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.

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.

Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses.

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.

Bad news for Paxlovid? Resistance may be coming.

In lab studies, SARS-CoV-2 finds ways to evade key drug. Some of the viral mutations are already found in people.

Discovery of 4'-O-methylscutellarein as a potent SARS-CoV-2 main protease inhibitor.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and seriously threatened public health and safety. Despite COVID-19 vaccines being readily popularized worldwide, targeted therapeutic agents for the treatment of this disease remain very limited. Here, we studied the inhibitory activity of the scutellarein and its methylated derivatives against SARS-CoV-2 main protease (Mpro) by the fluorescence resonance energy transfer (FRET) assay. Among all the methylated derivatives we studied, 4'-O-methylscutellarein exhibited the most promising enzyme inhibitory activity in vitro, with the half-maximal inhibitory concentration value (IC50) of 0.40 ± 0.03 µM. Additionally, the mechanism of action of the hits was further characterized through enzyme kinetic studies and molecular docking. Overall, our results implied that 4'-O-methylscutellarein could be a primary lead compound with clinical potential for the development of inhibitors against the SARS-CoV-2 Mpro.

Stephen Oroszlan and Retroviral Proteins.

Stephen Oroszlan received his early education in Hungary, graduating in 1950 from the Technical University in Budapest with a degree in chemical engineering [...].

There is nothing exempt from the peril of mutation - The Omicron spike.

The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.

Mechanism of Caspase-1 Inhibition by Four Anti-inflammatory Drugs Used in COVID-19 Treatment.

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Binding mechanism of inhibitors to SARS-CoV-2 main protease deciphered by multiple replica molecular dynamics simulations.

The outbreak caused by SARS-CoV-2 has received extensive worldwide attention. As the main protease (Mpro) in SARS-CoV-2 has no human homologues, it is feasible to reduce the possibility of targeting the host protein by accidental drugs. Thus, Mpro has been an attractive target of efficient drug design for anti-SARS-CoV-2 treatment. In this work, multiple replica molecular dynamics (MRMD) simulations, principal component analysis (PCA), free energy landscapes (FELs), and the molecular mechanics-generalized Born surface area (MM-GBSA) method were integrated together to decipher the binding mechanism of four inhibitors masitinib, O6K, FJC and GQU to Mpro. The results indicate that the binding of four inhibitors clearly affects the structural flexibility and internal dynamics of Mpro along with dihedral angle changes of key residues. The analysis of FELs unveils that the stability in the relative orientation and geometric position of inhibitors to Mpro is favorable for inhibitor binding. Residue-based free energy decomposition reveals that the inhibitor-Mpro interaction networks involving hydrogen bonding interactions and hydrophobic interactions provide significant information for the design of potent inhibitors against Mpro. The hot spot residues including H41, M49, F140, N142, G143, C145, H163, H164, M165, E166 and Q189 identified by computational alanine scanning are considered as reliable targets of clinically available inhibitors inhibiting the activities of Mpro.

Isolation and characterization of ACE-I inhibitory peptides from ribbonfish for a potential inhibitor of the main protease of SARS-CoV-2: An in silico analysis.

Recently, multifunctional fish peptides (FWPs) have gained a lot of attention because of their different biological activities. In the present study, three angiotensin-I converting enzyme (ACE-I) inhibitory peptides [Ala-Pro-Asp-Gly (APDG), Pro-Thr-Arg (PTR), and Ala-Asp (AD)] were isolated and characterized from ribbonfish protein hydrolysate (RFPH) and described their mechanism of action on ACE activity. As per the results, peptide PTR showed ≈ 2 and 2.5-fold higher enzyme inhibitory activity (IC50 = 0.643 ± 0.0011 µM) than APDG (IC50 = 1.061 ± 0.0127 µM) and AD (IC50 = 2.046 ± 0.0130 µM). Based on experimental evidence, peptides were used for in silico analysis to check the inhibitory activity of the main protease (PDB: 7BQY) of SARS-CoV-2. The results of the study reveal that PTR (-46.16 kcal/mol) showed higher binding affinity than APDG (-36.80 kcal/mol) and AD (-30.24 kcal/mol) compared with remdesivir (-30.64 kcal/mol). Additionally, physicochemical characteristics of all the isolated peptides exhibited appropriate pharmacological properties and were found to be nontoxic. Besides, 20 ns molecular dynamic simulation study confirms the rigid nature, fewer confirmation variations, and binding stiffness of the peptide PTR with the main protease of SARS-CoV-2. Therefore, the present study strongly suggested that PTR is the perfect substrate for inhibiting the main protease of SARS-CoV-2 through the in silico study, and this potential drug candidate may promote the researcher for future wet lab experiments.

Precursors of Viral Proteases as Distinct Drug Targets.

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

In Silico Evaluation of Iranian Medicinal Plant Phytoconstituents as Inhibitors against Main Protease and the Receptor-Binding Domain of SARS-CoV-2.

The novel coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which initially appeared in Wuhan, China, in December 2019. Elderly individuals and those with comorbid conditions may be more vulnerable to this disease. Consequently, several research laboratories continue to focus on developing drugs to treat this infection because this disease has developed into a global pandemic with an extremely limited number of specific treatments available. Natural herbal remedies have long been used to treat illnesses in a variety of cultures. Modern medicine has achieved success due to the effectiveness of traditional medicines, which are derived from medicinal plants. The objective of this study was to determine whether components of natural origin from Iranian medicinal plants have an antiviral effect that can prevent humans from this coronavirus infection using the most reliable molecular docking method; in our case, we focused on the main protease (Mpro) and a receptor-binding domain (RBD). The results of molecular docking showed that among 169 molecules of natural origin from common Iranian medicinal plants, 20 molecules (chelidimerine, rutin, fumariline, catechin gallate, adlumidine, astragalin, somniferine, etc.) can be proposed as inhibitors against this coronavirus based on the binding free energy and type of interactions between these molecules and the studied proteins. Moreover, a molecular dynamics simulation study revealed that the chelidimerine-Mpro and somniferine-RBD complexes were stable for up to 50 ns below 0.5 nm. Our results provide valuable insights into this mechanism, which sheds light on future structure-based designs of high-potency inhibitors for SARS-CoV-2.

Hispidin and Lepidine E: Two Natural Compounds and Folic Acid as Potential Inhibitors of 2019-novel Coronavirus Main Protease (2019- nCoVMpro), Molecular Docking and SAR Study.

BACKGROUND: 2019-nCoVis, a novel coronavirus was isolated and identified in 2019 in the city of Wuhan, China. On February 17, 2020 and according to the World Health Organization, 71, 429 confirmed cases worldwide were identified, among them 2162 new cases were recorded in the last 24 hours. One month later, the confirmed cases jumped to 179111, with 11525 new cases in the last 24 hours, with 7426 total deaths. No drug or vaccine is present at the moment for human and animal coronavirus. METHODS: The inhibition of 3CL hydrolase enzyme provides a promising therapeutic principle for developing treatments against CoViD-19. The 3CLpro (Mpro) is known for involving in counteracting the host innate immune response. RESULTS: This work presents the inhibitory effect of some natural compounds against 3CL hydrolase enzyme, and explains the main interactions in inhibitor-enzyme complex. Molecular docking study was carried out using Autodock Vina. By screening several molecules, we identified three candidate agents that inhibit the main protease of coronavirus. Hispidin, lepidine E, and folic acid are bound tightly in the enzyme, therefore strong hydrogen bonds have been formed (1.69-1.80Å) with the active site residues. CONCLUSION: This study provides a possible therapeutic strategy for CoViD-19.

Fragment-based in silico design of SARS-CoV-2 main protease inhibitors.

3CLpro is essential for SARS-CoV-2 replication and infection; its inhibition using small molecules is a potential therapeutic strategy. In this study, a comprehensive crystallography-guided fragment-based drug discovery approach was employed to design new inhibitors for SARS-CoV-2 3CLpro. All small molecules co-crystallized with SARS-CoV-2 3CLpro with structures deposited in the Protein Data Bank were used as inputs. Fragments sitting in the binding pocket (87) were grouped into eight geographical types. They were interactively coupled using various synthetically reasonable linkers to generate larger molecules with divalent binding modes taking advantage of two different fragments' interactions. In total, 1,251 compounds were proposed, and 7,158 stereoisomers were screened using Glide (standard precision and extra precision), AutoDock Vina, and Prime MMGBSA. The top 22 hits having conformations approaching the linear combination of their constituent fragments were selected for MD simulation on Desmond. MD simulation suggested 15 of these did adopt conformations very close to their constituent pieces with far higher binding affinity than either constituent domain alone. These structures could provide a starting point for the further design of SARS-CoV-2 3CLpro inhibitors with improved binding, and structures are provided.

Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro.

The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is of urgent demand to cure the huge number of patients. Virus-encoded proteases are considered potential drug targets. The human immunodeficiency virus protease inhibitors (lopinavir/ritonavir) has been recommended in the global Solidarity Trial in March launched by World Health Organization. However, there is currently no experimental evidence to support or against its clinical use. We evaluated the antiviral efficacy of lopinavir/ritonavir along with other two viral protease inhibitors in vitro, and discussed the possible inhibitory mechanism in silico. The in vitro to in vivo extrapolation was carried out to assess whether lopinavir/ritonavir could be effective in clinical. Among the four tested compounds, lopinavir showed the best inhibitory effect against the novel coronavirus infection. However, further in vitro to in vivo extrapolation of pharmacokinetics suggested that lopinavir/ritonavir could not reach effective concentration under standard dosing regimen [marketed as Kaletra®, contained lopinavir/ritonavir (200 mg/50 mg) tablets, recommended dosage is 400 mg/10 mg (2 tablets) twice daily]. This research concluded that lopinavir/ritonavir should be stopped for clinical use due to the huge gap between in vitro IC50 and free plasma concentration. Nevertheless, the structure-activity relationship analysis of the four inhibitors provided further information for de novel design of future viral protease inhibitors of SARS-CoV-2.

Screening potential FDA-approved inhibitors of the SARS-CoV-2 major protease 3CLpro through high-throughput virtual screening and molecular dynamics simulation.

It has been confirmed that the new coronavirus SARS-CoV-2 caused the global pandemic of coronavirus disease 2019 (COVID-19). Studies have found that 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for virus replication, and could be used as a potential target to inhibit SARS-CoV-2. In this work, 3CLpro was used as the target to complete the high-throughput virtual screening of the FDA-approved drugs, and Indinavir and other 10 drugs with high docking scores for 3CLpro were obtained. Studies on the binding pattern of 3CLpro and Indinavir found that Indinavir could form the stable hydrogen bond (H-bond) interactions with the catalytic dyad residues His41-Cys145. Binding free energy study found that Indinavir had high binding affinity with 3CLpro. Subsequently, molecular dynamics simulations were performed on the 3CLpro and 3CLpro-Indinavir systems, respectively. The post-dynamic analyses showed that the conformational state of the 3CLpro-Indinavir system transformed significantly and the system tended to be more stable. Moreover, analyses of the residue interaction network (RIN) and H-bond occupancy revealed that the residue-residue interaction at the catalytic site of 3CLpro was significantly enhanced after binding with Indinavir, which in turn inactivated the protein. In short, through this research, we hope to provide more valuable clues against COVID-19.

Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.

Structure-Based Virtual Screening to Discover Potential Lead Molecules for the SARS-CoV-2 Main Protease.

The COVID-19 disease is caused by a new strain of the coronavirus family (SARS-CoV-2), and it has affected at present millions of people all over the world. The indispensable role of the main protease (Mpro) in viral replication and gene expression makes this enzyme an attractive drug target. Therefore, inhibition of SARS-CoV-2 Mpro as a proposition to halt virus ingression is being pursued by scientists globally. Here we carried out a study with two objectives: the first being to perform comparative protein sequence and 3D structural analysis to understand the effect of 12 point mutations on the active site. Among these, two mutations, viz., Ser46 and Phe134, were found to cause a significant change at the active sites of SARS-CoV-2. The Ser46 mutation present at the entrance of the S5 subpocket of SARS-CoV-2 increases the contribution of other two hydrophilic residues, while the Phe134 mutation, present in the catalytic cysteine loop, can cause an increase in catalytic efficiency of Mpro by facilitating fast proton transfer from the Cys145 to His41 residue. It was observed that active site remained conserved among Mpro of both SARS-CoVs, except at the entrance of the S5 subpocket, suggesting sustenance of substrate specificity. The second objective was to screen the inhibitory effects of three different data sets (natural products, coronaviruses main protease inhibitors, and FDA-approved drugs) using a structure-based virtual screening approach. A total of 73 hits had a combo score >2.0. Eight different structural scaffold classes were identified, such as one/two tetrahydropyran ring(s), dipeptide/tripeptide/oligopeptide, large (approximately 20 atoms) cyclic peptide, and miscellaneous. The screened hits showed key interactions with subpockets of the active site. Further, molecular dynamics studies of selected screened compounds confirmed their perfect fitting into the subpockets of the active site. This study suggests promising structures that can fit into the SARS-CoV-2 Mpro active site and also offers direction for further lead optimization and rational drug design.