Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

Protein-ligand interactions from a quantum fragmentation perspective: The case of the SARS-CoV-2 main protease interacting with α-ketoamide inhibitors.

We present a hybrid, multi-method, computational scheme for protein/ligand systems well suited to be used on modern and forthcoming massively parallel computing systems. The scheme relies on a multi-scale polarizable molecular modeling, approach to perform molecular dynamics simulations, and on an efficient Density Functional Theory (DFT) linear scaling method to post-process simulation snapshots. We use this scheme to investigate recent α-ketoamide inhibitors targeting the main protease of the SARS-CoV-2 virus. We assessed the reliability and the coherence of the hybrid scheme, in particular, by checking the ability of MM and DFT to reproduce results from high-end ab initio computations regarding such inhibitors. The DFT approach enables an a posteriori fragmentation of the system and an investigation into the strength of interaction among identified fragment pairs. We show the necessity of accounting for a large set of plausible protease/inhibitor conformations to generate reliable interaction data. Finally, we point out ways to further improve α-ketoamide inhibitors to more strongly interact with particular protease domains neighboring the active site.

AI-Driven De Novo Design and Molecular Modeling for Discovery of Small-Molecule Compounds as Potential Drug Candidates Targeting SARS-CoV-2 Main Protease.

Over the past three years, significant progress has been made in the development of novel promising drug candidates against COVID-19. However, SARS-CoV-2 mutations resulting in the emergence of new viral strains that can be resistant to the drugs used currently in the clinic necessitate the development of novel potent and broad therapeutic agents targeting different vulnerable spots of the viral proteins. In this study, two deep learning generative models were developed and used in combination with molecular modeling tools for de novo design of small molecule compounds that can inhibit the catalytic activity of SARS-CoV-2 main protease (Mpro), an enzyme critically important for mediating viral replication and transcription. As a result, the seven best scoring compounds that exhibited low values of binding free energy comparable with those calculated for two potent inhibitors of Mpro, via the same computational protocol, were selected as the most probable inhibitors of the enzyme catalytic site. In light of the data obtained, the identified compounds are assumed to present promising scaffolds for the development of new potent and broad-spectrum drugs inhibiting SARS-CoV-2 Mpro, an attractive therapeutic target for anti-COVID-19 agents.

Evaluating the ability of some natural phenolic acids to target the main protease and AAK1 in SARS COV-2.

Researchers are constantly searching for drugs to combat the coronavirus pandemic caused by SARS-CoV-2, which has lasted for over two years. Natural compounds such as phenolic acids are being tested against Mpro and AAK1, which are key players in the SARS-CoV-2 life cycle. This research work aims to study the ability of a panel of natural phenolic acids to inhibit the virus's multiplication directly through Mpro and indirectly by affecting the adaptor-associated protein kinase-1 (AAK1). Pharmacophore mapping, molecular docking, and dynamic studies were conducted over 50 ns and 100 ns on a panel of 39 natural phenolic acids. Rosmarinic acid (16) on the Mpro receptor (- 16.33 kcal/mol) and tannic acid (17) on the AAK1 receptor (- 17.15 kcal/mol) exhibited the best docking energy against both receptors. These favourable docking score values were found to be superior to those of the co-crystallized ligands. Preclinical and clinical research is required before using them simultaneously to halt the COVID-19 life cycle in a synergistic manner.

Geraniin as a potential inhibitor of SARS-CoV-2 3CLpro.

Geraniin is a polyphenolic compound first isolated from Geranium thunbergii. The major protease (Mpro), namely 3 C-like protease (3CLpro), of coronaviruses is considered an attractive drug target as it is essential for the processing and maturation of viral polyproteins. Thus, our primary goal is to explore the efficiency of geraniin on 3CLpro of SARS-CoV-2 using the computational biology strategy. In this work, we studied the anti-coronavirus effect of geraniin in vitro and its potential inhibitory mode against the 3CLpro of SARS-CoV-2. We found that geraniin inhibited HCoV-OC43 coronavirus-infected cells during the attachment and penetration phases. Molecular docking and dynamics simulations exhibited that geraniin had a strong binding affinity and high stable binding to 3CLpro of SARS-CoV-2. Geraniin showed a strong inhibitory activity on coronavirus and may be a potential inhibitor of SARS-CoV-2 3CLpro.

SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

Easy access to α-ketoamides as SARS-CoV-2 and MERS Mpro inhibitors via the PADAM oxidation route.

SARS-CoV-2 caused worldwide the current outbreak called COVID-19. Despite multiple countermeasures implemented, there is an urgent global need for new potent and efficient antiviral drugs against this pathogen. In this context, the main protease (Mpro) of SARS-CoV-2 is an essential viral enzyme and plays a pivotal role in viral replication and transcription. Its specific cleavage of polypeptides after a glutamine residue has been considered as a key element to design novel antiviral drugs. Herein, we reported the design, synthesis and structure-activity relationships of novel α-ketoamides as covalent reversible inhibitors of Mpro, exploiting the PADAM oxidation route. The reported compounds showed µM to nM activities in enzymatic and in the antiviral cell-based assays against SARS-CoV-2 Mpro. In order to assess inhibitors' binding mode, two co-crystal structures of SARS-CoV-2 Mpro in complex with our inhibitors were solved, which confirmed the covalent binding of the keto amide moiety to the catalytic Cys145 residue of Mpro. Finally, in order to interrogate potential broad-spectrum properties, we assessed a selection of compounds against MERS Mpro where they showed nM inhibitory potency, thus highlighting their potential as broad-spectrum coronavirus inhibitors.

Quantum Chemical Interaction Analysis between SARS-CoV-2 Main Protease and Ensitrelvir Compared with Its Initial Screening Hit.

A non-covalent oral drug targeting SARS-CoV-2 main protease (Mpro), ensitrelvir (Xocova), has been developed using structure-based drug design (SBDD). To elucidate the factors responsible for enhanced inhibitory activities from an in silico screening hit compound to ensitrelvir, we analyzed the interaction energies of the inhibitors with each residue of Mpro using fragment molecular orbital (FMO) calculations. This analysis reveals that functional group conversion for P1' and P1 parts in the inhibitors increases the strength of existing interactions with Mpro and also provides novel interactions for ensitrelvir; the associated changes in the conformation of Mpro induce further interactions for ensitrelvir in other parts, including hydrogen bonds, a halogen bond, and π-orbital interactions. Thus, we illuminate the promising strategies of SBDD for leading ensitrelvir to get higher activity against Mpro by elucidating microscopic interactions through FMO-based analysis. These detailed mechanism findings, including water cross-linkings, will help to design novel inhibitors in SBDD.

A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants.

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Identification of and Mechanistic Insights into SARS-CoV-2 Main Protease Non-Covalent Inhibitors: An In-Silico Study.

The indispensable role of the SARS-CoV-2 main protease (Mpro) in the viral replication cycle and its dissimilarity to human proteases make Mpro a promising drug target. In order to identify the non-covalent Mpro inhibitors, we performed a comprehensive study using a combined computational strategy. We first screened the ZINC purchasable compound database using the pharmacophore model generated from the reference crystal structure of Mpro complexed with the inhibitor ML188. The hit compounds were then filtered by molecular docking and predicted parameters of drug-likeness and pharmacokinetics. The final molecular dynamics (MD) simulations identified three effective candidate inhibitors (ECIs) capable of maintaining binding within the substrate-binding cavity of Mpro. We further performed comparative analyses of the reference and effective complexes in terms of dynamics, thermodynamics, binding free energy (BFE), and interaction energies and modes. The results reveal that, when compared to the inter-molecular electrostatic forces/interactions, the inter-molecular van der Waals (vdW) forces/interactions are far more important in maintaining the association and determining the high affinity. Given the un-favorable effects of the inter-molecular electrostatic interactions-association destabilization by the competitive hydrogen bond (HB) interactions and the reduced binding affinity arising from the un-compensable increase in the electrostatic desolvation penalty-we suggest that enhancing the inter-molecular vdW interactions while avoiding introducing the deeply buried HBs may be a promising strategy in future inhibitor optimization.

An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.

The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.

Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses.

Since end of 2019, the global and unprecedented outbreak caused by the coronavirus SARS-CoV-2 led to dramatic numbers of infections and deaths worldwide. SARS-CoV-2 produces two large viral polyproteins which are cleaved by two cysteine proteases encoded by the virus, the 3CL protease (3CLpro) and the papain-like protease, to generate non-structural proteins essential for the virus life cycle. Both proteases are recognized as promising drug targets for the development of anti-coronavirus chemotherapy. Aiming at identifying broad spectrum agents for the treatment of COVID-19 but also to fight emergent coronaviruses, we focused on 3CLpro that is well conserved within this viral family. Here we present a high-throughput screening of more than 89,000 small molecules that led to the identification of a new chemotype, potent inhibitor of the SARS-CoV-2 3CLpro. The mechanism of inhibition, the interaction with the protease using NMR and X-Ray, the specificity against host cysteine proteases and promising antiviral properties in cells are reported.

Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Cotton flower metabolites inhibit SARS-CoV-2 main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally for over 2 years, causing serious contagious disease and incalculable damage. The introduction of vaccines has slowed the spread of SARS-CoV-2 to some extent, but there remains a need for specific and effective treatment. The high chemical diversity and safety profiles of natural products make them a potential source of effective anti-SARS-CoV-2 drugs. Cotton plant is one of the most important economic and medical crops and is the source of a large number of antiviral phytochemicals. In this work, we used SARS-CoV-2 main protein (Mpro ) as the target to identify potential anti-SARS-CoV-2 natural products in cotton. An in vitro assay showed that of all cotton tissues examined, cotton flower extracts (CFs) exhibited optimal inhibitory effects against Mpro . We proceeded to use the CF metabolite database to screen natural Mpro inhibitors by combining virtual screening and biochemical assays. We identified that several CF natural products, including astragalin, myricitrin, and astilbin, significantly inhibited Mpro with half-maximal inhibitory concentrations (IC50s) of 0.13, 10.73, and 7.92 µm, respectively. These findings may serve as a basis for further studies into the suitability of cotton as a source of potential therapeutics for SARS-CoV-2.

Discovery of novel SARS-CoV-2 3CL protease covalent inhibitors using deep learning-based screen.

SARS-CoV-2 3CL protease is one of the key targets for drug development against COVID-19. Most known SARS-CoV-2 3CL protease inhibitors act by covalently binding to the active site cysteine. Yet, computational screens against this enzyme were mainly focused on non-covalent inhibitor discovery. Here, we developed a deep learning-based stepwise strategy for selective covalent inhibitor screen. We used a deep learning framework that integrated a directed message passing neural network with a feed-forward neural network to construct two different classifiers for either covalent or non-covalent inhibition activity prediction. These two classifiers were trained on the covalent and non-covalent 3CL protease inhibitors dataset, respectively, which achieved high prediction accuracy. We then successively applied the covalent inhibitor model and the non-covalent inhibitor model to screen a chemical library containing compounds with covalent warheads of cysteine. We experimentally tested the inhibition activity of 32 top-ranking compounds and 12 of them were active, among which 6 showed IC50 values less than 12 µM and the strongest one inhibited SARS-CoV-2 3CL protease with an IC50 of 1.4 µM. Further investigation demonstrated that 5 of the 6 active compounds showed typical covalent inhibition behavior with time-dependent activity. These new covalent inhibitors provide novel scaffolds for developing highly active SARS-CoV-2 3CL covalent inhibitors.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. M^pro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of M^pro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against M^pro with IC₅₀ of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 M^pro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of M^pro, and ACF has been previously reported as a PL^pro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

Human Superantibodies to 3CLpro Inhibit Replication of SARS-CoV-2 across Variants.

Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CLpro) is a highly conserved enzyme of betacoronaviruses. The enzyme plays pivotal role in the virus replication cycle. Thus, it is a good target of a broadly effective anti-Betacoronavirus agent. In this study, human single-chain antibodies (HuscFvs) of the SARS-CoV-2 3CLpro were generated using phage display technology. The 3CLpro-bound phages were used to infect Escherichia coli host for the production the 3CLpro-bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected-E. coli clones that produced HuscFvs with the 3CLpro inhibitory potential. HuscFvs of three phage infected-E. coli clones were predicted to form contact interface with residues for 3CLpro catalytic activity, substrate binding, and homodimerization. These HuscFvs were linked to a cell-penetrating peptide to make them cell-penetrable, i.e., became superantibodies. The superantibodies blocked the 3CLpro activity in vitro, were not toxic to human cells, traversed across membrane of 3CLpro-expressing cells to co-localize with the intracellular 3CLpro and most of all, they inhibited replication of authentic SARS-CoV-2 Wuhan wild type and α, ß, δ, and Omicron variants that were tested. The superantibodies should be investigated further towards clinical application as a safe and broadly effective anti-Betacoronavirus agent.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Fullerene derivatives as inhibitors of the SARS-CoV-2 main protease.

COVID-19 is an ongoing worldwide pandemic. Even today, there is a need for the development of effective therapeutic agents. SARS-CoV-2 is known as the causative virus of COVID-19, and its main protease is one of the enzymes essential for its growth and is considered a drug discovery target. In this study, we evaluated the inhibitory activities of a variety of fullerene derivatives, including newly synthesized derivatives, against the main protease of SARS-CoV-2. As a result, the malonic acid-type fullerene derivatives showed the strongest inhibitory activity.