potential of four bioactive gut microbiota metabolites of the natural polyphenols ellagitannins (ETs) and ellagic acid (EA), also known as urolithins (UROs). In order to evaluate their ability to counter the effect of oxidative stress caused by reactive oxygen species (ROS), such as the hydroperoxyl radical (•OOH), different reaction mechanisms were investigated, considering water and lipid-like environments. Through our in silico results, it emerged that at physiological pH, the scavenging activity of all urolithins, except urolithin B, are higher than that of trolox and other potent antioxidants existing in nature, such as EA, α-mangostin, allicin, caffeine and melatonin. These findings were confirmed by experimental assays.

Insight into non-nucleoside triazole-based systems as viral polymerases inhibitors.

Viruses have been recognized as the etiological agents responsible for many pathological conditions ranging from asymptomatic infections to serious diseases, even leading to death. For this reason, many efforts have been made to identify selective viral targets with the aim of developing efficient therapeutic strategies, devoid of drug-resistance issues. Considering their crucial role in the viral life cycle, polymerases are very attractive targets. Among the classes of compounds explored as viral polymerases inhibitors, here we present an overview of non-nucleoside triazole-based compounds identified in the last fifteen years. Furthermore, the structure-activity relationships (SAR) of the different chemical entities are described in order to highlight the key chemical features required for the development of effective antiviral agents.

Selection of Natural Compounds with HMGA-Interfering Activities and Cancer Cell Cytotoxicity.

HMGA proteins are intrinsically disordered (ID) chromatin architectural factors characterized by three DNA binding domains (AT-hooks) that allow them to bind into the DNA minor groove of AT-rich stretches. HMGA are functionally involved in regulating transcription, RNA processing, DNA repair, and chromatin remodeling and dynamics. These proteins are highly expressed and play essential functions during embryonic development. They are almost undetectable in adult tissues but are re-expressed at high levels in all cancers where they are involved in neoplastic transformation and cancer progression. We focused on identifying new small molecules capable of binding into the minor groove of AT-rich DNA sequences that could compete with HMGA for DNA binding and, thus, potentially interfere with their activities. Here, a docking-based virtual screening of a unique high diversity in-house library composed of around 1000 individual natural products identified 16 natural compounds as potential minor groove binders that could inhibit the interaction between HMGA and DNA. To verify the ability of these selected compounds to compete with HMGA proteins, we screened them using electrophoretic mobility shift assays. We identified Sorocein C, a Diels-Alder (D-A)-type adducts, isolated from Sorocea ilicifolia and Sorocea bonplandii with an HMGA/DNA-displacing activity and compared its activity with that of two structurally related compounds, Sorocein A and Sorocein B. All these compounds showed a cytotoxicity effect on cancer cells, suggesting that the Sorocein-structural family may provide new and yet unexplored chemotypes for the development of minor groove binders to be evaluated as anticancer agents.

In Silico-Guided Rational Drug Design and Synthesis of Novel 4-(Thiophen-2-yl)butanamides as Potent and Selective TRPV1 Agonists.

We describe an in silico-guided rational drug design and the synthesis of the suggested ligands, aimed at improving the TRPV1-ligand binding properties and the potency of N-(4-hydroxy-3-methoxybenzyl)-4-(thiophen-2-yl) butanamide I, a previously identified TRPV1 agonist. The docking experiments followed by molecular dynamics simulations and thermodynamic analysis led the drug design toward both the introduction of a lipophilic iodine and a flat pyridine/benzene at position 5 of the thiophene nucleus. Most of the synthesized compounds showed high TRPV1 efficacy and potency as well as selectivity. The molecular modeling analysis highlighted crucial hydrophobic interactions between Leu547 and the iodo-thiophene nucleus, as in amide 2a, or between Phe543 and the pyridinyl moiety, as in 3a. In the biological evaluation, both compounds showed protective properties against oxidative stress-induced ROS formation in human keratinocytes. Additionally, while 2a showed neuroprotective effects in both neurons and rat brain slices, 3a exhibited potent antinociceptive effect in vivo..

Design, Synthesis, and In Vitro, In Silico and In Cellulo Evaluation of New Pyrimidine and Pyridine Amide and Carbamate Derivatives as Multi-Functional Cholinesterase Inhibitors.

Alzheimer disease is an age-linked neurodegenerative disorder representing one of the greatest medical care challenges of our century. Several drugs are useful in ameliorating the symptoms, even if none could stop or reverse disease progression. The standard approach is represented by the cholinesterase inhibitors (ChEIs) that restore the levels of acetylcholine (ACh) by inhibiting the acetylcholinesterase (AChE). Still, their limited efficacy has prompted researchers to develop new ChEIs that could also reduce the oxidative stress by exhibiting antioxidant properties and by chelating the main metals involved in the disease. Recently, we developed some derivatives constituted by a 2-amino-pyrimidine or a 2-amino-pyridine moiety connected to various aromatic groups by a flexible amino-alkyl linker as new dual inhibitors of AChE and butyrylcholinesterase (BChE). Following our previous studies, in this work we explored the role of the flexible linker by replacing the amino group with an amide or a carbamic group. The most potent compounds showed higher selectivity against BChE in respect to AChE, proving also to possess a weak anti-aggregating activity toward Aß42 and tau and to be able to chelate Cu2+ and Fe3+ ions. Molecular docking and molecular dynamic studies proposed possible binding modes with the enzymes. It is noteworthy that these compounds were predicted as BBB-permeable and showed low cytotoxicity on the human brain cell line.