Search Results (347)

SARS-CoV-2 main protease (M^pro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on computational predictions or cell-free biochemical approaches that provide limited functional context and often require specialized instrumentation, while mammalian cell-based models remain costly and require high biosafety levels. Accordingly, there remains a shortage of simple, rapid, and biosafe functional screening tools suitable for early-stage prioritization of potential M^pro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter assay was developed that directly links SARS-CoV-2 M^pro activity to β-galactosidase function in Escherichia coli. To the best of our knowledge, the developed assay represents the first bacterial colorimetric model for functional detection of SARS-CoV-2 M^pro inhibition based on a phenotypic readout. The assay enables the rapid visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective and biosafe platform for prioritizing candidate inhibitors, under standard laboratory conditions, prior to further validation. Due to its bacterial expression context, this assay is intended for functional screening to provide the most promising candidate compounds and/or extracts for subsequent biochemical or mammalian cell-based validation; it is not intended to determine quantitative potency or to replace further validation approaches. It should be noted that the selective compound uptake in E. coli restricts the range of chemical compositions that can be evaluated using this method. Therefore, proof-of-concept application was demonstrated using pomegranate juice, a representative natural inhibitor source, rather than most currently known specific M^pro inhibitors. In addition, other plant-derived preparations, including rhubarb, grape, and red/black currant juices, were tested demonstrating the assay’s applicability to diverse natural matrices. Full article

Significant advances in coronavirus immunoprophylaxis have enabled the control of the SARS-CoV-2 pandemic. However, the continued emergence of SARS-CoV-2 variants with immune escape potential highlights the need for effective direct-acting antivirals targeting conserved viral enzymes. The SARS-CoV-2 main protease (M^pro) remains one of the most promising antiviral drug targets due to its essential role in viral replication and the high conservation of its active site across coronavirus variants. Building upon the established GC373 scaffold, we designed, synthesized, and biochemically evaluated two novel GC373-like peptidomimetic inhibitors incorporated modified glutamine-mimic residues. These analogs were designed to enhance solubility and metabolic resilience while retaining key recognition features within the M^pro active site. Both compounds demonstrated micromolar inhibitory activity in enzymatic assays, supported by molecular docking and MM-PBSA analyses consistent with stable binding. The proposed inhibitors represent viable scaffolds for further optimization of electrophilic warheads and S1/S2 residue interactions. These findings contribute to the rational design of next-generation M^pro inhibitors and align with ongoing efforts to expand the chemical space of SARS-CoV-2 antiviral agents. Full article

The clinical durability of SARS-CoV-2 main protease (M^pro) inhibitors depends on their resilience to emerging resistance mutations. Recent genomic surveillance and functional reports have highlighted substitutions at positions 49, 165, and 301, raising questions about the robustness of the noncovalent inhibitor WU-04 in variant backgrounds. Here, we combined μs-scale, triplicate molecular dynamics simulations with end-state binding free energy estimates and a network-rewiring inference (NRI) framework that maps long-range dynamical communication across the full protease dimer. We evaluated wild type (WT), single mutants M49K, M165V, S301P, and selected double mutants (M49K & M165V, M49K & S301P). Relative to WT, single substitutions produced reductions in computed binding affinity of up to ~12kcal/mol, accompanied by loss or reshaping of the S2 subsite and altered ligand burial. Notably, the M49K/S301P double mutant partially restored WU-04 engagement, narrowing the ΔΔG_restore gap to within ΔΔG_restore of WT and re-establishing key hydrophobic and hydrogen-bond contacts. NRI analysis revealed that distal residue 301 participates in a communication corridor linking the C-terminal helical domain to the active-site cleft; its substitution rewires inter-domain coupling that can compensate for local disruptions at residue 49. Together, these results identify structural hotspots and network pathways that may inform the design of next-generation M^pro inhibitors with improved mutation tolerance—specifically by strengthening interactions that do not rely solely on the mutable S2 pocket and by engaging conserved backbone features near the 165–166 region. Full article

The continuous emergence of SARS-CoV-2 variants, especially the Omicron strain with its heightened transmissibility, has posed ongoing challenges to the efficacy of existing vaccine and drug regimens. This situation highlights the pressing demand for antiviral drugs employing novel mechanisms of action. Pentacyclic triterpenoids (PTs), a structurally varied group of compounds derived from plants, exhibit both antiviral and anti-inflammatory activities, making them attractive candidates for further therapeutic development. These natural products, along with their saponin derivatives, show broad-spectrum inhibitory effects against multiple SARS-CoV-2 variants (from Alpha to Omicron) via interactions with multiple targets, such as the spike protein, main protease (Mpro), RNA-dependent RNA polymerase (RdRp), and inflammatory signaling pathways. This review consolidates recent findings on PTs and their saponins, emphasizing their influence on the key structural features required for inhibiting viral attachment, membrane fusion, reverse transcription, and protease function. We systematically summarized the structure–activity relationships and their antiviral results of PTs based on different target proteins in existing studies. Furthermore, this work points toward new strategies for designing multi-target PT-based inhibitors with improved efficacy against Omicron and future variants. Full article

A promising direction for the creation of new biologically active derivatives of the alkaloid lupinine is the synthesis of “hybrid molecules” that combine a fragment of the alkaloid and the pharmacophore of 1,2,3-triazole in their structure. From a biological perspective, this work presents the first X-ray diffraction study of a single crystal of (1S,9aR)-1-({4-[4-(Benzyloxy)-3-methoxyphenyl]-1H-1,2,3-triazol-1-yl}methyl)octahydro-2H-quinolizine, a new, recently synthesized 1,2,3-triazole derivative of lupinine. A comparison of theoretically predicted and experimentally observed structural parameters was carried out. The FTIR spectroscopy study and vibrational properties calculations allowed us to interpret the FTIR absorption spectrum and localize specific vibrational modes in quinolizidine, 1,2,3-triazole, and benzene rings. Such information can be fruitful for further characterization of the synthesis process and products. The molecular docking of the compound was performed. It was shown that the studied molecules are capable of interacting with the Mpro binding site via non-covalent and hydrophobic interactions with subsites S3 (Met165, Glu166, Leu167, Pro168) and S5 (Gln189, Thr190, Gln192), which ensure the stabilization of the Mpro substrate. Blocking of the active site of the enzyme in the region of the oxyanion hole does not occur, but stable stacking interactions with the π-system of one of the catalytic amino acids, His41, are observed. Full article

Background: Probiotic supplementation for very preterm infants is a common practice in many neonatal units. Assessing the effects of early postnatal exposure to probiotics on long-term neurodevelopment, growth, and atopy-related outcomes is important. Extremely preterm (EP: <28 weeks) infants enrolled in our previously reported randomized trial (SiMPro) comparing short-term effects of single (SS: B. breve M-16V) versus triple-strain (TS: B. breve M-16V, B. longum subsp. infantis-M63, B. longum subsp. longum-BB536) probiotic provided a unique opportunity to study this issue. Methods: This follow-up study assessed the five-year outcomes of SiMPro trial infants, including neurodevelopment (cognition (Full Scale Intelligence Quotient/ FSIQ using WPPSI-IV), behavior (Strengths and Difficulties Questionnaire), executive function (BRIEF–P)), growth (anthropometry) and blood pressure (BP). Atopy-related outcomes were evaluated at six to seven years using the ISAAC questionnaire. A linear mixed model was used for longitudinal outcomes. Impairment indicators were modeled using logistic regression and adjusted for Socio-Economic Indexes for Areas (SEIFA) centiles. Results: Follow-up rates (SS: 89.2% versus TS: 95%), neurodevelopmental outcomes [severe impairment (FSIQ < 70): SS: 7.4% versus TS: 4.3%; p = 0.68], growth, BMI, and BP were comparable between the SS and TS groups. The total difficulty score or BRIEF–P executive indices, disability rates (none: 66.7% versus 55.4%), and atopy-related outcomes were comparable between groups. Conclusions: Both TS and SS Bifidobacterium probiotic formulations were safe, with comparable neurodevelopmental, growth, and atopy-related outcomes at school age. Full article

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose major global health challenges despite extensive vaccination efforts. Variant escape, waning immunity, and reduced vaccine efficacy in immunocompromised populations underscore the urgent need for complementary antiviral therapeutics. Here, we report the design, synthesis, and biological evaluation of precision-engineered dermatan sulfate (DS)-mimetic glycopolymers as multi-targeted inhibitors of SARS-CoV-2. Guided by molecular docking and virtual screening, sulfation at the C2 and C4 positions of iduronic acid was identified as critical for binding to the viral spike protein and inhibiting host and viral enzymes, including heparanase (HPSE) and main protease (M^pro). Chemically synthesized DS disaccharides were covalently grafted onto polymer scaffolds via a post-modification strategy, yielding glycopolymers with well-defined assembly that form uniform nanoparticles under physiological conditions. Surface plasmon resonance and pseudovirus assays revealed strong binding to the viral spike protein (K_D ≈ 177 nM), potent viral neutralization, and minimal cytotoxicity. Cellular uptake studies further demonstrated efficient internalization of nanoparticles and intracellular inhibition of HPSE and M^pro. These results establish a modular, non-anticoagulant, and glycosaminoglycan-mimetic platform for the development of broad-spectrum antiviral agents to complement vaccination and enhance preparedness against emerging coronavirus variants. Full article

SARS-CoV-2 virus contains two highly conserved domains, the papain-like protease (PLpro) and main protease (Mpro), which play important roles in virus replication, immune suppression, and the induction of inflammation in host tissue. In this study, we applied small-molecule chip screening, enzymatic assays, SARS-CoV-2 spike pseudotyped virus detection and molecular docking to find potential Mpro or PLpro inhibitors. Two small molecules, oxytocin and risedronate sodium, stood out in drug repurposing. Oxytocin and risedronate sodium were shown to influence the activities of Mpro and PLpro, thereby preventing the virus from replication, which may alleviate SARS-CoV-2 infection. Thus, oxytocin, risedronate sodium, and cephalosporins may expand the drug library for treating coronavirus infection. Full article

Coronaviruses include various strains that reside in natural animal reservoirs, with zoonotic transmission posing risks to both domesticated animals and human health. Recent efforts to address coronavirus infections have focused on developing inhibitors targeting the main protease (M^pro), some of which exhibit potential broad-spectrum efficacy. This study presents crystal structures of four clinically relevant inhibitors—GC376, PF-00835231, nirmatrelvir, and ibuzatrelvir—bound to M^pro from the feline coronavirus strain FECV-UU23. Structural analysis identified distinct FECV-specific features within the active site where these inhibitors bind and revealed S4 loop as a susceptible structural region essential for the enhanced binding of inhibitors in UU23 M^pro. We therefore propose to incorporate sterically constrained, functionally tailored heterocyclic moieties at the P3 site of known inhibitors which can optimally engage Q187, P188, and S189 residues of the S4 loop. The findings presented enhance understanding of inhibitor specificity and reinforce the promise of these inhibitor scaffolds for developing antivirals against feline coronavirus strains, with possible applications in broad-spectrum coronavirus therapy. Full article

Human adenovirus infections are typically self-limiting but can become life-threatening in pediatric populations and immunocompromised individuals. Despite this clinical importance, efforts to develop antiviral drugs against adenoviruses remain limited. A promising strategy is to target the adenovirus protease (AVP), an enzyme essential for viral maturation and infectivity. Yet, research on AVP has lagged far behind that on other viral proteases. In this work, we aimed to reposition AVP as a viable target for antiviral therapy. We first discuss why AVP research has fallen behind and emphasize the need to redirect attention toward this protease. Building on advances in SARS-CoV-2 drug discovery, we evaluated the potential of repurposing inhibitors of the main protease (M^pro) and papain-like protease (PL^pro) as modulators of AVP. Additionally, we examined the untapped potential of phytochemicals as novel scaffolds. These analyses were supported by original molecular docking studies. Our results revealed that previously reported SARS-CoV-2 inhibitors, such as the M^pro inhibitor ensitrelvir and the PL^pro inhibitor (compound) 19, engage the catalytic site of AVP and may serve as starting scaffolds for inhibitor design. Screening of phytochemicals further identified promising candidates, including apigenin, camptothecin, kaempferol, and piperine. Together, these findings highlight AVP’s druggability and suggest that both repurposed antivirals and natural products provide complementary avenues for inhibitor development. Finally, we provide some recommendations to facilitate efforts in the discovery of novel AVP inhibitors. Full article

Human immunodeficiency virus (HIV-1) and SARS-CoV-2 continue to co-burden global health, motivating discovery of broad-spectrum small molecules. Conessine, a steroidal alkaloid, has reported membrane-active and antimicrobial properties but remains underexplored as a dual antiviral chemotype. To interrogate conessine’s multi-target antiviral potential against key enzymatic and entry determinants of HIV-1 and SARS-CoV-2 and to benchmark performance versus approved comparators. Eight targets were modeled: HIV-1 reverse transcriptase (RT, 3V81), protease (PR, 1HVR), integrase (IN, 3LPT), gp120–gp41 trimer (4NCO); and SARS-CoV-2 main protease (M^pro, 6LU7), papain-like protease (PL^pro, 6W9C), RNA-dependent RNA polymerase (RdRp, 7BV2), spike RBD (6M0J). Ligands (conessine; positive controls: dolutegravir for HIV-1, nirmatrelvir for SARS-CoV-2) were prepared with standard protonation, minimized, and docked using AutoDock Vina v 1.2.0exhaustiveness 4; 20 poses). Binding modes were profiled in 2D/3D. Protocol robustness was verified by re-docking co-crystallized ligands (RMSD ≤ 2.0 Å). Atomistic MD (explicit TIP3P, OPLS4, 300 K/1 atm, NPT; 50–100 ns) assessed pose stability (RMSD/RMSF), pocket compaction (Rg, volume), and interaction persistence; MM/GBSA provided qualitative energy decomposition. ADMET was predicted in silico. Conessine showed coherent, hydrophobically anchored binding across both viral panels. Best docking scores (kcal·mol⁻¹) were: HIV-1—PR −6.910, RT −6.672, IN −5.733; SARS-CoV-2—spike RBD −7.025, M^pro −5.745, RdRp −5.737, PL^pro −5.024. Interaction maps were dominated by alkyl/π-alkyl packing to catalytic corridors (e.g., PR Ile50/Val82, RT Tyr181/Val106; M^pro His41/Met49; RBD L455/F486/Y489) with occasional carbon-/water-mediated H-bonds guiding orientation. MD sustained low ligand RMSD (typically ≤1.6–2.2 Å) and damped RMSF at catalytic loops, indicating pocket rigidification; MM/GBSA trends (≈ −30 to −40 kcal·mol⁻¹, dispersion-driven) supported persistent nonpolar stabilization. Benchmarks behaved as expected: dolutegravir bound strongly to IN (−6.070) and PR (−7.319) with stable MD; nirmatrelvir was specific for M^pro and displayed weaker, discontinuous engagement at PL^pro/RdRp/RBD under identical settings. ADMET suggested conessine has excellent permeability/BBB access (high logP), but liabilities include poor aqueous solubility, predicted hERG risk, and CYP2D6 substrate dependence.Conessine operates as a hydrophobic, multi-target wedge with the most favorable computed engagement at HIV-1 PR/RT and the SARS-CoV-2 spike RBD, while maintaining stable poses at M^pro and RdRp. The scaffold merits medicinal-chemistry optimization to improve solubility and de-risk cardiotoxicity/CYP interactions, followed by biochemical and cell-based validation against prioritized targets. Full article

Background: Derivatives of Pyrido[2,3-d]pyrimidine-6-carboxylate are promising multi-target scaffolds. This study focused on synthesizing 16 amino-functionalized derivatives and evaluating their dual anticancer and antibacterial activities, supported by mechanistic and computational analyses. Objectives: Design and synthesize derivatives, evaluate cytotoxicity against HeLa, HepG-2, and MCF-7 (selectivity against WI-38), investigate EGFR^WT and EGFR^T790M inhibition, assess cell cycle, apoptosis, and migration effects, antibacterial efficacy against E. coli and P. aeruginosa, and perform in silico ADMET, docking, molecular dynamics, DFT, and antiviral predictions. Methods: Synthesized 16 derivatives; tested for cytotoxicity, EGFR inhibition, cell cycle, apoptosis, migration; assessed antibacterial activity; performed ADMET profiling, molecular docking, molecular dynamics, and DFT calculations. Results: Derivatives 1, 2, and 7 showed highest cytotoxicity (IC₅₀ = 3.98–17.52 μM; WI-38 IC₅₀ = 64.07–81.65 μM). Compound 1 potently inhibited EGFRWT (IC₅₀ = 0.093 μM) and EGFRT790M (IC₅₀ = 0.174 μM), induced G0/G1 arrest (74.86%) and apoptosis (26.37%), and reduced MCF-7 migration (69.63%). Moderate antibacterial activity observed (MIC = 50 μg/mL). ADMET indicated favorable pharmacokinetics, low CYP inhibition, negative mutagenicity, and oral toxicity class III. Molecular dynamics confirmed stable binding (EGFRWT RMSD 3 Å; EGFRT790M 3.5–4.6 Å) with persistent hydrogen bonds. In silico antiviral evaluation suggested strong binding to HCV NS5A (–9.36 kcal/mol), SARS-CoV-2 Mpro (–9.82 kcal/mol), and E.coli DNA gyrase (–10.25 kcal/mol). Conclusions: Compound 1 exhibits dual anticancer and antibacterial activity, supported by mechanistic and computational analyses, highlighting pyrido[2,3-d]pyrimidines as promising multi-target therapeutic scaffolds. Full article

Thiol isomerases are a family of enzymes that participate in oxidative protein folding. They contain highly reactive vicinal thiols in a CXXC motif within their catalytic domains to mediate thiol-disulfide switching as part of their reductase, oxidase, and isomerase activity. In addition, they participate in chaperone function by binding to partially folded or misfolded proteins and preventing aggregation, thereby facilitating correct protein folding. The CXXC motif is conducive to oxidative influence based on the sulfur nucleophilicity. Redox modification of the CXXC motif may influence the enzymatic function. In this review we briefly discuss the family of thiol isomerases as it relates to thrombotic disorders. We then discuss the chemical mechanisms of making and breaking disulfides by the enzymes. Enzymatic and chemical models of oxidizing the CXXC motif are proposed. Lastly, we highlight evidence that natural galloylated polyphenols can inhibit both the coronavirus main protease Mpro and thiol isomerases, supporting a therapeutic strategy for COVID-19-associated coagulopathy and thrombosis by targeting the CXXC motif with these anti-oxidative compounds. Full article

Background/Objectives: SARS-CoV-2 pandemic led to the identification of peptide-based main protease (M^pro) inhibitors. The overwhelming majority of them carry an electrophilic warhead and a γ-lactam at the P1 position. During the selectivity assessment of an in-house Michael acceptors targeting SARS-CoV-2 M^pro, we unexpectedly observed a significant inhibition of human cathepsin S (hCatS). Methods: The biological investigation of three compounds (i.e., SPR38, SPR39, and SPR41) against hCatS was performed. The binding mode of SPRs was investigated by docking and molecular dynamics simulations. Results: Biological investigation has corroborated that hCatS is sensitive to peptide-based analogues harbouring γ-lactam at the P1 position and a vinyl methyl ketone warhead. In silico studies revealed that despite being solvent exposed, the γ-lactam at P1 might be involved in water-mediated H-bonds that could be optimized to gain inhibition potency and selectivity. Conclusions: The molecules repurposing of peptide-based SARS-CoV-2 M^pro inhibitors carrying the γ-lactam at the P1 site could pave the way for the identification of novel potent and selective hCatS ligands. Full article

A focused library of 1,2,4-thiadiazolidin-3,5-diones (THIA-1–10), previously characterized as hydrogen sulfide (H₂S) donors, was evaluated for inhibitory activity against cysteine proteases. We included two key cysteine proteases aiming at antiviral drug development—SARS-CoV-2 3CLpro (Mpro) and PLpro—alongside reference enzymes Papain and Cathepsin L. The compounds exhibited distinct selectivity profiles and inhibition mechanisms. The ability to act as covalent inhibitors of 3CLpro in the nanomolar range is of particular interest, with compounds THIA-6, -7, and -10 proving to be the most potent inhibitors of the series, and compounds THIA-1, -2, and -8 proving to be the most selective with respect to the other proteases. We explored the molecular bases of the observed activity profile of THIA-1–10 through computational studies, which supported and complemented the experimental findings, paving the way for future structure optimization. The results highlight that inhibitory potency depends not only on electrophilicity but also on the ability to access the catalytic cysteine within the active site. The dual functionality of THIA-1–10 as H₂S donors and selective cysteine protease inhibitors underscores its potential as a promising lead for therapeutic development. Full article