Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.

IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors.

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

N-sulfonyl peptide-hybrids as a new class of dengue virus protease inhibitors.

Dengue virus (DENV) from the Flaviviridae family causes an epidemic disease that seriously threatens human life. The viral serine protease NS2B-NS3 is a promising target for drug development against DENV and other flaviviruses. We here report the design, synthesis, and in-vitro characterization of potent peptidic inhibitors of DENV protease with a sulfonyl moiety as N-terminal cap, thereby creating sulfonamide-peptide hybrids. The in-vitro target affinities of some synthesized compounds were in the nanomolar range, with the most promising derivative reaching a Ki value of 78 nM against DENV-2 protease. The synthesized compounds did not have relevant off-target activity nor cytotoxicity. The metabolic stability of compounds against rat liver microsomes and pancreatic enzymes was remarkable. In general, the integration of sulfonamide moieties at the N-terminus of peptidic inhibitors proved to be a promising and attractive strategy for further drug development against DENV infections.

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.

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.

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. Mpro 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 Mpro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against Mpro 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 Mpro 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 Mpro, and ACF has been previously reported as a PLpro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of 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.

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

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.

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.

Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay.

SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.

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 [...].

Biochemical Screening of Potent Zika Virus Protease Inhibitors.

As the Zika virus protease is an essential and well-established target for the development of antiviral agents, we biochemically screened for inhibitors using a purified recombinantly expressed form of this enzyme. As a result, we were able to identify 10 new Zika virus protease inhibitors. These compounds are natural products and showed strong inhibition in the biochemical assays. Inhibitory constants values for the compounds ranged from 5 nM to 8 µM. Among the most potent inhibitors are flavonoids like irigenol hexa-acetate (Ki =0.28 µM), katacine (Ki =0.26 µM), theaflavin gallate (Ki =0.40 µM) and hematein (Ki =0.33 µM). Inhibitors from other groups of natural products include sennoside A (Ki =0.19 µM) and gossypol (Ki =0.70 µM). Several of the obtained compounds are known for their beneficial health effects and have acceptable pharmacokinetic characteristics. Thus, they could be of interest as lead compounds for the development of important and essential Zika antiviral drugs.

Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19.

BACKGROUND: Nirmatrelvir is an orally administered severe acute respiratory syndrome coronavirus 2 main protease (Mpro) inhibitor with potent pan-human-coronavirus activity in vitro. METHODS: We conducted a phase 2-3 double-blind, randomized, controlled trial in which symptomatic, unvaccinated, nonhospitalized adults at high risk for progression to severe coronavirus disease 2019 (Covid-19) were assigned in a 1:1 ratio to receive either 300 mg of nirmatrelvir plus 100 mg of ritonavir (a pharmacokinetic enhancer) or placebo every 12 hours for 5 days. Covid-19-related hospitalization or death from any cause through day 28, viral load, and safety were evaluated. RESULTS: A total of 2246 patients underwent randomization; 1120 patients received nirmatrelvir plus ritonavir (nirmatrelvir group) and 1126 received placebo (placebo group). In the planned interim analysis of patients treated within 3 days after symptom onset (modified intention-to treat population, comprising 774 of the 1361 patients in the full analysis population), the incidence of Covid-19-related hospitalization or death by day 28 was lower in the nirmatrelvir group than in the placebo group by 6.32 percentage points (95% confidence interval [CI], -9.04 to -3.59; P<0.001; relative risk reduction, 89.1%); the incidence was 0.77% (3 of 389 patients) in the nirmatrelvir group, with 0 deaths, as compared with 7.01% (27 of 385 patients) in the placebo group, with 7 deaths. Efficacy was maintained in the final analysis involving the 1379 patients in the modified intention-to-treat population, with a difference of -5.81 percentage points (95% CI, -7.78 to -3.84; P<0.001; relative risk reduction, 88.9%). All 13 deaths occurred in the placebo group. The viral load was lower with nirmatrelvir plus ritonavir than with placebo at day 5 of treatment, with an adjusted mean difference of -0.868 log10 copies per milliliter when treatment was initiated within 3 days after the onset of symptoms. The incidence of adverse events that emerged during the treatment period was similar in the two groups (any adverse event, 22.6% with nirmatrelvir plus ritonavir vs. 23.9% with placebo; serious adverse events, 1.6% vs. 6.6%; and adverse events leading to discontinuation of the drugs or placebo, 2.1% vs. 4.2%). Dysgeusia (5.6% vs. 0.3%) and diarrhea (3.1% vs. 1.6%) occurred more frequently with nirmatrelvir plus ritonavir than with placebo. CONCLUSIONS: Treatment of symptomatic Covid-19 with nirmatrelvir plus ritonavir resulted in a risk of progression to severe Covid-19 that was 89% lower than the risk with placebo, without evident safety concerns. (Supported by Pfizer; ClinicalTrials.gov number, NCT04960202.).

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.

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern.

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.