Discovery of novel bicyclic[3.3.0]proline peptidyl α-ketoamides as potent 3CL-protease inhibitors for SARS-CoV-2.

The outbreak of SARS-CoV-2 has caused global crisis on health and economics. The multiple drug-drug interaction risk associated with ritonavir warrants specialized assessment before using Paxlovid. Here we report a multiple-round SAR study to provide a novel bicyclic[3.3.0]proline peptidyl α-ketoamide compound 4a, which is endowed with excellent antiviral activities and pharmacokinetic properties. Also, in vivo HCoV-OC43 neonatal mice model demonstrated compound 4a has good in vivo efficacy. Based on these properties, compound 4a worth further SAR optimization with the goal to develop compounds with better pharmacokinetic properties and finally to realize single agent efficacy in human.

COVID-19 therapeutics: Small-molecule drug development targeting SARS-CoV-2 main protease.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the causative factor behind the 2019 global coronavirus pandemic (COVID-19). The main protease, known as Mpro, is encoded by the viral genome and is essential for viral replication. It has also been an effective target for drug development. In this review, we discuss the rationale for inhibitors that specifically target SARS-CoV-2 Mpro. Small molecules and peptidomimetic inhibitors are two types of inhibitor with various modes of action and we focus here on novel inhibitors that were only discovered during the COVID-19 pandemic highlighting their binding modes and structures.

Targeting the I7L Protease: A Rational Design for Anti-Monkeypox Drugs?

The latest monkeypox virus outbreak in 2022 showcased the potential threat of this viral zoonosis to public health. The lack of specific treatments against this infection and the success of viral protease inhibitors-based treatments against HIV, Hepatitis C, and SARS-CoV-2, brought the monkeypox virus I7L protease under the spotlight as a potential target for the development of specific and compelling drugs against this emerging disease. In the present work, the structure of the monkeypox virus I7L protease was modeled and thoroughly characterized through a dedicated computational study. Furthermore, structural information gathered in the first part of the study was exploited to virtually screen the DrugBank database, consisting of drugs approved by the Food and Drug Administration (FDA) and clinical-stage drug candidates, in search for readily repurposable compounds with similar binding features as TTP-6171, the only non-covalent I7L protease inhibitor reported in the literature. The virtual screening resulted in the identification of 14 potential inhibitors of the monkeypox I7L protease. Finally, based on data collected within the present work, some considerations on developing allosteric modulators of the I7L protease are reported.

Human coronavirus 3CL proteases cleave septins and disrupt Hedgehog signaling, causing ciliary dysfunction.

Coronaviruses target ciliate cells causing the loss of cilia, acute rhinorrheas, and other ciliopathies. The loss of ciliary function may help the virus infect, replicate, and spread. However, the molecular mechanisms by which coronaviruses cause ciliary defects are still unclear. Herein we demonstrate how coronavirus infection and severe acute respiratory syndrome coronavirus2 3CL protease induce cilia dysfunction by targeting a host protein septin that is required for the structure and function of cilia. Further, we demonstrate that coronaviruses and 3CL protease lead to the cleavage of several septin proteins (SEPT2, -6, and -9), producing cleaved obstructive fragments. Furthermore, ectopic expression of cleaved SEPT2 fragments shows defective ciliogenesis, disoriented septin filaments, and ablated Sonic Hedgehog (SHH) signaling in a protease activity-dependent manner. We present that the 3CLpro inhibitors are potent and prevent abnormal ciliary structures and SHH signaling. These results provide useful insights into the general mechanisms underlying ciliary defects caused by coronaviruses, which, in turn, facilitate virus spread and prove that preclinical and clinical 3CL protease inhibitors may prove useful as therapeutics for treating ciliary defects of coronaviruses.

Highlights From CROI, the Conference on Retroviruses and Opportunistic Infections-Postexposure Prophylaxis for Sexually Transmitted Infections, a New Protease Inhibitor for COVID-19, Goals for Preventing HIV Transmission, and More.

This Medical News Q&A discusses research highlights from the recent Conference on Retroviruses and Opportunistic Infections.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

Efficacy comparison of 3CL protease inhibitors ensitrelvir and nirmatrelvir against SARS-CoV-2 in vitro and in vivo.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become established in the human population, making the need to develop safe and effective treatments critical. We have developed the small-molecule antiviral ensitrelvir, which targets the 3C-like (3CL) protease of SARS-CoV-2. This study evaluated the in vitro and in vivo efficacy of ensitrelvir compared with that of another SARS-CoV-2 3CL PI, nirmatrelvir. METHODS: Cultured cells, BALB/cAJcl mice and Syrian hamsters were infected with various SARS-CoV-2 strains, including the ancestral strain WK-521, mouse-adapted SARS-CoV-2 (MA-P10) strain, Delta strain and Omicron strain. Ensitrelvir efficacy was compared with that of nirmatrelvir. Effective concentrations were determined in vitro based on virus-induced cytopathic effects, viral titres and RNA levels. Lung viral titres, nasal turbinate titres, body-weight changes, and animal survival were also monitored. RESULTS: Ensitrelvir and nirmatrelvir showed comparable antiviral activity in multiple cell lines. Both ensitrelvir and nirmatrelvir reduced virus levels in the lungs of mice and the nasal turbinates and lungs of hamsters. However, ensitrelvir demonstrated comparable or better in vivo efficacy than that of nirmatrelvir when present at similar or slightly lower unbound-drug plasma concentrations. CONCLUSIONS: Direct in vitro and in vivo efficacy comparisons of 3CL PIs revealed that ensitrelvir demonstrated comparable in vitro efficacy to that of nirmatrelvir in cell culture and exhibited equal to or greater in vivo efficacy in terms of unbound-drug plasma concentration in both animal models evaluated. The results suggest that ensitrelvir may become an important resource for treating individuals infected with SARS-CoV-2.

Computer-aided drug design for the pain-like protease (PLpro) inhibitors against SARS-CoV-2.

A new coronavirus, known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a highly contagious virus and has caused a massive worldwide health crisis. While large-scale vaccination efforts are underway, the management of population health, economic impact and asof-yet unknown long-term effects on physical and mental health will be a key challenge for the next decade. The papain-like protease (PLpro) of SARS-CoV-2 is a promising target for antiviral drugs. This report used pharmacophore-based drug design technology to identify potential compounds as PLpro inhibitors against SARS-CoV-2. The optimal pharmacophore model was fully validated using different strategies and then was employed to virtually screen out 10 compounds with inhibitory. Molecular docking and non-bonding interactions between the targeted protein PLpro and compounds showed that UKR1129266 was the best compound. These results provided a theoretical foundation for future studies of PLpro inhibitors against SARS-CoV-2.

Treatment-Experienced Patients on Third-Line Therapy: A Retrospective Cohort of Treatment Outcomes at the HIV Advanced Treatment Centre, University Teaching Hospital, Zambia.

Antiretroviral therapy (ART) uptake continues to increase across sub-Saharan Africa and emergence of drug-resistant HIV mutations poses significant challenges to management of treatment-experienced patients with virologic failure. In Zambia, new third-line ART (TLART) guidelines including use of dolutegravir (DTG) were introduced in 2018. We assessed virologic suppression, immunologic response, and HIV drug-resistant mutations (DRMs) among patients on TLART at the University Teaching Hospital (UTH) in Lusaka, Zambia. We conducted a retrospective review of patients enrolled at UTH on TLART for >6 months between January 2010 and June 30, 2021. CD4 and HIV viral load (VL) at TLART initiation and post-initiation were assessed to determine virologic and immunologic outcomes. Regression analysis using bivariate and multivariate methods to describe baseline characteristics, virologic, and immunologic response to TLART was performed. A total of 345 patients met inclusion criteria; women comprised 57.6% (199/345) of the cohort. Median age at HIV diagnosis was 30 (interquartile range: 17.3-36.8). In 255 (73.8%) patients with at least two VLs, VL decreased from mean of 3.45 log10 copies/mL (standard deviation [SD]: 2.02) to 1.68 log10 copies/mL (SD: 1.79). Common ARVs prescribed included DTG (89.9%), tenofovir disoproxil fumarate (68.7%), and darunavir boosted with ritonavir (66.4%); 170 (49.3%) patients had genotypes; mutations consisted of 88.8% nucleoside reverse transcriptase inhibitor, 86.5% non-nucleoside reverse transcriptase inhibitor, and 55.9% protease inhibitor. VL suppression to <1,000 copies/mL was achieved in 225 (78.9%) patients. DRM frequency ranged from 56% to 89% depending on drug class. Treatment-experienced patients receiving TLART in Zambia achieved high rates of suppression despite high proportions of HIV mutations illustrating TLART effectiveness in the DTG era.

Structure-Based Design of a Dual-Targeted Covalent Inhibitor Against Papain-like and Main Proteases of SARS-CoV-2.

The two proteases, PLpro and Mpro, of SARS-CoV-2 are essential for replication of the virus. Using a structure-based co-pharmacophore screening approach, we developed a novel dual-targeted inhibitor that is equally potent in inhibiting PLpro and Mpro of SARS-CoV-2. The inhibitor contains a novel warhead, which can form a covalent bond with the catalytic cysteine residue of either enzyme. The maximum rate of the covalent inactivation is comparable to that of the most potent inhibitors reported for the viral proteases and covalent inhibitor drugs currently in clinical use. The covalent inhibition appears to be very specific for the viral proteases. The inhibitor has a potent antiviral activity against SARS-CoV-2 and is also well tolerated by mice and rats in toxicity studies. These results suggest that the inhibitor is a promising lead for development of drugs for treatment of COVID-19.

Dual Inhibitors of Main Protease (MPro) and Cathepsin L as Potent Antivirals against SARS-CoV2.

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Ensitrelvir as a potential treatment for COVID-19.

INTRODUCTION: First-generation therapeutics have improved clinical outcomes in patients infected with SARS-CoV-2. However, viral evolution has produced variants and subvariants capable of resisting many of these drugs and novel treatment strategies are urgently needed. AREAS COVERED: A corporate compound library screen identified ensitrelvir (formerly S-217622), a non-covalent, non-peptidic, orally bioavailable small-molecule protease inhibitor as a potential treatment for SARS-CoV-2. Ensitrelvir cleaves the active site of the 3C-like protease (3CLpro), which is conserved across SARS-CoV-2 variants and subvariants, with no human cell protease with similar specificity. EXPERT OPINION: Ensitrelvir demonstrates strong in vitro antiviral activity against the SARS-CoV-2 Omicron subvariants BA.4 and BA.5, which have driven new waves of infection throughout 2022, suggesting a potential therapeutic option for patients with COVID-19. This manuscript reviews what is known about ensitrelvir and explores how this drug may be used in the future to address the SARS-CoV-2 pandemic.

In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2.

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC50 = 0.39 ± 0.01 µM, EC90 = 0.75 ± 0.01 µM; B.1.351 (Beta): EC50 = 0.28 ± 0.11 µM, EC90 = 0.57 ± 0.21 µM; B.1.617.2 (Delta): EC50 = 0.27 ± 0.05 µM, EC90 = 0.81 ± 0.20 µM; B.1.1.529 (Omicron): EC50 = 0.26 ± 0.06 µM and EC50 = 0.042 ± 0.007 µM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed Ctrough ss in plasma and predicted Ctrough ss of lung total concentration were 0.163 and 2.5 µg/mL, which were approximately 9 and 132-fold higher than the EC50 of 0.019 µg/mL (0.042 µM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).

Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for Severe Acute Respiratory Syndrome Coronavirus 2 3CL Protease.

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.

Targeting SARS-CoV-2 Main Protease for Treatment of COVID-19: Covalent Inhibitors Structure-Activity Relationship Insights and Evolution Perspectives.

The viral main protease is one of the most attractive targets among all key enzymes involved in the SARS-CoV-2 life cycle. Covalent inhibition of the cysteine145 of SARS-CoV-2 MPRO with selective antiviral drugs will arrest the replication process of the virus without affecting human catalytic pathways. In this Perspective, we analyzed the in silico, in vitro, and in vivo data of the most representative examples of covalent SARS-CoV-2 MPRO inhibitors reported in the literature to date. In particular, the studied molecules were classified into eight different categories according to their reactive electrophilic warheads, highlighting the differences between their reversible/irreversible mechanism of inhibition. Furthermore, the analyses of the most recurrent pharmacophoric moieties and stereochemistry of chiral carbons were reported. The analyses of noncovalent and covalent in silico protocols, provided in this Perspective, would be useful for the scientific community to discover new and more efficient covalent SARS-CoV-2 MPRO inhibitors.

S-217622, a 3CL Protease Inhibitor and Clinical Candidate for SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has highlighted the ability of scientists to quickly react to the immense challenge presented to the world. The orally available 3CL protease inhibitor S-217622 is currently progressing through clinical trials and its discovery via structure-based drug design, screening and optimization by Shionogi and Hokkaido University is presented here.

Proteases and Their Potential Role as Biomarkers and Drug Targets in Dry Eye Disease and Ocular Surface Dysfunction.

Dry eye disease (DED) is a multifactorial disorder that leads to ocular discomfort, visual disturbance, and tear film instability. DED is accompanied by an increase in tear osmolarity and ocular surface inflammation. The diagnosis and treatment of DED still present significant challenges. Therefore, novel biomarkers and treatments are of great interest. Proteases are present in different tissues on the ocular surface. In a healthy eye, proteases are highly regulated. However, dysregulation occurs in various pathologies, including DED. With this review, we provide an overview of the implications of different families of proteases in the development and severity of DED, along with studies involving protease inhibitors as potential therapeutic tools. Even though further research is needed, this review aims to give suggestions for identifying novel biomarkers and developing new protease inhibitors.

Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites.

The SARS-CoV-2 3CL protease (3CLpro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CLpro and validate a subset of our results within authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CLpro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CLpro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CLpro and for drug development against coronaviruses.

Paxlovid: Mechanism of Action, Synthesis, and In Silico Study.

In this work, the discovery and description of PF-07321332, a major bioavailable oral SARS-CoV-2 protease inhibitor with in vitro human coronavirus antiviral activity, and excellent selection of off-target and in vivo immune profiles are reported. Various drugs and novel compound candidates for the treatment of the COVID-19 pandemic have been developed. PF-07321332 (or nirmatrelvir) is a new oral antiviral drug developed by Pfizer. In response to the pandemic, Pfizer has developed the COVID vaccine and in 2022 will launch its new major anti-SARS-Cov-2 protease inhibitor (PI). The combination of ritonavir and nirmatrelvir is under study in phase III of the clinical trial with a brand name Paxlovid. Paxlovid is an active 3Cl protease inhibitor. Paxlovid exerts its antiviral efficacy by inhibiting a necessary protease in the viral replication procedure. Proteases of coronavirus cleave several sites in the viral polyprotein where pyrrolidone was replaced by flexible glutamine. Due to the coronavirus pandemic, there is high demand for synthesis and development of this novel drug. Herein, we report the synthetic route and the mechanism of action was recently published on nirmatrelvir. Also, a comparison of the performance of two new oral antiviruses (molnupiravir and nirmatrelvir) for the treatment of COVID-19 is described. This review will be helpful for different disciplines such as biochemistry, organic chemistry, medicinal chemistry, and pharmacology.

Indole alkaloids as potential candidates against COVID-19: an in silico study.

COVID-19 has recently grown to be pandemic all around the world. Therefore, efforts to find effective drugs for the treatment of COVID-19 are needed to improve humans' life quality and survival. Since the main protease (Mpro) of SARS-CoV-2 plays a crucial role in viral replication and transcription, the inhibition of this enzyme could be a promising and challenging therapeutic target to fight COVID-19. The present study aims to identify alkaloid compounds as new potential inhibitors for SARS-CoV-2 Mpro by the hybrid modeling analyses. The docking-based virtual screening method assessed a collection of alkaloids extracted from over 500 medicinal plants and sponges. In order to validate the docking process, classical molecular dynamic simulations were applied on selected ligands, and the calculation of binding free energy was performed. Based on the proper interactions with the active site of the SARS-CoV-2 Mpro, low binding energy, few side effects, and the availability in the medicinal market, two indole alkaloids were found to be potential lead compounds that may serve as therapeutic options to treat COVID-19. This study paves the way for developing natural alkaloids as stronger potent antiviral agents against the SARS-CoV-2.