Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations.

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

X-Ray Film Gel Contact Print Method for Analyzing Differential Expression of Plant Protease Inhibitors Under Abiotic Stress.

The regulation of proteolytic enzymes by protease inhibitors is crucial for maintaining the balance between protein synthesis and degradation, preventing uncontrolled proteolysis and fine-tuning cellular processes essential for optimal function and survival of the plants. It is known that the plant protease inhibitors activities are induced in defense of biotic as well as abiotic stresses. Thus, beyond their fundamental physiological functions, their involvement in stress responses, such as drought, cold, and salinity, is of equally significant. The X-ray film contact print method is an effective method for assessing various protease inhibitors exposed to stress conditions. In this approach, initially plant protease inhibitors will be separated using electrophoresis, and then the gel is treated with trypsin, which inhibits protease inhibitors. This gel when placed on X-ray film, the trypsin will digest the gelatin layer present on the film and the gelatinolytic activity stalled at the premises of protease inhibitors. This will provide the impression of the differentially expressed protease inhibitors in stress-treated plants.

Exploring the Therapeutic Potential of Petiveria alliacea L. Phytochemicals: A Computational Study on Inhibiting SARS-CoV-2's Main Protease (Mpro).

The outbreak of SARS-CoV-2, also known as the COVID-19 pandemic, is still a critical risk factor for both human life and the global economy. Although, several promising therapies have been introduced in the literature to inhibit SARS-CoV-2, most of them are synthetic drugs that may have some adverse effects on the human body. Therefore, the main objective of this study was to carry out an in-silico investigation into the medicinal properties of Petiveria alliacea L. (P. alliacea L.)-mediated phytocompounds for the treatment of SARS-CoV-2 infections since phytochemicals have fewer adverse effects compared to synthetic drugs. To explore potential phytocompounds from P. alliacea L. as candidate drug molecules, we selected the infection-causing main protease (Mpro) of SARS-CoV-2 as the receptor protein. The molecular docking analysis of these receptor proteins with the different phytocompounds of P. alliacea L. was performed using AutoDock Vina. Then, we selected the three top-ranked phytocompounds (myricitrin, engeletin, and astilbin) as the candidate drug molecules based on their highest binding affinity scores of -8.9, -8.7 and -8.3 (Kcal/mol), respectively. Then, a 100 ns molecular dynamics (MD) simulation study was performed for their complexes with Mpro using YASARA software, computed RMSD, RMSF, PCA, DCCM, MM/PBSA, and free energy landscape (FEL), and found their almost stable binding performance. In addition, biological activity, ADME/T, DFT, and drug-likeness analyses exhibited the suitable pharmacokinetics properties of the selected phytocompounds. Therefore, the results of this study might be a useful resource for formulating a safe treatment plan for SARS-CoV-2 infections after experimental validation in wet-lab and clinical trials.

Screening of Mpro Protease (SARS-CoV-2) Covalent Inhibitors from an Anthocyanin-Rich Blueberry Extract Using an HRMS-Based Analytical Platform.

BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site. RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

3D-QSAR and molecular docking studies of peptide-hybrids as dengue virus NS2B/NS3 protease inhibitors.

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R1-R2-Lys-R3-NH2) by the 3D-QSAR technique. Comparative molecular field analysis (q2 = 0.613, r2 = 0.938, r2pred = 0.820) and comparative molecular similarity indices analysis (q2 = 0.640, r2 = 0.928, r2pred = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R1 position, minor, electropositive, H-bond donor groups at the R2 position, and bulky, hydrophobic groups at the R3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease.

Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Synthesis and in silico inhibitory action studies of azo-anchored imidazo[4,5-b]indole scaffolds against the COVID-19 main protease (Mpro).

The present work elicits a novel approach to combating COVID-19 by synthesizing a series of azo-anchored 3,4-dihydroimidazo[4,5-b]indole derivatives. The envisaged methodology involves the L-proline-catalyzed condensation of para-amino-functionalized azo benzene, indoline-2,3-dione, and ammonium acetate precursors with pertinent aryl aldehyde derivatives under ultrasonic conditions. The structures of synthesized compounds were corroborated through FT-IR, 1H NMR, 13C NMR, and mass analysis data. Molecular docking studies assessed the inhibitory potential of these compounds against the main protease (Mpro) of SARS-CoV-2. Remarkably, in silico investigations revealed significant inhibitory action surpassing standard drugs such as Remdesivir, Paxlovid, Molnupiravir, Chloroquine, Hydroxychloroquine (HCQ), and (N3), an irreversible Michael acceptor inhibitor. Furthermore, the highly active compound was also screened for cytotoxicity activity against HEK-293 cells and exhibited minimal toxicity across a range of concentrations, affirming its favorable safety profile and potential suitability. The pharmacokinetic properties (ADME) of the synthesized compounds have also been deliberated. This study paves the way for in vitro and in vivo testing of these scaffolds in the ongoing battle against SARS-CoV-2.

Synthesis, characterisation and in vitro anticancer activity of conjugated protease inhibitor-silver nanoparticles (AgNPs-PI) against human breast MCF-7 and prostate PC-3 cancer cell lines.

The conjugated silver nanoparticles using biomolecules have attracted great attention of researchers because physical dimensions and surface chemistry play important roles in toxicity and biocompatibility of AgNPs. Hence, in the current study, synthesis of bio-conjugated AgNPs with protein protease inhibitor (PI) isolated from Streptomyces spp. is reported. UV-visible spectra of PI and AgNPs showed stronger peaks at 280 and 405 nm, confirming the synthesis of conjugated AgNPs-PI. TEM and SEM images of AgNPs-PI showed spherical-shaped nanoparticles with a slight increase in particle size and thin amorphous layer around the surface of silver nanomaterial. Circular dichroism, FT-IR and fluorescence spectral studies confirmed AgNPs-PI conjugation. Conjugated AgNPs-PI showed excellent anticancer potential than AgNPs and protease inhibitor separately on human breast MCF-7 and prostate PC-3 cell lines. The findings revealed that surface modification of AgNPs with protein protease inhibitor stabilised the nanomaterial and increased its anticancer activity.

Phytoconstituents of Artemisia Annua as potential inhibitors of SARS CoV2 main protease: an in silico study.

BACKGROUND: In November 2019, the world faced a pandemic called SARS-CoV-2, which became a major threat to humans and continues to be. To overcome this, many plants were explored to find a cure. METHODS: Therefore, this research was planned to screen out the active constituents from Artemisia annua that can work against the viral main protease Mpro as this non-structural protein is responsible for the cleavage of replicating enzymes of the virus. Twenty-five biocompounds belonging to different classes namely alpha-pinene, beta-pinene, carvone, myrtenol, quinic acid, caffeic acid, quercetin, rutin, apigenin, chrysoplenetin, arteannunin b, artemisinin, scopoletin, scoparone, artemisinic acid, deoxyartemisnin, artemetin, casticin, sitogluside, beta-sitosterol, dihydroartemisinin, scopolin, artemether, artemotil, artesunate were selected. Virtual screening of these ligands was carried out against drug target Mpro by CB dock. RESULTS: Quercetin, rutin, casticin, chrysoplenetin, apigenin, artemetin, artesunate, sopolin and sito-gluside were found as hit compounds. Further, ADMET screening was conducted which represented Chrysoplenetin as a lead compound. Azithromycin was used as a standard drug. The interactions were studied by PyMol and visualized in LigPlot. Furthermore, the RMSD graph shows fluctuations at various points at the start of simulation in Top1 (Azithromycin) complex system due to structural changes in the helix-coil-helix and beta-turn-beta changes at specific points resulting in increased RMSD with a time frame of 50 ns. But this change remains stable after the extension of simulation time intervals till 100 ns. On other side, the Top2 complex system remains highly stable throughout the time scale. No such structural dynamics were observed bu the ligand attached to the active site residues binds strongly. CONCLUSION: This study facilitates researchers to develop and discover more effective and specific therapeutic agents against SARS-CoV-2 and other viral infections. Finally, chrysoplenetin was identified as a more potent drug candidate to act against the viral main protease, which in the future can be helpful.

Discovery of meisoindigo derivatives as noncovalent and orally available Mpro inhibitors: their therapeutic implications in the treatment of COVID-19.

The progressive emergence of SARS-CoV-2 variants has necessitated the urgent exploration of novel therapeutic strategies to combat the COVID-19 pandemic. The SARS-CoV-2 main protease (Mpro) represents an evolutionarily conserved therapeutic target for drug discovery. This study highlights the discovery of meisoindigo (Mei), derived from the traditional Chinese medicine (TCM) Indigo naturalis, as a novel non-covalent and nonpeptidic Mpro inhibitor. Substantial optimizations and structure-activity relationship (SAR) studies, guided by a structure-based drug design approach, led to the identification of several Mei derivatives, including S5-27 and S5-28, exhibiting low micromolar inhibition against SARS-CoV-2 Mpro with high binding affinity. Notably, S5-28 provided significant protection against wild-type SARS-CoV-2 in HeLa-hACE2 cells, with EC50 up to 2.66 µM. Furthermore, it displayed favorable physiochemical properties and remarkable gastrointestinal and metabolic stability, demonstrating its potential as an orally bioavailable drug for anti-COVID-19 therapy. This research presents a promising avenue for the development of new antiviral agents, offering hope in the ongoing battle against COVID-19.

Sacubitril/valsartan has an underestimated impact on the right ventricle in patients with sleep-disordered breathing, especially central sleep apnoea syndrome.

BACKGROUND: Sacubitril/valsartan has been demonstrated to significantly improve left ventricular performance and remodelling in patients with heart failure. However, its effects on the right ventricle in patients with chronic heart failure and sleep-disordered breathing (SDB) have not been studied. AIM: To investigate the impact of sacubitril/valsartan treatment on right ventricular function in patients with SDB. METHODS: This was a subanalysis of an observational prospective multicentre study involving 101 patients. At inclusion, patients were evaluated by echocardiography and nocturnal ventilatory polygraphy, which allowed patients to be divided into three groups: "central-SDB"; "obstructive-SDB"; and "no-SDB". RESULTS: After 3 months of sacubitril/valsartan therapy, a positive impact on right ventricular function was observed. In the general population, tricuspid annular plane systolic excursion increased by +1.32±4.74mm (P=0.024) and systolic pulmonary artery pressure decreased by -3.1±10.91mmHg (P=0.048). The central-SDB group experienced the greatest echocardiographic improvement, with a significant increase in tricuspid annular plane systolic excursion of +2.1±4.9mm (P=0.045) and a significant reduction in systolic pulmonary artery pressure of -8.4±9.7mmHg (P=0.001). CONCLUSIONS: Sacubitril/valsartan improved right ventricular function in patients with heart failure and SDB after only 3 months of treatment. The greatest improvement in right ventricular function was observed in the central-SDB group.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Glycyrrhizic acid conjugates with amino acid methyl esters target the main protease, exhibiting antiviral activity against wild-type and nirmatrelvir-resistant SARS-CoV-2 variants.

COVID-19 pandemic is predominantly caused by SARS-CoV-2, with its main protease, Mpro, playing a pivotal role in viral replication and serving as a potential target for inhibiting different variants. In this study, potent Mpro inhibitors were identified from glycyrrhizic acid (GL) derivatives with amino acid methyl/ethyl esters. Out of the 17 derivatives semisynthesized, Compounds 2, 6, 9, and 15, with methionine methyl esters, D-tyrosine methyl esters, glutamic acid methyl esters, and methionines in the carbohydrate moiety, respectively, significantly inhibited wild-type SARS-CoV-2 Mpro-mediated proteolysis, with IC50 values ranging from 0.06 µM to 0.84 µM. They also demonstrated efficacy in inhibiting trans-cleavage by mutant Mpro variants (Mpro_P132H, Mpro_E166V, Mpro_P168A, Mpro_Q189I), with IC50 values ranging from 0.05 to 0.92 µM, surpassing nirmatrelvir (IC50: 1.17-152.9 µM). Molecular modeling revealed stronger interactions with Valine166 in the structural complex of Mpro_E166V with the compounds compared to nirmatrelvir. Moreover, these compounds efficiently inhibited the post-entry viral processes of wild-type SARS-CoV-2 single-round infectious particles (SRIPs), mitigating viral cytopathic effects and reducing replicon-driven GFP reporter signals, as well as in vitro infectivity of wild-type, Mpro_E166V, and Mpro_Q189I SRIPs, with EC50 values ranging from 0.02 to 0.53 µM. However, nirmatrelvir showed a significant decrease in inhibiting the replication of mutant SARS-CoV-2 SRIPs carrying Mpro_E166V (EC50: >20 µM) and Mpro_Q189I (EC50: 13.2 µM) compared to wild-type SRIPs (EC50: 0.06 µM). Overall, this study identifies four GL derivatives as promising lead compounds for developing treatments against various SARS-CoV-2 strains, including Omicron, and nirmatrelvir-resistant variants.

Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.