Main Protease Research Articles

Noncovalent Inhibitors of SARS-CoV-2 Main Protease: A Rescaffolding Attempt

AbstractOut of the results of the sole large-scale screening for inhibitors of SARS-CoV-1 main protease reported in 2013, attempts to improve the identified 3-pyridyl-bearing hits have been conducted in research laboratories, either on this enzyme or more recently on the closely related SARS-CoV-2 main protease. From the resulting structural information reported, we sought to design analogues featuring some of the components providing an affinity for the active site of these proteases along with a different scaffold, which would allow for further structure-activity relationship studies and/or pharmacological improvements. We describe here the introduction of a bridging component with the aim of stabilizing the ligand conformation adopted when bound to these proteases. Accordingly, this led us to prepare 3,3-disubstituted piperazin-2-ones from an array of ketones, via either a Bargellini reaction or a multistage condensation/cyclization/hydrolysis involving ethylene diamine and potassium cyanide. However, even the most elaborate and lipophilic biphenyl-bearing analogues displayed only a weak effect in a bioluminescence-based SARS-CoV-2 main protease inhibition assay.

SARS-CoV-2 Mpro Dihedral Angles Reveal Allosteric Signaling.

In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state of the art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the protein's C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedral angles in transmitting allosteric signals.

Revealing the impact of active site residues in modeling the inhibition mechanism of SARS-Cov-2 main protease by GC373.

Revealing the impact of active site residues in modeling the inhibition mechanism of SARS-Cov-2 main protease by GC373.

Identification of anti-SARS-CoV-2 compounds from Qingwen Zhike prescription and exploration of their underlying mechanism by UPLC-Q-Exactive Orbitrap MS, high-throughput screening assays and transmission electron microscopy.

Identification of anti-SARS-CoV-2 compounds from Qingwen Zhike prescription and exploration of their underlying mechanism by UPLC-Q-Exactive Orbitrap MS, high-throughput screening assays and transmission electron microscopy.

Design, synthesis, and antiviral activity of fragmented-lapatinib aminoquinazoline analogs towards SARS-CoV-2 inhibition.

Design, synthesis, and antiviral activity of fragmented-lapatinib aminoquinazoline analogs towards SARS-CoV-2 inhibition.

Discovery of Nirmatrelvir (PF-07321332): A Potent, Orally Active Inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) Main Protease.

In early 2020, severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infections leading to COVID-19 disease reached a global level leading to the World Health Organization (WHO) declaration of a pandemic. Scientists around the globe rapidly responded to try and discover novel therapeutics and repurpose extant drugs to treat the disease. This work describes the preclinical discovery efforts that led to the invention of PF-07321332 (nirmatrelvir, 14), a potent and orally active inhibitor of the SARS CoV-2 main protease (Mpro) enzyme. At the outset we focused on modifying PF-00835231 (1) discovered in 2004 as a potent inhibitor of the SARS CoV-1 Mpro with poor systemic exposure. Our effort was focused on modifying 1 with the goal of engineering in oral bioavailability by design, while maintaining cellular potency and low metabolic clearance. Modifications of 1 ultimately led to the invention of nirmatrelvir 14, the Mpro inhibitor component in PAXLOVID.

Unveiling the antiviral inhibitory activity of ebselen and ebsulfur derivatives on SARS-CoV-2 using machine learning-based QSAR, LB-PaCS-MD, and experimental assay

Ebsulfur and ebselen derivatives that were proven to be potent inhibitors against the main protease (MPro) of SARS-CoV-2 which is an essential enzyme for viral replication were chosen to study the quantitative structure–activity relationship (QSAR) analysis using a classical multiple linear regression (MLR) and a machine learning approach of random forest (RF) and artificial neural network (ANN) in order to find the relationship between molecular structural properties and biological inhibitory activities. With the statistical criteria, the R2 values of MLR, RF, and ANN models for the training set were 0.83, 0.82, and 0.92, respectively. The RMSE values of the test were considered for model evaluation, and the results were 0.27, 0.18, and 0.09 for MLR, RF, and ANN models, respectively. Therefore, the ANN model was the best-obtained model for predicting the MPro inhibitory activity of thirteen new synthetic ebselen analogs that haven’t tested the biological assay before. Notably, our predicted inhibitory activities against SARS-CoV-2 were then examined using enzyme-based assays and cytotoxicity tests, which found that compound P8 resulted in a good potential candidate for SARS-CoV-2 MPro inhibitory activity. Furthermore, the molecular dynamics simulations were performed to study the dynamic interaction of ligand and binding site; the results showed a binding pathway and mechanism of compound P8 with key residues surrounding the active site of SARS-CoV-2 MPro, which is useful for further development of ebselen derivatives.

Chondroitin sulfate binds to main protease of SARS-CoV-2 and efficaciously inhibits its activity.

Chondroitin sulfate binds to main protease of SARS-CoV-2 and efficaciously inhibits its activity.

Battling COVID-19 leveraging nanobiotechnology: Gold and silver nanoparticle-B-escin conjugates as SARS-CoV-2 inhibitors.

The COVID-19 pandemic, an unprecedented global health crisis, has thrust humanity into a relentless battle with a variety of treatments and vaccines against the SARS-CoV-2 virus. Recent developments in nanotechnology have garnered significant interest in the application of metallic nanoparticles (NPs); specifically, silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) have demonstrated antimicrobial and antiviral properties. This study investigates the molecular interactions between the receptor binding domains of five SARS-CoV-2 spike protein variants (Alpha, Beta, Delta, Omicron, and Gamma) and the angiotensin-converting enzyme 2 (ACE2) receptor, followed by the docking of AuNPs and AgNPs and the natural compound Beta-escin onto these complexes. As well as the inspection of both NPs against the virus main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). Comprehensive computational simulations utilizing Autodock 4.2 and HDOCK server were employed to evaluate the binding affinities of these NPs toward key viral targets, SARS-CoV-2 Mpro, RdRp, and the spike glycoprotein. The results revealed that both AgNPs and AuNPs exhibited successful binding to the active pockets of SARS-CoV-2 Mpro, with slightly varying binding energies. In contrast, for RdRp, AgNPs demonstrated superior binding affinity compared to AuNPs, with differences in the residues involved in the binding pocket. AuNPs exhibited stronger binding affinities in the spike protein pocket. We also determined robust binding affinities between ACE2 and the spike variants, with the Omicron variant exhibiting the highest affinity. Subsequent docking of AuNPs and AgNPs revealed strong interactions with all ACE2-spike complexes, with AuNPs showing slightly higher affinities. The findings contribute to a deeper understanding of the interactions between NPs and viral proteins, shedding light on their mechanisms of action and their potential to offer innovative solutions for combating infectious diseases, particularly those caused by SARS-CoV-2.

Evaluating the structure-based virtual screening performance of SARS-CoV-2 main protease: A benchmarking approach and a multistage screening example against the wild-type and Omicron variants.

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.

Dihydropyrimidine-2-thione derivatives as SARS-CoV-2 main protease inhibitors: synthesis, SAR and in vitro profiling.

Despite the passage of approximately five years since the outbreak, an efficacious remedy for SARS-CoV-2 remains elusive, highlighting the urgent imperative for developing SARS-CoV-2 potent inhibitors. In our current study, we have unmasked the hitherto unrealized potential of dihydropyrimidine-2-thiones against the Main Protease (Mpro) of SARS-CoV-2. Employing a predictive docking tool, we identified promising lead compounds and optimized them via comprehensive Structural Activity Relationship (SAR) studies. Key design elements included proton donor/acceptor groups, six-membered rings, and fluorinated moieties to enhance interactions. These leads underwent in vitro inhibition assays to enhance their interaction with key Mpro amino acid residues. Our findings indicated that all synthesized compounds exhibited significant inhibition of the Mpro. Compounds 12j (IC50 = 0.063 μM), and 12l (IC50 = 0.054 μM) displayed exceptional in vitro binding affinities. In addition to their string inhibitory activity, CC50 values were assessed, confirming acceptable cytotoxicity profiles for potent compounds. Molecular dynamic simulation substantiated the binding mechanism revealing that compound 12l maintains robust stability with the target protein. Furthermore, compounds predicted to have minimal oral toxicity and high intestinal absorption make them promising candidates for drug development. These findings paved the way for the potent clinical application of these dihydropyrimidine-2-thiones as efficient SARS-CoV-2 therapeutics.

T-ALPHA: A Hierarchical Transformer-Based Deep Neural Network for Protein-Ligand Binding Affinity Prediction with Uncertainty-Aware Self-Learning for Protein-Specific Alignment.

There is significant interest in targeting disease-causing proteins with small molecule inhibitors to restore healthy cellular states. The ability to accurately predict the binding affinity of small molecules to a protein target in silico enables the rapid identification of candidate inhibitors and facilitates the optimization of on-target potency. In this work, we present T-ALPHA, a novel deep learning model that enhances protein-ligand binding affinity prediction by integrating multimodal feature representations within a hierarchical transformer framework to capture information critical to accurately predicting binding affinity. T-ALPHA outperforms all existing models reported in the literature on multiple benchmarks designed to evaluate protein-ligand binding affinity scoring functions. Remarkably, T-ALPHA maintains state-of-the-art performance when utilizing predicted structures rather than crystal structures, a powerful capability in real-world drug discovery applications where experimentally determined structures are often unavailable or incomplete. Additionally, we present an uncertainty-aware self-learning method for protein-specific alignment that does not require additional experimental data and demonstrate that it improves T-ALPHA's ability to rank compounds by binding affinity to biologically significant targets such as the SARS-CoV-2 main protease and the epidermal growth factor receptor. To facilitate implementation of T-ALPHA and reproducibility of all results presented in this paper, we made all of our software available at https://github.com/gregory-kyro/T-ALPHA.

Ensitrelvir for the Treatment of Nonhospitalized Adults with COVID-19: Results from the SCORPIO-HR, Phase 3, Randomized, Double-blind, Placebo-Controlled Trial.

Ensitrelvir, a severe acute respiratory syndrome coronavirus-2 main protease inhibitor, has demonstrated clinical and virologic efficacy in previous studies. In this global phase 3 trial, nonhospitalized adults with mild-to-moderate coronavirus disease 2019 (COVID-19) and symptom onset within 5 days were randomized (1:1) to receive once-daily ensitrelvir (375 mg day 1, 125 mg days 2-5) or blinded matching placebo. The primary endpoint was the restricted mean time to sustained (≥2 days) resolution of 15 COVID-19 symptoms, recorded in participant daily diaries, through day 29 in participants starting treatment within 3 days after symptom onset. Virologic efficacy and safety were assessed. Of 2093 participants, 1888 started treatment within 3 days after symptom onset. Mean time to symptom resolution was 12.5 and 13.1 days with ensitrelvir and placebo, respectively (difference, -0.6 days; 95% confidence interval, -1.38 to 0.19; P = .14). On day 4, ensitrelvir reduced least-squares mean RNA by 0.72 log10 copies/mL more than placebo (95% confidence interval, 0.55-0.90). Among those with positive viral cultures at enrollment, 274/287 (95.5%) ensitrelvir-treated versus 210/280 (75.0%) placebo-treated participants had negative cultures on day 4. RNA rebound was similar (<1.5%) between groups. The proportion of participants with ≥1 adverse event was similar with ensitrelvir (61.5%) and placebo (60.6%). No treatment-related serious adverse events or deaths occurred. Three (0.3%) ensitrelvir-treated and 1 (0.1%) placebo-treated participants had COVID-19-related hospitalizations by day 29. Despite the evidence of antiviral activity with ensitrelvir, this trial did not demonstrate a significant difference in time to sustained symptom resolution.

Nickel(II) Complexes of Benzoylpyridine Diaminoguanidinedihydrazone via In Situ One‐Pot Method: Structural, Spectral, and Catalytic Studies

ABSTRACTSeven Ni(II) complexes (1–7) designed from a new nitrogen‐rich ligand 1,3‐bis(phenyl(pyridin‐2‐yl)methylideneamino)guanidine hydrochloride (H3L‧ HCl) are reported. The complexes were synthesized using in situ one‐pot method and characterized by elemental analyses, FT‐IR, Far‐IR, electronic solution‐phase spectra, solid‐state electronic diffuse reflectance spectra (UV‐DRS), conductivity measurements, and room temperature magnetic moment calculations. The structures of H3L‧ HCl and complex 7 were confirmed via SCXRD analysis. Additionally, complex 4 on crystallization yielded a new mononuclear complex, [Ni(H2L)]Cl‧ 2H2O (4a), which was confirmed by SCXRD analysis. The molecular packing of the compounds is affected by the counter anions and stabilized by various noncovalent interactions within the crystal lattice, which were analyzed quantitatively by Hirshfeld surface analysis. The band gaps of the compounds indicate semiconductor characteristics, which were evaluated experimentally and subsequently analyzed by DFT calculations. The catalytic investigation reveals that the Ni(II) complex 7 facilitates a cinnamyl alcohol conversion of 90% with a selectivity of 92% for cinnamaldehyde under the optimal reaction conditions of 80°C for 24 h, employing TBHP in decane as the oxidizing agent. Similarly, all other complexes are also found to exhibit comparable conversions in the range of 81%–91% under optimal conditions. Furthermore, in silico molecular docking studies demonstrated the binding affinity of the ligand and complex 4 with SARS‐CoV‐2 main protease active site (Mpro), which makes them suitable candidates for further biological exploration.

In Silico Discovery of SARS-CoV-2 Main Protease Inhibitors Using Docking, Molecular Dynamics, and Fragment Molecular Orbital Calculations.

The 3C-like protease of severe acute respiratory syndrome coronavirus 2, known as the main protease (Mpro), is an attractive drug target for the treatment of coronavirus disease 2019. This study reports the discovery of novel Mpro 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 Mpro inhibitory activity with an IC50 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 Mpro. The Mpro inhibitory activity of SH-4-54, a derivative without the aforementioned hydroxyl group, was investigated and observed to be significantly reduced, with an IC50 of 81.5 μM. This result strongly supports the suggested interaction mechanism.

Structure-Based Optimization of Pyridone α-Ketoamides as Inhibitors of the SARS-CoV-2 Main Protease.

The main protease Mpro is a clinically validated target to treat infections by the coronavirus SARS-CoV-2. Among the first reported Mpro inhibitors was the peptidomimetic α-ketoamide 13b, whose cocrystal structure with Mpro 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 Mpro with IC50s 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 EC50 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.

Galloylated polyphenols represent a new class of antithrombotic agents with broad activity against thiol isomerases.

Galloylated polyphenols represent a new class of antithrombotic agents with broad activity against thiol isomerases.

Cathepsin F alters viral acquisition, retention, and transmission of TYLCV and ToCV by Bemisia tabaci MED.

Tomato yellow leaf curl virus (TYLCV) and Tomato chlorosis virus (ToCV) are plant-infecting viruses that are mainly transmitted by Bemisia tabaci Gennadius. In addition to their significant individual impacts on agricultural production, TYLCV and ToCV co-infections are increasingly common and can cause devastating losses in tomato and other crops. Cathepsins, the main proteases in lysosomes, affect both immune responses and the digestion of plant proteins and may help mediate Bemisia-virus-plant interactions. We conducted research exploring the role of cathepsin in the interaction between B. tabaci MED and the plant viruses TYLCV and ToCV, both singly and in combination, on tomato. Levels of cathepsin F increased sharply in B. tabaci MED after feeding on TYLCV-infected, ToCV-infected, and co-infected plants and remained elevated for several days after feeding cessation. In all cases, levels were higher in co-infected B. tabaci MED than in singly infected individuals. Viral loads of each virus were also higher in co- versus singly infected B. tabaci MED, suggesting a synergistic relationship between TYLCV and ToCV. We next studied how dosing B. tabaci MED with a cathepsin inhibitor, inducer, or control affected viral acquisition, retention, and transmission. Viral acquisition and retention were lower in B. tabaci MED treated with cathepsin inducer than in controls; B. tabaci MED treated with cathepsin inhibitor had higher rates of viral acquisition and retention. Viral transmission was highest in the inhibitor treatment and lowest in the inducer treatment. Our results provide more opportunities for the design of novel control strategies to manage insect vectors and their transmitted viruses.

Nucleoside reverse transcriptase inhibition enhances platelet production and megakaryocyte maturation in patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is a systemic infection frequently involving the haematopoietic system. Thrombocytopenia is associated with increased risks of severe disease progression and mortality. Antiviral agents have shown much promise in decreasing viral load and shortening the time to the resolution of symptoms. However, their effect on platelet counts in patients with COVID-19 remains unexplored. Therefore, we first performed a retrospective study to evaluate the variation in platelet mass of hospitalized patients with high-risk COVID-19 who were given either SARS-CoV-2 main protease inhibitor, nucleoside reverse transcriptase inhibitor or no antiviral agents. A total of 177 patients were included, among which 64 received azvudine, 12 received nirmatrelvir-ritonavir and 101 patients received none. Compared to those without antiviral treatment, significantly higher platelet counts' increments were observed in patients receiving azvudine or nirmatrelvir-ritonavir (p = 0.001). Of note, this elevation was significantly more profound in the azvudine group than that in the control group (p < 0.001) or the nirmatrelvir-ritonavir group (p = 0.042). Subsequently, invitro experiments were conducted to investigate the mechanism of platelet elevation underlying the activity of azvudine. Results showed that azvudine promoted the polyploidization and platelet production of the MEG-01 cell line. Although azvudine had minimal effect on megakaryopoiesis, it could significantly trigger platelet release of megakaryocytes in the presence of SARS-CoV-2 spike-membrane recombinant fusion protein or not. Finally, RNA-sequencing demonstrated that azvudine-treated MEG-01 exhibited a marked increase in VWF, TUBB1 and GP1BA, and upregulated genes associated with the PI3K/AKT and JAK/STAT signalling pathways. In conclusion, our findings indicated that nucleoside reverse transcriptase inhibition potentially enhances platelet production and megakaryocyte maturation in patients with COVID-19, suggesting a new therapeutic option for thrombocytopenia.

Identifying Inhibitor-SARS-CoV2-3CLpro Binding Mechanism Through Molecular Docking, GaMD Simulations, Correlation Network Analysis and MM-GBSA Calculations.

The main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as 3CLpro, is crucial in the virus's life cycle and plays a pivotal role in COVID-19. Understanding how small molecules inhibit 3CLpro'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 3CLpro 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 3CLpro 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 3CLpro'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 3CLpro'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 3CLpro inhibitors.