Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase.

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage-a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues-bis(2-carbamoylaryl)phenyl diselenides-in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.

Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.

Therapeutic strategies for COVID-19: progress and lessons learned.

The coronavirus disease 2019 (COVID-19) pandemic has stimulated tremendous efforts to develop therapeutic strategies that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or human proteins to control viral infection, encompassing hundreds of potential drugs and thousands of patients in clinical trials. So far, a few small-molecule antiviral drugs (nirmatrelvir-ritonavir, remdesivir and molnupiravir) and 11 monoclonal antibodies have been marketed for the treatment of COVID-19, mostly requiring administration within 10 days of symptom onset. In addition, hospitalized patients with severe or critical COVID-19 may benefit from treatment with previously approved immunomodulatory drugs, including glucocorticoids such as dexamethasone, cytokine antagonists such as tocilizumab and Janus kinase inhibitors such as baricitinib. Here, we summarize progress with COVID-19 drug discovery, based on accumulated findings since the pandemic began and a comprehensive list of clinical and preclinical inhibitors with anti-coronavirus activities. We also discuss the lessons learned from COVID-19 and other infectious diseases with regard to drug repurposing strategies, pan-coronavirus drug targets, in vitro assays and animal models, and platform trial design for the development of therapeutics to tackle COVID-19, long COVID and pathogenic coronaviruses in future outbreaks.

Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (Mpro, 3CLpro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. Mpro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 Mpro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC50 of 120 nM against the Mpro and EC50 values of 0.8-3.4 µM for antiviral activity in different cell types. Crystal structures have been elucidated for the Mpro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.

Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts.

Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.

Identification of a Dual Inhibitor of Secreted Phospholipase A2 (GIIA sPLA2) and SARS-CoV-2 Main Protease.

The development of novel agents to combat COVID-19 is of high importance. SARS-CoV-2 main protease (Mpro) is a highly attractive target for the development of novel antivirals and a variety of inhibitors have already been developed. Accumulating evidence on the pathobiology of COVID-19 has shown that lipids and lipid metabolizing enzymes are critically involved in the severity of the infection. The purpose of the present study was to identify an inhibitor able to simultaneously inhibit both SARS-CoV-2 Mpro and phospholipase A2 (PLA2), an enzyme which plays a significant role in inflammatory diseases. Evaluating several PLA2 inhibitors, we demonstrate that the previously known potent inhibitor of Group IIA secretory PLA2, GK241, may also weakly inhibit SARS-CoV-2 Mpro. Molecular mechanics docking and molecular dynamics calculations shed light on the interactions between GK241 and SARS-CoV-2 Mpro. 2-Oxoamide GK241 may represent a lead molecular structure for the development of dual PLA2 and SARS-CoV-2 Mpro inhibitors.

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the Mpro. Starting from crystal structures of the Mpro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the Mpro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC50 of 13 nM versus the recombinant Mpro, and similar potency was observed for its P1' N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 Mpro inhibition, we also explored the activity of MG-78 against the Mpro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 Mpro), moderate (1.45 µM, Coxsackievirus 3Cpro), and relatively poor (6.7 µM, enterovirus A71 3Cpro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus Mpros but further optimization would be needed to target enterovirus 3Cpros efficiently.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation.

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.

Design, Synthesis, and Biological Evaluation of Peptidomimetic Aldehydes as Broad-Spectrum Inhibitors against Enterovirus and SARS-CoV-2.

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3Cpro) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound 18p demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3Cpro in complex with 18p determined at a resolution of 1.2 Å revealed that 18p covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CLpro and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound 18p (IC50 = 0.034 µM, EC50 = 0.29 µM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088, compound 18p also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.

SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging.

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.

Processing of the SARS-CoV pp1a/ab nsp7-10 region.

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.