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Human norovirus (HuNoV) is a leading cause of gastroenteritis worldwide and is associated with significant morbidity, mortality, and economic impact. There are currently no licensed antiviral drugs for the treatment of HuNoV-associated gastroenteritis. The HuNoV protease plays a critical role in the initiation of virus replication by cleaving the polyprotein. Thus, it is an ideal target for developing antiviral small-molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by the H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases., Importance: Human noroviruses are a significant cause of sporadic and epidemic gastroenteritis worldwide. There are no vaccines or antiviral drugs currently available to treat infections. Our work elucidates the structural differences between GI.1 and GII proteases in response to inhibitor binding and will inform the future development of broad-spectrum norovirus protease inhibitors., Competing Interests: The authors declare no conflict of interest.

Here, we predicted that Plasmepsin II (PlmII) can explore open-flap conformations not sampled for human aspartic proteases: Cathepsin D, Renin, and Pepsin were used in molecular dynamics simulations. We combined 24 independent (50 ns) MD runs to improve the conformational sampling of each system. We discovered two PlmII noncompetitive selective inhibitors: SPB07935 and RH01201, with K i values in the μM range by targeting the open-flap conformations. Both compounds did not inhibit human Cathepsin D (hCatD) at high concentrations. We predicted that SPB07935 and RH01201 bind stably to the flap cryptic pocket, keeping this hairpin in an open or semiopen conformation along the MD simulations, respectively. Significantly, SPB07935 inhibited the P . falciparum chloroquine-resistant strain FcB1 growth in vitro, with an IC 50 value of 8 μM while having a lower toxicity for HEK-293 human cells (CC 50 = 189 μM).

COVID-19 still poses a worldwide health threat due to continuous viral mutations and potential resistance to vaccination. SARS-CoV-2 viral multiplication hindrance by inhibiting the viral main protease (Mpro) deemed propitious. Structure-based virtual screening (SBVS) is a conventional strategy for discovering new inhibitors. Nonetheless, the SBVS efforts against Mpro variants needed to be benchmarked. Herein, in the first stage of the study, we evaluated four docking tools (FRED, PLANTS, AutoDock Vina and CDOCKER) via an in-depth benchmarking approach against SARS-CoV2 Mpro of both the wild type (WTMpro) and the deadly Omicron P132H variant (OMpro). We started by compiling an active dataset of non-covalent small molecule inhibitors of the WTMpro from literature and the COVID-Moonshot database along with generating a high-quality benchmark set via DEKOIS 2.0. pROC-Chemotype plots revealed superior performance for AutoDock Vina against WTMpro, while both FRED and AutoDock Vina demonstrated excellent performance for OMPro. In the second stage, VS was performed on a focused library of 636 compounds transformed from the early-enriched cluster related to perampanel via a scaffold hopping approach. Subsequently, molecular dynamics (MD) simulation and MM GBSA calculations validated the binding potential of the recommended hits against both explored targets. This study provides an example of how to conduct an in-depth benchmarking approach for both WTMPro and OMPro variants and offering an evaluated SBVS protocol for them both., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Galal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We herein examined the inhibitory effects of tea catechins on the SARS-CoV-2 main protease (M pro ). Among the catechins analyzed, epigallocatechin 3-(3″- O -methyl)gallate, epigallocatechin gallate (EGCG), gallocatechin, gallocatechin gallate, and epigallocatechin inhibited recombinant M pro in a dose-dependent manner. Peptide mapping revealed that catechins preferentially formed covalent bonds with five sequences with the strongest activity at the C145 active site. Fragmentation analysis indicated 184 cleavages from peptides containing C145, corresponding to the D ring, suggesting that the B ring was attached to C145. When 10 bottled teas were incubated with M pro , four green teas inhibited the enzyme by over 80%, whereas the blended and barley teas showed no effect. EGCG reacted covalently with SARS-CoV-2 M pro within cells when incubated with cultured cells expressing M pro . This is the first study to report direct covalent binding between tea catechins and M pro in cells. This suggests that catechins from green tea can inhibit M pro in infected cells.

The 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), and RNA-dependent RNA polymerase (RdRp) are key enzymes in SARS-CoV-2 replication and serve as critical targets for an antiviral drug. Currently, Paxlovid® and Lagevrio™ specifically target 3CLpro and RdRp, respectively, for COVID-19 treatment. However, no antivirals target for the SARS-CoV-2 PLpro enzyme, essential for viral replication and suppression of the host antiviral immune response. This study identified 9-aminominocycline (9-AMN) as a potent inhibitor of SARS-CoV-2 PLpro. Unlike the parent compound minocycline, 9-AMN inhibits PLpro's proteolytic and deubiquitinase activities by approximately 90%, with IC 50 values of 4.15 µM and 4.55 µM, respectively, while showing no effect on the enzymatic activity of 3CLpro or RdRp. Enzyme kinetics reveal that 9-AMN functions as a mixed PLpro inhibitor and binds to its active site, disrupting its function as predicted by computer modeling. Furthermore, 9-AMN demonstrates, efficacy against the Delta and Omicron variants, with EC 50 values of 1.04 µM and 2.35 µM, respectively. When combined with EIDD-1931 (an active form of molnupiravir) or nirmatrelvir (PF-332), 9-AMN exhibits synergistic effects, significantly reducing the doses required to inhibit the Omicron variant. In conclusion, 9-AMN inhibits SARS-CoV-2 replication, and PLpro activity, highlighting its potential as a promising candidate for COVID-19 treatment strategies., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The ever-evolving SARS-CoV-2 variants necessitate the development of additional oral antivirals. This study presents the systematic design of quinoline-containing SARS-CoV-2 papain-like protease (PL pro ) inhibitors as potential oral antiviral drug candidates. By leveraging the recently discovered Val70 Ub binding site in PL pro , we designed a series of quinoline analogs demonstrating potent PL pro inhibition and antiviral activity. Notably, the X-ray crystal structures of 6 lead compounds reveal that the 2-aryl substitution can occupy either the Val70 Ub site as expected or the BL2 groove in a flipped orientation. The in vivo lead Jun13296 exhibits favorable pharmacokinetic properties and potent inhibition against SARS-CoV-2 variants and nirmatrelvir-resistant mutants. In a mouse model of SARS-CoV-2 infection, oral treatment with Jun13296 significantly improves survival, reduces body weight loss and lung viral titers, and prevents lung tissue damage. These results underscore the potential of quinoline PL pro inhibitors as promising oral SARS-CoV-2 antiviral candidates, instilling hope for the future of SARS-CoV-2 treatment., Competing Interests: Competing interests: Rutgers, the State University of New Jersey, has applied for a patent US2024382494A1 covering the PLpro inhibitors reported in this study and related compounds, which has been published and is pending. J.W. is listed as an inventor. The remaining authors declare no competing interests., (© 2025. The Author(s).)

The 3C-like protease of severe acute respiratory syndrome coronavirus 2, known as the main protease (M pro ), is an attractive drug target for the treatment of coronavirus disease 2019. This study reports the discovery of novel M pro inhibitors using several in silico techniques, including docking, molecular dynamics (MD), and fragment molecular orbital (FMO) calculations. We performed docking calculations on 5950 compounds with bioactivity, and 12 compounds were selected. An enzymatic assay was conducted, revealing that BP-1-102 exhibits significant M pro inhibitory activity with an IC 50 of 11.1 μM. The identification of seed compounds from the experiments on a few compounds demonstrates the effectiveness of our docking calculations. Furthermore, the detailed analyses using MD and FMO calculations suggested an interaction mechanism in which the hydroxyl group of BP-1-102 forms a hydrogen bond with E166 of M pro . The M pro inhibitory activity of SH-4-54, a derivative without the aforementioned hydroxyl group, was investigated and observed to be significantly reduced, with an IC 50 of 81.5 μM. This result strongly supports the suggested interaction mechanism.

The main protease (M pro ) of SARS-CoV-2 is a key drug target for the development of antiviral therapeutics. Here, we designed and synthesized a series of small-molecule peptidomimetics with various cysteine-reactive electrophiles. Several compounds were identified as potent SARS-CoV-2 M pro inhibitors, including compounds 8n (IC 50 = 0.0752 μM), 8p (IC 50 = 0.0887 μM), 8r (IC 50 = 0.0199 μM), 10a (IC 50 = 0.0376 μM), 10c (IC 50 = 0.0177 μM), and 10f (IC 50 = 0.0130 μM). Most of them additionally inhibited cathepsin L and were also active against SARS-CoV-1 and MERS-CoV M pro . In Calu-3 cells, several inhibitors, including 8r , 10a , and 10c , displayed high antiviral activity in the nanomolar range without showing cellular toxicity. The cocrystal structure of SARS-CoV-2 M pro in complex with 8p revealed covalent binding to the enzyme's catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds 10c and especially 8n were effective against a panel of naturally occurring nirmatrelvir-resistant mutants, particularly E166V, and showed metabolic stability and additional favorable pharmacokinetic properties, making it a suitable candidate for further preclinical development.

The main protease M pro is a clinically validated target to treat infections by the coronavirus SARS-CoV-2. Among the first reported M pro inhibitors was the peptidomimetic α-ketoamide 13b , whose cocrystal structure with M pro paved the way for multiple lead-finding studies. We established structure-activity relationships for the 13b series by modifying residues at the P1', P3, and P4 sites. Guided by cocrystal structures, we reduced the P1' substituent size to better fill the pocket and added a fluorine substituent to the pyridone ring, enabling a new hydrogen bond with Gln189 in P3. Among 22 novel analogues, 6d and 12d inhibited M pro with IC 50 s of 110 nM and 40 nM, improving the potency of 13b by up to 9.5-fold. Compound 6d had pronounced antiviral activity with an EC 50 of 1.6 μM and was stable in plasma and microsomes. The study illustrates the potential of structure-based design to systematically improve peptidomimetic α-ketoamides.

The main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as 3CL pro , is crucial in the virus's life cycle and plays a pivotal role in COVID-19. Understanding how small molecules inhibit 3CL pro 's activity is vital for developing anti-COVID-19 therapeutics. To this end, we employed Gaussian accelerated molecular dynamics (GaMD) simulations to enhance the sampling of 3CL pro conformations and conducted correlation network analysis (CNA) to explore the interactions between different structural domains. Our findings indicate that a CNA-identified node in domain II of 3CL pro acts as a conduit, transferring conformational changes from the catalytic regions in domains I and II, triggered by the binding of inhibitors (7YY, 7XB, and Y6G), to domain III, thereby modulating 3CL pro 's activity. Normal mode analysis (NMA) and principal component analysis (PCA) revealed that inhibitor binding affects the structural flexibility and collective movements of the catalytic sites and domain III, influencing 3CL pro 's function. The binding free energies, predicted by both MM-GBSA and QM/MM-GBSA methods, showed a high correlation with experimental data, validating the reliability of our analyses. Furthermore, residues L27, H41, C44, S46, M49, N142, G143, S144, C145, H163, H164, M165, and E166, identified through residue-based free energy decomposition, present promising targets for the design of anti-COVID-19 drugs and could facilitate the development of clinically effective 3CL pro inhibitors.

The global impact of SARS-CoV-2 highlights the need for treatments beyond vaccination, given the limited availability of effective medications. While Pfizer introduced Paxlovid , an FDA-approved antiviral targeting the SARS-CoV-2 main protease (Mpro), this study focuses on designing new antivirals against another protease, papain-like protease (PLpro), which is crucial for viral replication and immune suppression. NCATS/NIH performed a high-throughput screen of ∼15,000 molecules from an internal molecular library, identifying initial hits with a 0.5% success rate. To improve the hit rate and identify potent inhibitors, machine learning-based virtual screens were applied to ∼150,000 compounds, yielding 125 top predicted hits. Biochemical evaluation revealed 25 promising compounds, with a 20% hit-rate and IC 50 values from 1.75 μM to <36 μM across 13 chemotypes. Further analog screening of those chemotypes, as part of the structure-activity relationships, led to 20 additional hits. Additionally, the hit-to-lead optimization of chemotype 7 produced 10 more analogs. These PLpro inhibitors provide promising templates for antiviral development against COVID-19.

Cancer management has traditionally depended on chemotherapy as the mainstay of treatment; however, recent advancements in targeted therapies and immunotherapies have offered new options. Ubiquitin-specific proteases (USPs) have emerged as promising therapeutic targets in cancer treatment due to their crucial roles in regulating protein homeostasis and various essential cellular processes. This review covers the following: (1) the structural and functional characteristics of USPs, highlighting their involvement in key cancer-related pathways, and (2) the discovery, chemical structures, mechanisms of action, and potential clinical implications of USP inhibitors in cancer therapy. Particular attention is given to the role of USP inhibitors in enhancing cancer immunotherapy, e.g., modulation of the tumor microenvironment, effect on regulatory T cell function, and influence on immune checkpoint pathways. Furthermore, this review summarizes the current progress and challenges of clinical trials involving USP inhibitors as cancer therapy. We also discuss the complexities of achieving target selectivity, the ongoing efforts to develop more specific and potent USP inhibitors, and the potential of USP inhibitors to overcome drug resistance and synergize with existing cancer treatments. We finally provide a perspective on future directions in targeting USPs, including the potential for personalized medicine based on specific gene mutations, underscoring their significant potential for enhancing cancer treatment. By elucidating their mechanisms of action, clinical progress, and potential future applications, we hope that this review could serve as a useful resource for both basic scientists and clinicians in the field of cancer therapeutics.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for 'direct-to-biology' (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (M Pro ), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a 'switch' to a reversible inhibitor series. This example of ligand discovery for M Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a potentially lethal infection that presents a substantial threat to health, especially in Middle East nations. Given that no FDA-approved specific therapy for MERS infection exists, designing and discovering a potent antiviral therapy for MERS-CoV is crucial. One pivotal strategy for inhibiting MERS replication is to focus on the viral main protease (M pro ). In this study, we identify potential novel M pro inhibitors employing structure-based virtual screening of our recently reported Ugi reaction-derived library (URDL) consisting of cherry-picked molecules from the literature. The key features of the URDL library include synthetic tractability (1-2 pot synthesis) of the molecules scaffold and unexplored chemical space. The hits were ranked based on the docking score, MM-GBSA free energy of binding, and the interaction pattern with the active site residues. A molecular dynamics (MD) simulation study was performed for the first two top-ranked compounds to analyze the stability and free binding energy based on the molecular mechanics Poisson-Boltzmann surface area. The potential mean force calculated from the steered molecular dynamics (SMD) simulations of the hits indicates improved H-bond potential, enhanced conformational stability, and binding affinity toward the target, compared to the cocrystallized ligand. The discovered hits represent novel synthetically tractable scaffolds as potential MERS-CoV M pro inhibitors.Communicated by Ramaswamy H. Sarma.

To make investigational drug candidates available to patients sooner, timelines for drug development are becoming shorter. Synthesis route scouting for active pharmaceutical ingredients (API) and drug product development often must occur simultaneously, requiring formulators to make decisions regarding drug product process selection before commercial API route finalization. Alternatively, the formulation strategy may be locked, thereby constraining drug substance processes with strict API attribute requirements. Critical quality attributes of the drug product can depend heavily on the API, yet final physical attributes may not be known early on in development. Furthermore, the desire to reduce pill burden means higher drug loading in formulations, leaving little room for excipients to compensate for suboptimal API performance. The opposing challenges of API synthetic route and drug product formulation development typically lead to elongated development timelines requiring an iterative approach. In this work, a coordinated strategy was designed and implemented to deliberately range API attributes via crystallization and milling techniques to enable robust assessment of downstream manufacturing and significantly reduce the time for final process selection. The study presented was conducted on a protease inhibitor targeted for treatment of Covid-19. Given the emergent need for treatment options, this dramatically accelerated approach was crucial for potential emergency use authorization (EUA)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The papain-like protease (PLpro) is a highly conserved domain encoded by the coronavirus (CoV) genome and it plays an essential role in the replication and maturation of the virus in addition to weakening host immune response. Due to the virus's reliance on PLpro for survival and propagation, small-molecule inhibitors of PLpro serve as an attractive model for direct-acting antiviral therapeutic agents against SARS-CoV-2. Building upon existing work aimed at designing covalent inhibitors against PLpro, we report the synthesis and structure-activity relationship of analogs based on the known covalent inhibitor 1 (Sanders, et al.2023). To evaluate the efficacy of synthesized derivatives, we conducted enzymatic inhibition assays, SARS-CoV-2/HeLa-ACE2 cellular potency and toxicity assays, and profiled the most promising analogs via in vitro ADME and in vivo pharmacokinetic studies. Additionally, we describe computational docking of profiled compounds bound to PLpro to elucidate the structure-activity relationship of compounds based on 1 and offer suggestions for optimizing the potency and selectivity of the electrophilic warhead and improving ADME and PK properties for this chemotype. Relative to the parent compound, new designs demonstrate comparable potency and target selectivity for PLpro. The accomplished SAR campaign provides novel insight for future development of antivirals against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was supported by Center for Antiviral Medicines & Pandemic Preparedness (CAMPP; grant number U19AI1443)., (Copyright © 2024 The Scripps Research Institute. Published by Elsevier Ltd.. All rights reserved.)

Hyperexpression of cathepsin B caused by an imbalance of endogenous inhibitors is involved in multiple pathologies, hence making it a key therapeutic target. Protease inhibitors are effective biomolecules that regulate protease activities and are considered potential therapeutic agents in various diseases. Plant protease inhibitors have been reported as an effective complementary alternative drug. A proteinaceous cathepsin B inhibitor (CBI-BP) has been isolated from Musa acuminata Colla (banana) peel with a molecular weight of 27.9 kDa on SDS-PAGE. The purity of the CBI-BP was confirmed on the native- PAGE. The isolated CBI-BP showed an IC 50 value of 8.14 μg and a K i value of 10.59 μg (0.19 μM). Cathepsin B inhibition kinetics indicated that CBI-BP follows a mixed-type of cathepsin B inhibition. Its inhibition activity was also confirmed by reverse zymography. The inhibitor was stable from pH 2.6-10.0 with maximum activity at pH 7.2, temperature 25-100 °C and exhibited thermostability for 60 min at 70 °C. MALDI/TOF/MS analysis of CBI-BP showed 40 % similarity to the GH18 domain-containing protein (A0A4S8JRM9) from Musa balbisiana. Although in-silico docking studies showed binding of A0A4S8JRM9 to cathepsin B affects the binding energy of the substrate to cathepsin B but is not reported for any anti-cathepsin B activity. This suggests that isolated CBI-BP might be a novel protein with anti-cathepsin B activity. Thus the isolated CBI-BP may be further explored as possible anti-cathepsin B drug., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

COVID-19 is a highly infectious disease caused by a new type of extremely contagious coronavirus called SARS-CoV-2. The virus's main protease enzyme, SARS-CoV-2 Mpro, is essential for its replication and transcription processes. Targeting this enzyme presents a promising avenue for antiviral drug development. Researchers have explored the intricate three-dimensional configurations of the enzyme, analyzing its interactions with various inhibitors. These findings provide a foundation for designing specific and powerful inhibitors targeting SARS-CoV-2 Mpro. Certain plants possess medicinal attributes due to the presence of bioactive compounds that inhibit pathogens. The olive tree ( Olea europaea ) has served as a source of food and medicine, containing bioactive compounds in its leaves that hinder the proliferation of various pathogens including viruses. This study explores the potential of bioactive compounds from olive leaf extract (OLE) to inhibit SARS-CoV-2 Mpro. In-silico study was conducted to predict the pharmacokinetic and toxicity profiles of these compounds. Molecular docking was utilized to assess their binding affinity to SARS-CoV-2 Mpro and their potential interference with its function. The top three compounds, apigenin (Api), luteolin-7-O-glucoside (Lut) and rutin (Rut), were chosen based on their favorable drug-like properties and strong binding affinities to Mpro. Detailed molecular dynamics simulations demonstrated the stability of SARS-CoV-2 Mpro in conjunction with these compounds, showing minimal structural alterations over the simulation period. Particularly, Lut and Rut formed bonds with critical amino acid residues His41 and Cys145 of Mpro, suggesting their potential inhibitory effect. These findings suggest that these compounds hold promise as natural drug candidates for combating COVID-19.Communicated by Ramaswamy H. Sarma.

The COVID-19 pandemic, driven by the novel coronavirus SARS-CoV-2, has drastically reshaped global health and socioeconomic landscapes. The papain-like protease (PLpro) plays a critical role in viral polyprotein cleavage and immune evasion, making it a prime target for therapeutic intervention. Numerous compounds have been identified as inhibitors of SARS-CoV-2 PLpro, with many characterized through crystallographic studies. To date, over 70 three-dimensional (3D) structures of PLpro complexed ligands have been deposited in the Protein Data Bank, offering valuable insight into ligand-binding features that could aid the discovery and development of effective COVID-19 treatments targeting PLpro. In this study, we reviewed and analyzed these 3D structures, focusing on the key residues involved in ligand interactions. Our analysis revealed that most inhibitors bind to PLpro's substrate recognition sites S3/S4 and SUb2. While these sites are highly attractive and have been extensively explored, other potential binding regions, such as SUb1 and the Zn(II) domain, are less explored and may hold untapped potential for future COVID-19 drug discovery and development. Our structural analysis provides insights into the molecular features of PLpro that could accelerate the development of novel therapeutics targeting this essential viral enzyme.

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a major challenge to global health. Targeting the main protease of the virus (Mpro), which is essential for viral replication and transcription, offers a promising approach for therapeutic intervention. In this study, advanced computational techniques such as molecular docking and molecular dynamics simulations were used to screen a series of antiviral compounds for their potential inhibitory effect on the SARS-CoV-2 Mpro. A comprehensive analysis of compounds from the ChemDiv and PubChem databases was performed. The physicochemical properties, pharmacokinetics, and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiles were evaluated to determine drug similarity and safety. Compound 4896 - 4038 proved to be the most promising candidate. It exhibited a favorable balance between molecular weight (491.06) and lipophilicity (logP 3.957), high intestinal absorption (92.119%), and broad tissue distribution (VDss of 0.529), indicating good oral bioavailability and therapeutic potential. Molecular docking studies showed that 4896 - 4038 has a strong binding affinity to the active site of Mpro and forms key interactions, such as hydrogen bonds, carbon-hydrogen bonds, pi-sulfur, and multiple van der Waals and pi-pi stacked bonds. The binding energy was comparable to that of the reference drug X77, indicating potential efficacy. Molecular dynamics simulations over 300 ns confirmed the stability of the Mpro/4896 - 4038 complex of protein-ligand. Free energy landscape mapping and MM/PBSA calculations further substantiated the favorable binding and stability of the complex. Importantly, 4896 - 4038 exhibited a comparatively favorable safety profile. In summary, compound 4896 - 4038 shows significant potential as a potent SARS-CoV-2 Mpro inhibitor, combining potent inhibitory activity with favorable pharmacokinetic and safety profiles. These results support the further development of 4896 - 4038 as a promising therapeutic agent in the fight against COVID-19 that warrants experimental validation and clinical investigation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

In the wake of the pandemic, peptidyl protease inhibitors with Pro-based rigid Leu mimetics at the P 2 position have emerged as potent drug candidates against the SARS-CoV-2 main protease. This success is intuitively attributed to the enhanced hydrophobic interactions and rigidity of Pro-based rigid Leu mimetics in the literature. However, the tertiary amide of proline P 2 derivatives, which hinders the formation of a critical hydrogen bond with the enzyme active site, and the constrained PP II conformation, which contradicts the protease preferred β-strand conformation, represent two overlooked disadvantages associated with these inhibitors over traditional inhibitors and, theoretically, should adversely affect their potency. Interestingly, despite these major disadvantages, they maintain or display improved potency compared to traditional peptidyl protease inhibitors. In this study, we uncover a previously unnoticed preference for P 2 residues of the protease inhibitors to adopt the PP II conformation, regardless of residue identity, in the main protease-bound form of key RNA viruses, deviating from the traditional β-strand conformation. We also demonstrate that Pro-based rigid Leu mimetics at P 2 enhance binding affinity by favoring the enzyme-preferred PP II conformation and significantly reducing configurational entropy loss upon binding, comparable to that of a typical hydrogen bond. This work also highlights the importance of a multidisciplinary approach to enhance the understanding of structure-activity relationships beyond traditional medicinal chemistry intuition. We believe these findings provide new, deep insights and address a major knowledge gap in the area of peptidyl protease inhibitor design, identifying key drivers behind the success of Pro-based peptidyl protease inhibitors beyond mere rigidity and hydrophobicity.

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P 2 position of a peptidomimetic inhibitor. At the P 1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir , and PF-835321 . Our inhibitors contain glutamine isosteres at the P 1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from K i = 0.6-18 nM (cathepsin L) and K i = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC 50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys 145 , and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P 3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

The emergence of new SARS-CoV-2 variants of concern (VOC) is a propulsion for accelerated potential therapeutic discovery. SARS-CoV-2's main protease (Mpro), essential for host cell viral replication, is a pre-eminent druggable protein target. Here, we perform extensive drug re-profiling of the comprehensive Excelra database, which compiles various under-trial drug candidates for COVID-19 treatment. For mechanistic understanding, the most promising screened-out molecules with targets are subjected to molecular dynamics (MD) simulations. Post-MD analyses demonstrate Darunavir, Ponatinib, and Tomivosertib forming a stable complex with Mpro, characterized by less fluctuation of Cα atoms, smooth and stable root-mean-square deviation (RMSD), and robust contact with the active site residues. Likewise, they all have lower binding free energy with Mpro, demonstrating strong affinity. In free energy landscape profiles, the distances from His41 and Cys145 exhibit a single energy minima basin, implying their preponderance in proximity to Mpro's catalytic dyad. Overall, the computational assessment earmarks promising candidates from the Excelra database, emphasizing on carrying out exhaustive biochemical experiments along with clinical trials. The work lays the foundation for potential therapeutic interventions in treating COVID-19., (© 2024 Wiley-VCH GmbH.)

Monkeypox is a zoonotic viral disease caused by the monkeypox virus, a member of the genus Orthopoxvirus within the family Poxviridae, which also includes the variola virus. On 14 August 2024, WHO Director-General declared monkeypox outbreak a public health emergency of international concern. Similar to variola virus core protease K7L, I7L could be identified as a promising target to fight against monkeypox virus. Our work provides a solid foundation as well as specific molecular tools (protease production methods, assay design, inhibitor design) that can now be used to probe the function of I7L in vitro. Notably, in this work, various reported covalent lead compounds for COVID-19 proteases were screened and A68, shikonin, and myricetin were identified as exhibiting high inhibitory activity against I7L. This work not only sheds light on effective inhibitors for the monkeypox virus core protease but also contributes to the broader search for antiviral agents targeting this enzyme., (© The Author(s) 2025. Published by Oxford University Press on behalf of FEMS.)

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (M pro ) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV M pro are essential for its dimerization, and are used to design inhibitors against SARS-CoV M pro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 M pro . To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated M pro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 M pro , with N8-A24 being the most efficient with an IC 50 value of 1.44 mM. We observed that N8-A24 reduced M pro dimerization with an IC 50 value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 M pro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19., (© 2024 Wiley-VCH GmbH.)

Variants of SARS-CoV-2 have continued to emerge across the world and cause hundreds of deaths each week. Due to the limited efficacy of vaccines against SARS-CoV-2 and resistance to current therapies, additional anti-viral therapeutics with pan-coronavirus activity are of high interest. Here, we screen 2.8 billion compounds from a DNA-encoded chemical library and identify small molecules that are non-covalent inhibitors targeting the conserved 3CL protease of SARS-CoV-2 and other coronaviruses. We perform structure-based optimization, leading to the creation of a series of potent, non-covalent SARS-CoV-2 3CL protease inhibitors, for coronavirus infections. To characterize their binding mechanism to the 3CL protease, we determine 16 co-crystal structures and find that optimized inhibitors specifically interact with both protomers of the native homodimer of 3CL protease. Since 3CL protease is catalytically competent only in the dimeric state, these data provide insight into the design of drug-like inhibitors targeting the native homodimer state. With a binding mode different from the covalent 3CL inhibitor nirmatrelvir, the protease inhibitor in the COVID drug Paxlovid, these compounds may overcome resistance reported for nirmatrelvir and complement its clinical utility., Competing Interests: Competing interests: S.I., H.L., A.Z., F.F., B.R.S., A.C., and D.D.H. are inventors on a patent application submitted based on this work. B.R.S. is an inventor on additional patents and patent applications related to small molecule therapeutics, and co-founded and serves as a consultant to Exarta Therapeutics, and ProJenX Inc., serves as a consultant to Weatherwax Biotechnologies and Akin Gump Strauss Hauer & Feld LLP, and holds equity in Sonata Therapeutics. S.I. and D.D.H. have sponsored research agreements with Enanta Pharmaceuticals, Shionogi & Co., Ltd., and Regeneron Pharmaceuticals. D.D.H. is a co-founder of TaiMed Biologics and RenBio, consultant to WuXi Biologics, Brii Biosciences, Apexigen, and Veru Inc., and board director for Vicarious Surgical. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: The main protease (M pro ) is a crucial enzyme for the life cycle of SARS-CoV-2 and a validated target for the treatment of COVID-19 infection. Natural products have been a proper alternative for treating viral diseases by modulating different steps of the life cycle of many viruses., Objective: This review article is designed to summarize the cumulative information of natural-derived M pro inhibitors that are validated by experimental biological testing., Methods: The natural-derived M pro inhibitors of SARS-CoV-2 that have been discovered since the emergence of the COVID-19 pandemic are reviewed in this article. Only natural products with experimental validation are reported in this article. Collected compounds are classified according to their chemical identity into flavonoids, phenolic acids, quinones, alkaloids, chromones, stilbenes, tannins, lignans, terpenes, and other polyphenolic and miscellaneous natural-derived M pro inhibitors., Conclusion: These compounds could serve as scaffolds for further lead-structure optimization for desirable potency, a larger margin of safety, and better oral activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The persistence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new mutant strains continue to present a substantial threat with potential for future pandemics. Safe, effective, and readily available COVID-19 therapeutics are urgently needed to prepare for future coronavirus pandemics. To help identify new antiviral agents, the present study focused on natural products in the extracts of Holy Basil, Ocimum tenuiflorum L., which show potential inhibitory effects against the SARS-CoV-2 main protease (M pro ). Bioassay-guided isolation of the MeOH extracts of O. tenuiflorum led to the identification of a sulfur-containing glyceroglycolipid, sulfoquinovosyl diacylglycerol (SQDG: 1), as a potent M pro inhibitor that effectively inhibited M pro activity (IC 50 : 0.42 µM). SQDG (1) also markedly suppressed SARS-CoV-2 replication (EC 50 , 51.2 µM) in vitro while displaying no cytotoxicity (CC 50 > 100 µM). Further inhibition kinetic studies and docking simulations clearly demonstrated that SQDG strongly inhibited SARS-CoV-2 M pro in a competitive and mixed-inhibition manner. These findings highlight SQDG as a promising lead compound for COVID-19 therapy and emphasize the need to explore new drugs from natural sources., Competing Interests: Declarations. Conflict of interest: The authors declare no competing financial interests., (© 2024. The Author(s).)

Plasmodium falciparum is the leading cause of malaria with 627,000 deaths annually. Invasion and egress are critical stages for successful infection of the host yet depend on proteins that are extensively pre-processed by various maturases. Plasmepsins (Plasmodium pepsins, abbreviated PM, I-X) are pepsin-like aspartic proteases that are involved in almost all stages of the life cycle. The goal of this study was to use de-novo generative modeling techniques to create novel potential PfPMX inhibitors. A total of 4325 compounds were virtually screened by structural-based docking methods. The obtained hits were utilized to refine a structure-based Ligand Neural Network (L-Net) generative model to generate related compounds. The obtained optimal L-Net Compounds with smina scores ≤ -5.00KCalmol-1 and QED ≥ 0.35 were further taken for amplification utilizing Ligand Based Transformer modeling using Deep generative learning (Drug Explorer/DrugEx). The resulting hits were then subjected to XP Glide conventional Molecular docking and QikProp ADMET screening; molecules with XP Docking score ≤ -7.00KCalmol-1 were retained. Based on their Glide ligand efficiency, originality, and uniqueness, 30 compounds were chosen for binding affinity and MM&#95;GBSA energy determination. Following Induced Fit docking (IFD), 7 compounds were taken for 50 ns MD simulations and FEP/MD calculations. This study reported novel potential PfPMX inhibitors with acceptable ADMET profiles and reasonable synthetic accessibility scores, as well as sufficient docking scores against other PMs were generated. The PfPMX inhibitors reported in this article are promising antimalarials for the next stages of drug development, and the first of their kind to be investigated thoroughly.Communicated by Ramaswamy H. Sarma.

In the last few years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the cause of a worldwide pandemic, highlighting the need for novel antiviral agents. The main protease (M pro ) of SARS-CoV-2 was immediately identified as a crucial enzyme for viral replication and has been validated as a drug target. Here, we present the design and synthesis of peptidomimetic M pro covalent inhibitors characterized by quinoline-based P 3 moieties. Structure-activity relationships (SARs) were also investigated at P 1 and P 2 , as well as for different warheads. The binding modes of the designed inhibitors were assessed using X-ray crystallographic and molecular docking studies. The identified M pro inhibitors were tested for their antiviral activities in cell-based assays, and the results were encouraging. The SAR studies presented here can contribute to the future design of improved inhibitors by addressing some of the current or prospective issues regarding M pro inhibitors currently used in therapy., (© 2025 Deutsche Pharmazeutische Gesellschaft.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like cysteine protease (PLpro) represents one of only two essential cysteine proteases involved in the regulation of viral replication. It, therefore, qualifies as a promising therapeutic target for the development of antiviral agents. We identified a previously synthesized protease inhibitor, resulting from an earlier project, as a PLpro inhibitor and crafted a structure-activity relationship around the hit, leading to the more potent inhibitors ZHAWOC6941 (17h) and ZHAWOC25153 (17o) displaying IC 50 values of 8 and 7 µM, respectively. The two compounds represent a new class of PLpro inhibitors and, with single-digit micromolar IC 50 values, are comparable to inhibitors found in the literature., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Background: Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain ( G. lingzhi ) and an Amazonian isolate ( Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study., Methods: Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR)., Results: Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDSPAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor., Conclusion: Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Plasmepsin II (PlmII) belongs to the aspartate proteases and is involved in hemoglobin degradation in Plasmodium falciparum. Due to its critical role in the survival of the Plasmodium, PlmII is considered as a potent drug target for antimalarial therapy. We have done recombinant protein production of pro-plasmepsin II (Pro-plmII). Pro-PlmII was further activated to mature form (mPlmII) and its activity was confirmed by enzyme kinetics studies. The fluorescence spectroscopic and isothermal titration calorimetric studies show that fluoroquinolone-based antibiotic drugs norfloxacin, ciprofloxacin, and sparfloxacin bind with mPlmII. Molecular docking results show that only norfloxacin and ciprofloxacin are able to bind at the active site of mPlmII via hydrogen binding and hydrophobic interactions. Enzyme kinetics analysis reveals that norfloxacin and ciprofloxacin effectively inhibit mPlmII activity, while sparfloxacin does not exhibit any inhibitory effect on the enzyme's catalytic function. The two methyl groups on the 3rd and 5th carbon atoms of the piperazine ring make sparfloxacin unable to go inside mPlmII and bind at its active site. Overall, the results here suggested that fluoroquinolone-based antibiotic drugs norfloxacin, and ciprofloxacin, can be repurposed as antimalarial inhibitors targeting aspartic proteases. These findings contribute to pave the way for potential therapeutic interventions targeted at malaria., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The mosquito-borne pathogenic alphavirus known as Chikungunya virus (CHIKV) is becoming a greater hazard to public health, which causes thousands of cases annually in both rural and urban areas of many different nations throughout the world. Finding and creating new leads for the CHIKV virus is crucial because there are currently no effective medications or vaccinations against it. The non-structural protein 2 (nsP2) protease has emerged as a promising target for therapeutic intervention due to its crucial role in viral replication., Purpose: This systematic review aims to evaluate recent advances in natural products as inhibitors of the CHIKV nsP2 protease, summarizing current research, identifying promising compounds, and highlighting gaps in the existing knowledge., Study Design: A comprehensive literature search was conducted between January 2006, and June 2024 using databases including PubMed, Scopus, Science Direct, and Google Scholar. Search terms included CHIKV, nsP2 protease, antivirals, natural products, phytochemicals, and inhibitors. Studies were selected based on predefined inclusion and exclusion criteria, focusing on original research articles examining natural products as inhibitors of CHIKV nsP2 protease., Methods: Relevant studies were screened, and data were extracted regarding the source of natural compounds, methods of extraction, chemical structures, mechanisms of action, potency, and efficacy in inhibiting nsP2 protease or CHIKV replication., Results: The review included 40 studies, revealing a variety of natural products and their derivatives with inhibitory effects on CHIKV nsP2 protease. Several compounds demonstrated promising inhibitory activity with EC50 values in the micromolar range. Mechanistic studies revealed diverse modes of action, including inhibition of protease activity or interference with viral replication processes., Conclusion: Natural products have gained attention for their diverse chemical structures and bioactivities, offering a rich source of compounds with antiviral potential. We summarize the current knowledge on natural products derived from various sources including flavonoids, alkaloids, terpenoids, polyphenols, and some derivative compounds that have demonstrated inhibitory effects against CHIKV through different mechanisms of action. Overall, this systematic review underscores the importance of exploring natural products as promising candidates for the development of effective therapeutics against Chikungunya fever, particularly through targeting the nsP2 protease., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor M pro . We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains M pro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall M pro inhibition and the fraction of uncleaved precursor form of M pro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature M pro . Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature M pro , indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of M pro for inhibition or premature activation of M pro ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Dengue, West Nile, Zika, Yellow fever, and Japanese encephalitis viruses persist as significant global health threats. The development of new therapeutic strategies based on inhibiting essential viral enzymes or viral-host protein interactions is problematic due to the fast mutation rate and rapid emergence of drug resistance. This study focuses on the NS2B-NS3 protease as a promising target for antiviral drug development. Promising allosteric binding sites were identified in two conformationally distinct inactive states and characterized for five flaviviruses and four Dengue virus subtypes. Their shapes, druggability, inter-viral similarity, sequence variation, and susceptibility to drug-resistant mutations have been studied. Two identified allosteric inactive state pockets appear to be feasible alternatives to a larger closed pocket near the active site, and they can be targeted with specific drug-like small-molecule inhibitors. Virus-specific sequence and structure implications and the feasibility of multi-viral inhibitors are discussed.

3-Chymotrypsin-like protease (3CL pro ) is a prominent target against pathogenic coronaviruses. Expert knowledge of the cysteine-targeted covalent reaction mechanism is crucial to predict the inhibitory potency of approved inhibitors against 3CL pro s of SARS-CoV-2 variants and perform structure-based drug design against newly emerging coronaviruses. We carried out an extensive array of classical and hybrid QM/MM molecular dynamics simulations to explore covalent inhibition mechanisms of five well-characterized inhibitors toward SARS-CoV-2 3CL pro and its mutants. The calculated binding affinity and reactivity of the inhibitors are highly consistent with experimental data, and the predicted inhibitory potency of the inhibitors against 3CL pro with L167F, E166V, or T21I/E166V mutant is in full agreement with IC 50 s determined by the accompanying enzymatic assays. The explored mechanisms unveil the impact of residue mutagenesis on structural dynamics that communicates to change not only noncovalent binding strength but also covalent reaction free energy. Such a change is inhibitor dependent, corresponding to varied levels of drug resistance of these 3CL pro mutants against nirmatrelvir and simnotrelvir and no resistance to the 11a compound. These results together suggest that the present simulations with a suitable protocol can efficiently evaluate the reactivity and potency of covalent inhibitors along with the elucidated molecular mechanisms of covalent inhibition.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, necessitates the development of effective therapeutics. The main protease of the virus, Mpro, is a key target due to its crucial role in viral replication. Our study presents a novel approach combining ligand-based pharmacophore modeling with structure-based advanced virtual screening to identify potential inhibitors of Mpro. We screened around 200 million compounds using this integrated methodology, resulting in a shortlist of promising compounds. These were further scrutinized through molecular dynamics simulations, revealing their interaction dynamics with Mpro. Subsequent in vitro assays using the Mpro enzyme identified two compounds exhibiting significant micromolar inhibitory activity. These findings provide valuable scaffolds for the development of advanced therapeutics targeting Mpro. The comprehensive nature of our approach, spanning computational predictions to experimental validations, offers a robust pathway for rapid and efficient identification of potential drug candidates against COVID-19., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All the authors have approved the manuscript for publication. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

This study investigated a library of known and novel glycyrrhizic acid (GL) conjugates with amino acids and dipeptide esters, as inhibitors of the DENV NS2B-NS3 protease. We utilized docking algorithms to evaluate the interactions of these GL derivatives with key residues (His51, Asp75, Ser135, and Gly153) within 10 Å of the DENV-2 NS2B-NS3 protease binding pocket (PDB ID: 2FOM). It was found that compounds 11 and 17 exhibited unique binding patterns, forming hydrogen bonds with Asp75, Tyr150, and Gly153. Based on the molecular docking data, conjugates 11 with L-glutamic acid dimethyl ester, 17 with β-alanine ethyl ester, and 19 with aminoethantic acid methyl ester were further demonstrated as potent inhibitors of DENV-2 NS3 protease, with IC50 values below 1 μM, using NS3-mediated cleavage assay. Compound 11 was the most potent, with EC50 values of 0.034 μM for infectivity, 0.042 μM for virus yield, and a selective index over 2000, aligning with its strong NS3 protease inhibition. Compound 17 exhibited better NS3 protease inhibition than compound 19 but showed weaker effects on infectivity and virus yield. While all compounds strongly inhibited viral infectivity post-entry, compound 19 also blocked viral entry. This study provided valuable insights into the interactions between active GL derivatives and DENV-2 NS2B-NS3 protease, offering a comprehensive framework for identifying lead compounds for further drug optimization and design as NS2B-NS3 protease inhibitors against DENV.

Proteases are a privileged class of enzymes due to their catalysis of an irreversible post translational modification, namely cleavage of substrate proteins. Protease activity is essential for human pathways including inflammation, blood clotting, and apoptosis. Proteases are also essential for the propagation of many viruses due to their role in cleavage of the viral polyprotein. For these reasons, proteases are an attractive and highly exploited class of drug targets. To fully harness the power of proteases as drug targets, it is essential that their presence and function are detectable throughout the course of the protease lifetime, from inactive zymogen to the fully cleaved (mature) protease. A number of methods for detection of proteases have been developed, however, many rely on catalytic activity, so are not useful throughout the proteolytic life cycle. Here, we build on our observation that the MH1 family of benzofuran-aminothiazolopyridine inhibitors of Zika virus protease (ZVP) undergo a unique FRET interaction with tryptophan residues in the protease. The full FRET signal is only observed in higher potency binding interactions. Moreover, this approach can distinguish two inactive variants of ZVP based on their folded or unfolded state. These studies also probe the physicochemical basis of the FRET signal. Exploiting these types of FRET interactions may offer an orthogonal approach for detection of this protease, which takes advantage of the relationship between the novel ligand and the core of the protein and is therefore useful throughout the protease maturation cycle. Depending on chemical properties, this approach may be applicable in other proteases and other protein classes.

The SARS-CoV-2 main protease (Mpro, also known as 3CLpro) is a key target for antiviral therapy due to its critical role in viral replication and maturation. This study investigated the inhibitory effects of Bofutrelvir, Nirmatrelvir, and Selinexor on 3CLpro through molecular docking, molecular dynamics (MD) simulations, and free energy calculations. Nirmatrelvir exhibited the strongest binding affinity across docking tools (AutoDock Vina: -8.3 kcal/mol; DiffDock: -7.75 kcal/mol; DynamicBound: 7.59 to 7.89 kcal/mol), outperforming Selinexor and Bofutrelvir. Triplicate 300 ns MD simulations revealed that the Nirmatrelvir-3CLpro complex displayed high conformational stability, reduced root mean square deviation (RMSD), and a modest decrease in solvent-accessible surface area (SASA), indicating enhanced structural rigidity. Gibbs free energy analysis highlighted greater flexibility in unbound 3CLpro, stabilized by Nirmatrelvir binding, supported by stable hydrogen bonds. MolProphet prediction tools, targeting the Cys145 residue, confirmed that Nirmatrelvir exhibited the strongest binding, forming multiple hydrophobic, hydrogen, and π-stacking interactions with key residues, and had the lowest predicted IC 50 /EC 50 (9.18 × 10 -8 mol/L), indicating its superior potency. Bofutrelvir and Selinexor showed weaker interactions and higher IC 50 /EC 50 values. MM/PBSA analysis calculated a binding free energy of -100.664 ± 0.691 kJ/mol for the Nirmatrelvir-3CLpro complex, further supporting its stability and binding potency. These results underscore Nirmatrelvir's potential as a promising therapeutic agent for SARS-CoV-2 and provide novel insights into dynamic stabilizing interactions through AI-based docking and long-term MD simulations.

Inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) by small-molecules is being eagerly investigated for the treatment of various autoimmune diseases and in the field of immuno-oncology after its active involvement in antigen presentation and processing. Currently, ERAP1 inhibitors are at different stages of clinical development, which highlights its significance as a promising drug target. In the present work, we describe the first-ever successful identification of several ERAP1 inhibitors derived from a fragment-based screening approach. We applied an enzymatic activity assay to a large library of ∼3000 fragment entries in order to retrieve 32 hits. After a multi-faceted selection process, we prioritized 3 chemotypes for SAR optimization and strategic modifications provided 2 series (2-thienylacetic acid and rhodanine scaffolds) with improved analogues at the low micromolar range of ERAP1 inhibition. We report also evidence of selectivity against homologous aminopeptidase IRAP, combined with complementary in silico docking studies to predict the binding mode and site of inhibition. Our compounds can be the starting point for future fragment growing and rational drug development, incorporating new chemical modalities., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rebecca Deprez-Poulain reports financial support was provided by European Commission. Vasileios Fougiaxis reports financial support was provided by European Commission. Rebecca Deprez-Poulain reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has not yet been eradicated. SARS-CoV-2 has two types of proteases, a main protease (M pro ) and a papain-like protease (PL pro ), which together process two translated non-structural polyproteins, pp1a and pp1ab, to produce functional viral proteins. In this study, effective inhibitors against PL pro of SARS-CoV-2 were designed and synthesized using GRL-0048 as a lead. A docking simulation of GRL-0048 and SARS-CoV-2 PL pro showed that GRL-0048 noncovalently interacts with PL pro , and there is a newly identified binding pocket in PL pro . Structure-activity relationship studies were next performed on GRL-0048, resulting in the development of several inhibitors, specifically compounds 1, 2b, and 3h, that have more potent inhibitory activity than GRL-0048., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Neprilysin is a cell surface metallo-endopeptidase, commonly identified as neutral endopeptidase (NEP), that plays a crucial role in the cleavage of peptides, for example, natriuretic peptides, angiotensin II, enkephalins, endothelin, bradykinin, substance P, glucagon-like peptide and amyloid beta. In the case of heart failure, a significant upsurge in NEP activity and expression enhances the degradation of natriuretic peptides. Therefore, NEP inhibitors have gained attention in the field of cardiology. NEP has been studied for over 40 years; however, it has recently gained attention with the US FDA approval of a fixed dose combination of sacubitril (NEP inhibitor) and valsartan (AT-1 inhibitor) for chronic heart failure treatment. The present review elucidates the role of neprilysin in cardiorenal disease, its pathophysiology, and how NEP inhibition benefits. It also summarizes the research advances in NEP inhibitors (NEPi) and their structure-activity relationships. Moreover, the review provides insight into NEPi effectiveness - alone or combined with other cardiorenal protective agents. It is expected to help medicinal chemists synthesize and develop novel NEPi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The COVID-19 pandemic, exacerbated by persistent viral mutations, underscored the urgent need for diverse inhibitors targeting multiple viral proteins. In this study, we utilized covalent DNA-encoded libraries to discover innovative triazine-based covalent inhibitors for the 3-chymotrypsin-like protease (3CL pro , Nsp5) and the papain-like protease (PL pro ) domains of Nsp3, as well as novel non-nucleoside covalent inhibitors for the nonstructural protein 12 (Nsp12, RdRp). Optimization through molecular docking and medicinal chemistry led to the development of LU9 , a nonpeptide 3CL pro inhibitor with an IC 50 of 0.34 μM, and LU10 , whose crystal structure showed a distinct binding mode within the 3CL pro active site. The X-ray cocrystal structure of SARS-CoV-2 PL pro in complex with XD5 uncovered a previously unexplored binding site adjacent to the catalytic pocket. Additionally, a non-nucleoside covalent Nsp12 inhibitor XJ5 achieved a potency of 0.12 μM following comprehensive structure-activity relationship analysis and optimization. Molecular dynamics revealed a potential binding mode. These compounds offer valuable chemical probes for target validation and represent promising candidates for the development of SARS-CoV-2 antiviral therapies.

In the postpandemic era, the emergence of "long COVID" from SARS-CoV-2 has brought ongoing negative impacts on individual health and society. The development of more efficient methods for drug screening and monitoring viral activity remains a critical need. The main protease (M pro ), due to its important role in the virus lifecycle, high conservation, and specificity, is considered an ideal biomarker for SARS-CoV-2. Herein, we have developed several chemiluminescence probes based on different substrates modified from covalent inhibitors targeted at M pro . Among these, the best probe, MPCL-2, exhibits a rapid response (<20 min), an extremely low limit of detection (LoD; 0.11 nM), great selectivity, and chemical stability. After validating the probe's mechanism of action, MPCL-2 can also be used for real-time, in-situ imaging of enzymes in cells infected with the authentic virus and has the potential for real-time, in-situ M pro imaging in vivo . Compared to other methods reported to date, the probe demonstrates superior performance and broader applicability, such as in drug screening or virus activity monitoring. Further, the unique design strategy for the substrate can be adopted to develop sensitive probes for other pathogens.

Profiling the substrate sequence preferences of proteases is important for understanding both biological functions as well as for designing protease inhibitors. Several methods are available for profiling the sequence specificity of proteases. However, there is currently no rapid and high-throughput method to profile specificity of proteases for noncanonical substrates. In this study, we described a strategy to use a DNA-encoded noncanonical substrate library to identify the protease substrates composed of both canonical and noncanonical amino acids. This approach uses a DNA-encoded peptide library and introduces a biotin molecule at the N-terminus to immobilize the library on a solid support. Upon protease hydrolysis, the released DNA tag of the substrate peptides can be sequenced to identify the substrate structures. Using this approach, we profiled trypsin and fibroblast activation protein α and discovered noncanonical substrates that were more efficiently cleaved than the commonly used substrates. The identified substrates of FAP were further used to design corresponding covalent inhibitors containing non-canonical sequences with high potency for the target protease. Overall, our approach can aid in the development of new protease substrates and inhibitors., (© 2024 Wiley-VCH GmbH.)

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases M pro and PL pro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC 50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC 50 against PL pro at approximately 10-fold higher micromolar concentrations. Although originally developed as PL pro inhibitors, the comparison between IC 50 and EC 50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

Background: The COVID-19 pandemic has created an urgent need for effective therapeutic agents. The SARS-CoV-2 Main Protease (M pro ) plays a crucial role in viral replication and immune evasion, making it a key target for drug development. While several studies have explored M pro inhibition, identifying FDA-approved drugs with potential efficacy remains a critical research focus., Purpose: This study aims to identify FDA-approved drugs that could inhibit SARS-CoV-2 M pro . Using computational screening, we seek compounds that share structural similarities with a known co-crystallized ligand (PRD&#95;002214) and exhibit strong binding affinity to the enzyme, providing viable candidates for COVID-19 treatment., Research Design: A systematic in silico approach was used, screening 3009 FDA-approved drugs. The initial screening focused on structural similarity to PRD&#95;002214 (PDB ID: 6LU7), followed by molecular docking studies to predict binding affinity. Promising compounds were further analyzed through molecular dynamics (MD) simulations to evaluate their stability and interactions with M pro over 100 ns., Study Sample: Of the 3009 FDA-approved drugs screened, 74 were selected for initial evaluation. After refinement, 28 compounds underwent docking analysis, with eight showing strong binding potential to M pro ., Analysis: Molecular docking assessed the binding affinity and interaction of the selected compounds with M pro . MD simulations were conducted on the top compound, Atazanavir, to study its dynamic interactions. MM-GBSA, PLIP, and PCAT analyses were used to validate binding affinity and interactions., Results: Eight compounds, including Carfilzomib, Atazanavir, Darunavir, and others, exhibited promising binding affinities. Among them, Atazanavir showed the highest binding strength and was selected for further MD simulation studies. These simulations revealed that Atazanavir forms stable interactions with M pro , demonstrating favorable binding and dynamic stability. The binding affinity was further confirmed through MM-GBSA, PLIP, and PCAT analyses, supporting Atazanavir's potential as an effective M pro inhibitor., Conclusions: In silico results suggest that Atazanavir is a promising candidate for targeting SARS-CoV-2 M pro , with strong binding affinity and dynamic stability. These findings support its potential as a lead compound for further preclinical and clinical testing, though in vitro and in vivo validation are needed to confirm its therapeutic efficacy against COVID-19., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.