Mpro Inhibition Research Articles

Benzocarbazoledinones as SARS-CoV-2 Replication Inhibitors: Synthesis, Cell-Based Studies, Enzyme Inhibition, Molecular Modeling, and Pharmacokinetics Insights

Endemic and pandemic viruses represent significant public health challenges, leading to substantial morbidity and mortality over time. The COVID-19 pandemic has underscored the urgent need for the development and discovery of new, potent antiviral agents. In this study, we present the synthesis and anti-SARS-CoV-2 activity of a series of benzocarbazoledinones, assessed using cell-based screening assays. Our results indicate that four compounds (4a, 4b, 4d, and 4i) exhibit EC50 values below 4 μM without cytotoxic effects in Calu-3 cells. Mechanistic investigations focused on the inhibition of the SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) have used enzymatic assays. Notably, compounds 4a and 4b showed Mpro inhibition activity with IC50 values of 0.11 ± 0.05 and 0.37 ± 0.05 µM, respectively. Furthermore, in silico molecular docking, physicochemical, and pharmacokinetic studies were conducted to validate the mechanism and assess bioavailability. Compound 4a was selected for preliminary drug-likeness analysis and in vivo pharmacokinetics investigations, which yielded promising results and corroborated the in vitro and in silico findings, reinforcing its potential as an anti-SARS-CoV-2 lead compound.

Sunflower Trypsin Monocyclic Inhibitor Selected for the Main Protease of SARS-CoV-2 by Phage Display.

Main protease (Mpro), also known as 3-chymotrypsin-like protease (3CLpro), is a nonstructural protein (NSP5) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the cleavage of virus polyproteins during viral replication at 11 sites, which generates 12 functional proteins. Mpro is a cysteine protease that presents specificity for the amino acid residue glutamine (Gln) at the P1 position of the substrate. Due to its essential role in processing the viral polyprotein for viral particle formation (assembly), Mpro inhibition has become an important tool to control coronavirus disease 2019 (COVID-19), since Mpro inhibitors act as antivirals. In this work, we proposed to identify specific inhibitors of the Mpro of SARS-CoV-2 using a monocyclic peptide (sunflower trypsin inhibitor (SFTI)) phage display library. Initially, we expressed, purified and activated recombinant Mpro. The screening of the mutant SFTI phage display library using recombinant Mpro as a receptor resulted in the five most frequent SFTI mutant sequences. Synthetized mutant SFTIs did not inhibit Mpro protease using the fluorogenic substrate. However, the mutant SFTI 4 efficiently decreased the cleavage of recombinant human prothrombin as a substrate by Mpro, as confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Additionally, SFTI 4 presented a dissociation constant (KD) of 21.66 ± 6.66 µM for Mpro by surface plasmon resonance. Finally, 0.1 µM SFTI 4 reduced VERO cell infection by SARS-CoV-2 wt after 24 and 48 h. In conclusion, we successfully screened a monocyclic peptide library using phage display for the Mpro of SARS-CoV-2, suggesting that this methodology can be useful in identifying new inhibitors of viral enzymes.

New insights in the mechanism of the SARS-CoV-2 Mpro inhibition by benzisoselenazolones and diselenides.

Although global vaccination campaigns alleviated the SARS-CoV-2 pandemic in terms of morbidity and mortality, the ability of the virus to originate mutants may reduce the efficacy of vaccines, posing a serious risk of a renewed pandemic. There is therefore a need to develop small molecules capable of targeting conserved viral targets, such as the main protease (Mpro). Here, a series of benzisoselenazolones and diselenides were tested for their ability to inhibit Mpro; then the most potent compounds were measured for antiviral activity in vitro, and the mechanism of action was investigated. Density functional theory calculations, molecular docking and molecular dynamics simulations were also used to elucidate the protein/drug interaction. Finally, a bio-organic model was established to study the reaction between selenorganic compounds and biologically relevant thiols to unveil possible metabolic pathways of such compounds. The overall results contribute to the identification of a series of novel Se-containing molecules active against SARS-CoV-2 and to the clarification of some important aspects in the mechanisms of action of such inhibitors targeting SARS-CoV-2 Mpro.

Flavonoids as potential SARS-CoV-2 main protease inhibitors: In vitro activity evaluation, molecular docking, 3D-QSAR investigation and synthesis

The damage to public health posed by the COVID-19 epidemic remains non-negligible, and research into highly effective antiviral drugs is key to addressing the epidemic. The main protease (Mpro) is critical for SARS-CoV-2 replication and acts as an effective target for drug development. To date, many flavonoid compounds have been reported to exhibit inhibition of Mpro, and flavonoid structures are informative for the development of new inhibitors. In the present study, we first evaluated the ability of 35 flavonoid compounds to bind Mpro and found that the hydrophobic interaction generated between the benzene ring and the residues may be the main source of binding force. The in vitro inhibitory activity of these compounds was tested by the FRET method, and most of them showed significant inhibition of Mpro. Preliminary analysis revealed that the presence of the glycosidic group was detrimental to the activity of the compounds, in addition the position of the hydroxyl substituent had an effect on the activity of the compounds. Reliable and predictive CoMFA (q2 = 0.541, r2 = 0.998, SEE = 0.039) and CoMISA (q2 = 0.554, r2 = 0.999, SEE = 0.034) models were developed, and the compounds were analyzed by 3D-QSAR with modeled steric, electrostatic, hydrogen bond donor, and hydrophobic fields. A series of compounds were synthesized and characterized, which showed similar experimental and predicted activities, with the activity of 15d (IC50 = 0.74 ± 0.04 uM) exceeding that of the template molecule 15 (0.98 ± 0.13 uM). Subsequently, molecular docking revealed the binding mode of 15d to Mpro and verified the predictions of steric, hydrogen bond donor, and hydrophobic field. In conclusion, these findings provide ideas for subsequent structural studies of flavonoids that could be used in the development of flavonoid-type SARS-CoV-2 Mpro inhibitors.

Viola stocksii: A rich source of antioxidant and anti-inflammatory flavonoid glycosides with converged therapeutic potential against SARS-CoV-2 MPro, spike trimer, and surface glycoproteins

This research presents the first comprehensive investigation into the phytochemical and nutraceutical properties of Viola stocksii. The study aimed to identify the therapeutic efficacy and bioactive constituents of this medicinal herb, traditionally used as an herbal tea for managing respiratory and abdominal issues in remote areas of Pakistan. The therapeutic efficacy of the plant was assessed as an anti-oxidant-rich health tonic coupled with anti-inflammatory and immunomodulatory activities against COVID-19 and its evolving variants. The study involved sequential extraction, bio-assay guided fractionation, isolation, characterization, and biological evaluation. Preliminary in vitro studies have demonstrated that polar fractions of the plant possess significant antioxidant and antiinflammatory potential. A diverse combination of chromatographic techniques (Diaion resin HP-20, Sephadex LH-20, ODS-C18, and RP-HPLC) facilitated the purification of active constituents of the plant. Comprehensive analysis of NMR (1H, 13C, and 2D NMR) and mass (EIMS, HR-EIMS, and FAB-HRMS) spectral data lead to characterize the isolated compounds (1–4) as flavonoid glycosides. Results from in vitro and in-silico evaluations revealed that the isolated glycosides exhibit high antioxidant, antiinflammatory, and immunomodulatory activities, which synergistically show promising potential in combating SARS-CoV-2 variants. Besides MPro, therapeutic potential against two unexplored proteins i.e. the spike trimer (7WZ1) and surface glycoproteins (7QTJ) was studied for the first time. Kaempferol (KamGluαRh) and quercetin (QurGluαRh) carrying rutinoside, were identified as the most active constituents of the plant, with the highest binding affinities in the range of −10 to −13 kcal/mol. Ten distinct active regions were explored on the spike trimer, including strong binding affinity with the essential RBD region, spike head, and tail of the protein. MD simulation validated the stability of Mpro-KamGluαRh and Mpro-QurGluαRh complexes evidenced by an average RMSD of 0.2 nm led to the mechanistic understanding of Mpro inhibition. ADMET and pharmacokinetic studies authenticated the therapeutic ability and drug-likeness profiling of all extracted compounds. Results revealed the beneficial role of glycosides in boosting the medicinal effectiveness of flavonoids and the potential of Viola stocksii as an immunity booster against respiratory infections like COVID-19.

Nonpeptidic Irreversible Inhibitors of SARS-CoV-2 Main Protease with Potent Antiviral Activity.

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (Mpro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including Mpro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC50 values in the low nanomolar range, kinact/Ki values of >100,000 M-1 s-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p-ethoxy-o-fluorobenzoyl chloropyridyl ester (PSB-21110, 29b, MW 296 g/mol; EC50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

A Structural Comparison of Oral SARS-CoV-2 Drug Candidate Ibuzatrelvir Complexed with the Main Protease (Mpro) of SARS-CoV-2 and MERS-CoV.

Ibuzatrelvir (1) was recently disclosed and patented by Pfizer for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has received fast-track status from the USA Food and Drug Administration (FDA) and has entered phase III clinical trials as a possible replacement for Paxlovid. Like nirmatrelvir (2) in Paxlovid, this orally active drug candidate is designed to target viral main proteases (Mpro) through reversible covalent interaction of its nitrile warhead with the active site thiol of the chymotrypsin-like cysteine protease (3CL protease). Inhibition of Mpro hinders the processing of the proteins essential for viral replication in vivo. However, ibuzatrelvir apparently does not require ritonavir (3), which is coadministered in Paxlovid to block human oxidative metabolism of nirmatrelvir. Here, we report the crystal structure of the complex of ibuzatrelvir with the active site of SARS-CoV-2 Mpro at 2.0 Å resolution. In addition, we show that ibuzatrelvir also potently inhibits the Mpro of Middle East respiratory syndrome-related coronavirus (MERS-CoV), which is fortunately not widespread but can be dangerously lethal (∼36% mortality). Co-crystal structures show that the binding mode of the drug to both active sites is similar and that the trifluoromethyl group of the inhibitor fits precisely into a critical S2 substrate binding pocket of the main proteases. However, our results also provide a rationale for the differences in potency of ibuzatrelvir for these two proteases due to minor differences in the substrate preferences leading to a weaker H-bond network in MERS-CoV Mpro. In addition, we examined the reversibility of compound binding to both proteases, which is an important parameter in reducing off-target effects as well as the potential immunogenicity. The crystal structures of the ibuzatrelvir complexes with Mpro of SARS-CoV-2 and of MERS-CoV will further assist drug design for coronaviral infections in humans and animals.

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

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

SARS-CoV-2 Main Protease Inhibitors That Leverage Unique Interactions with the Solvent Exposed S3 Site of the Enzyme.

The main protease (MPro) of SARS-CoV-2 is crucial for the virus's replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1-3') and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of MPro bound with dipeptide inhibitors containing a flexible N-terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N-terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in MPro inhibition. Our findings underscore the importance of the S3 site's unique interactions in the design of future MPro inhibitors as potential COVID-19 therapeutics.

In silico study of alkaloids with quercetin nucleus for inhibition of SARS-CoV-2 protease and receptor cell protease.

Covid-19 disease caused by the deadly SARS-CoV-2 virus is a serious and threatening global health issue declared by the WHO as an epidemic. Researchers are studying the design and discovery of drugs to inhibit the SARS-CoV-2 virus due to its high mortality rate. The main Covid-19 virus protease (Mpro) and human transmembrane protease, serine 2 (TMPRSS2) are attractive targets for the study of antiviral drugs against SARS-2 coronavirus. Increasing consumption of herbal medicines in the community and a serious approach to these drugs have increased the demand for effective herbal substances. Alkaloids are one of the most important active ingredients in medicinal plants that have wide applications in the pharmaceutical industry. In this study, seven alkaloid ligands with Quercetin nucleus for the inhibition of Mpro and TMPRSS2 were studied using computational drug design including molecular docking and molecular dynamics simulation (MD). Auto Dock software was used to evaluate molecular binding energy. Three ligands with the most negative docking score were selected to be entered into the MD simulation procedure. To evaluate the protein conformational changes induced by tested ligands and calculate the binding energy between the ligands and target proteins, GROMACS software based on AMBER03 force field was used. The MD results showed that Phyllospadine and Dracocephin-A form stable complexes with Mpro and TMPRSS2. Prolinalin-A indicated an acceptable inhibitory effect on Mpro, whereas it resulted in some structural instability of TMPRSS2. The total binding energies between three ligands, Prolinalin-A, Phyllospadine and Dracocephin-A and two proteins MPro and TMRPSS2 are (-111.235 ± 15.877, - 75.422 ± 11.140), (-107.033 ± 9.072, -84.939 ± 10.155) and (-102.941 ± 9.477, - 92.451 ± 10.539), respectively. Since the binding energies are at a minimum, this indicates confirmation of the proper binding of the ligands to the proteins. Regardless of some Prolinalin-A-induced TMPRSS2 conformational changes, it may properly bind to TMPRSS2 binding site due to its acceptable binding energy. Therefore, these three ligands can be promising candidates for the development of drugs to treat infections caused by the SARS-CoV-2 virus.

Primary Antioxidant Power and Mpro SARS-CoV-2 Non-Covalent Inhibition Capabilities of Miquelianin.

The antioxidant power of quercetin-3-O-glucuronide (miquelianin) has been studied, at the density functional level of theory, in both lipid-like and aqueous environments. In the aqueous phase, the computed pKa equilibria allowed the identification of the neutral and charged species present in solution that can react with the ⋅OOH radical. The Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET) and Radical Adduct Formation (RAF) mechanisms were considered, and the individual, total and fraction corrected rate constants were obtained. Potential non-covalent inhibition of Mpro from SARS-CoV-2 by miquelianin has been also evaluated.

Investigation of phytochemicals isolated from selected Saudi medicinal plants as natural inhibitors of SARS CoV-2 main protease: In vitro, molecular docking and simulation analysis

The escalation of many coronavirus variants accompanied by the lack of an effective cure has motivated the hunt for effective antiviral medicines. In this regard, 18 Saudi Arabian medicinal plants were evaluated for SARS CoV-2 main protease (Mpro) inhibition activity. Among them, Terminalia brownii and Acacia asak alcoholic extracts exhibited significant Mpro inhibition, with inhibition rates of 95.3 % and 95.2 %, respectively, at a concentration of 100 µg/mL. Bioassay-guided phytochemical study for the most active n-butanol fraction of T. brownii led to identification of eleven compounds, including two phenolic acids (1, and 2), seven hydrolysable tannins (3–10), and one flavonoid (11) as well as four flavonoids from A. asak (12–15). The structures of the isolated compounds were established using various spectroscopic techniques and comparison with known compounds. To investigate the chemical interactions between the identified compounds and the target Mpro protein, molecular docking was performed using AutoDock 4.2. The findings identified compounds 4, 5, 10, and 14 as the most potential inhibitors of Mpro with binding energies of −9.3, −8.5, −8.1, and −7.8 kcal mol−1, respectively. In order to assess the stability of the protein–ligand complexes, molecular dynamics simulations were conducted for a duration of 100 ns, and various parameters such as RMSD, RMSF, Rg, and SASA were evaluated. All selected compounds 4, 5, 10, and 14 showed considerable Mpro inhibiting activity in vitro, with compound 4 being the most powerful with an IC50 value of 1.2 µg/mL. MM-GBSA free energy calculations also revealed compound 4 as the most powerful Mpro inhibitor. None of the compounds (4, 5, 10, and 14) display any significant cytotoxic activity against A549 and HUVEC cell lines.

Novel 1,3-Indanedione-Thiazole Hybrids as Small-Molecule SARS-COV-2 Main Protease Inhibitors With Potential anti-Coronaviral Activity

Since arising in 2019, COVID 19 has been causing rapidly-increasing mortality and morbidity rates across the globe. Herein, novel 1,3-indanedione-thiazole hybrids were designed and synthesized as small-molecule SARS-COV-2 Main protease (Mpro) inhibitors with potential anti-covid activity. All target compounds were screened in vitro for their ability to inhibit SARS-COV-2 Mpro. Several compounds displayed potent SARS-COV-2 Mpro inhibition at one-digit IC50 values ranging from 4.3 to 9.9 µM, compared to ritonavir (IC50= 2.4 µM). Moreover, antiviral assay revealed the ability of compounds 12c, 12f, and 16a to significantly inhibit the replication of SARS-COV-2 in Vero cells at EC50 values of 7.79, 2.79 and 1.65 µM, respectively, relative to ritonavir (EC50 = 1.72 µM). Cytotoxicity assay was also conducted. None of the tested compounds exhibited significant cytotoxicity in Vero cells showing CC50 values from 171.77 to 299.96 µM and SI from 38.5 to 178.6 µM. In addition, a docking study revealed proper orientation and well-fitting of title compounds into the binding pocket of SARS-COV-2 Main protease.

Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate

The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.

Evaluation of natural products from virtual screenings as SARS-CoV-2 main protease inhibitors using combinational experiments

Recently, andrographolide, kaempferol, maslinic acid, rutin, and schaftoside have been identified as potent SARS-CoV-2 main protease (Mpro) inhibitors via molecular docking studies. However, no comprehensive in vitro testing of these compounds against Mpro has been conducted. In this study, we rigorously evaluated the in vitro inhibition of Mpro by these compounds using combinational experiments, including fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays. Our data revealed that these compounds are not Mpro inhibitors based on the results from a set of in vitro assays. These results suggest that an efficient combination of a molecular docking approach and an experimental assay is essential for the discovery of Mpro inhibitors in the future.

Noncovalent SARS-COV-2 main protease inhibitors: A virtual screening and molecular dynamic simulation study

The main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease (Mpro, also known as 3CLpro) plays a central role in virus replication, thereby constituting an appealing therapeutic target for coronavirus disease 2019 (COVID-19) treatment. In this study, we used recently reported crystal structures of SARS-CoV-2 Mpro complexed with small-molecule inhibitors. Using the established structure–activity relationships of small-molecule inhibitors of the Mpro of SARS-CoV-2, we used the Virtual Screening Workflow (with hydrogen bond constraints) implemented in Schrödinger Suite 2021 to screen approximately one million compounds in the ChemDiv compound library to identify potential novel noncovalent Mpro inhibitors. Our docking analysis yielded 65 promising lead compounds, the inhibitory activity of which against Mpro was assessed using a fluorescent SARS-CoV-2 Mpro inhibition assay. Notably, six compounds exhibited SARS-CoV-2 Mpro inhibition with IC50 values ranging 22.95–64.63 μM. These hit compounds exhibited good structural diversity, which prompted us to investigate their SARS-CoV-2 Mpro binding modes. We used a previously validated IFD-BPMD-MD workflow to predict the binding positions of these hit compounds. Our simulations showed that these hit complexes exhibited diverse and robust binding to the SARS-CoV-2 Mpro. Our study findings offer valuable insights into the development of novel noncovalent small-molecule protease inhibitor.

Discovery of a nasal spray steroid, tixocortol, as an inhibitor of SARS-CoV-2 main protease and viral replication.

Coronaviruses rely on the viral-encoded chymotrypsin-like main protease (Mpro or 3CLpro) for replication and assembly. Our previous research on Mpro of SARS-CoV-2 identified cysteine 300 (Cys300) as a potential allosteric site of Mpro inhibition. Here, we identified tixocortol (TX) as a covalent modifier of Cys300 which inhibits Mpro activity in vitro as well as in a cell-based Mpro expression assay. Most importantly TX inhibited SARS-CoV-2 replication in ACE2 expressing HeLa cells. Biochemical analysis and kinetic assays were consistent with TX acting as a non-competitive inhibitor. By contrast, TX was a weaker inhibitor and modifier of C300S Mpro, confirming a role for Cys300 in inhibition of WT Mpro but also providing evidence for an additional Cys target. TX pivalate (TP), a prodrug for TX that was previously marketed as a nasal spray, also inhibited SARS-CoV-2 replication in HeLa-ACE2 cells at low micromolar IC50s. These studies suggest that TX and/or TP could possibly be repurposed for the prevention and/or treatment of SARS-CoV-2 infection.

A SARS-CoV-2 Mpro fluorescent sensor for exploring pharmacodynamic substances from traditional Chinese medicine.

The main protease of SARS-CoV-2 (SARS-CoV-2 Mpro) plays a critical role in the replication and life cycle of the virus. Currently, how to screen SARS-CoV-2 Mpro inhibitors from complex traditional Chinese medicine (TCM) is the bottleneck for exploring the pharmacodynamic substances of TCM against SARS-CoV-2. In this study, a simple, cost-effective, rapid, and selective fluorescent sensor (TPE-S-TLG sensor) was designed with an AIE (aggregation-induced emission) probe (TPE-Ph-In) and the SARS-CoV-2 Mpro substrate (S-TLG). The TPE-S-TLG sensor was characterized using UV-Vis absorption spectroscopy, fluorescence spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential, and Fourier transform infrared (FTIR) spectroscopy techniques. The limit of detection of this method to detect SARS-CoV-2 Mpro was measured to be 5 ng mL-1. Furthermore, the TPE-S-TLG sensor was also successfully applied to screen Mpro inhibitors from Xuebijing injection using the separation and collection of the HPLC-fully automatic partial fraction collector (HPLC-FC). Six active compounds, including protocatechualdehyde, chlorogenic acid, hydroxysafflower yellow A, caffeic acid, isoquercetin, and pentagalloylglucose, were identified using UHPLC-Q-TOF/MS that could achieve 90% of the Mpro inhibition rate for the Xuebijing injection. Accordingly, the strategy can be broadly applied in the detection of disease-related proteases as well as screening active substances from TCM.

Crystal structure of SARS-CoV-2 main protease (Mpro) mutants in complex with the non-covalent inhibitor CCF0058981

SARS-CoV-2 constantly circulates and evolves worldwide, generating many variants and posing a menace to global health. It is urgently needed to discover effective medicines to treat the disease caused by SARS-CoV-2 and its variants. An established target for anti-SARS-CoV-2 drug discovery is the main protease (Mpro), since it exerts an irreplaceable action in viral life cycle. CCF0058981, derived from ML300, is a non-covalent inhibitor that exhibits low nanomolar potency against SARS-CoV-2 Mpro and submicromolar anti-SARS-CoV-2 activity, thereby providing a valuable starting point for drug design. However, structural basis underlying inhibition of SARS-CoV-2 Mpro by CCF0058981 remains undetermined. In this study, the crystal structures of CCF0058981 in complex with two SARS-CoV-2 Mpro mutants (M49I and V186F), which have been identified in the recently emerged Omicron subvariants, were solved. Structural analysis defined the pivotal molecular factors responsible for the interactions between CCF0058981 and these two Mpro mutants, and revealed the binding modes of CCF0058981 to Mpro M49I and V186F mutants. These data not only provide structural insights for SARS-CoV-2 Mpro inhibition by CCF0058981, but also add to develop effective broad-spectrum drugs against SARS-CoV-2 as well as its variants.