3CLpro Research Articles

Discovery of a novel benzimidazole conjugated quinazolinone derivative as a promising SARS-CoV-2 3CL protease inhibitor.

This report presents the design and synthesis of quinazolinone-based derivatives as promising SARS-CoV-2 3CL protease inhibitors. Two novel series, namely, febrifugine analogues 4a-i and quinazolinone conjugated benzimidazoles 9a-c, were successfully synthesized starting from isatoic anhydride. The synthesized quinazolinone derivatives were evaluated for their cytotoxicity against cancer cell lines and SARS-CoV-2 3CL inhibitory activity. The results showed that the synthesized compounds did not have significant toxicity for the non-cancer HEK293 cell line and MCF-7, MDA-MB-231, HEPG2 and HEPG2.2.15 cancer cell lines. Notably, compound 9b exhibited anti-3CL enzymatic activity in a dose-dependent manner, with the calculated IC50 value of 10.73 ± 1.17 μM. Docking results highlighted the interaction between 9b and 3CL protease through hydrogen bonding with key amino acids, including His41, Met49, Cys145, Met165, Arg188, His164, and Glu166, at the active site of the protease. Pharmacokinetic studies and ADME analyses provide valuable insights into the potential of compound 9b as a drug candidate. These findings support the new scaffold as a candidate for 3CLpro inhibition and advanced anti-coronavirus drug research.

Design and evaluation of peptide inhibitors targeting the dimerization of SARS-CoV-2 main protease.

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (Mpro) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV Mpro are essential for its dimerization, and are used to design inhibitors against SARS-CoV Mpro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 Mpro. To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated Mpro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 Mpro, with N8-A24 being the most efficient with an IC50 value of 1.44 mM. We observed that N8-A24 reduced Mpro dimerization with an IC50 value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 Mpro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19.

Design and biological evaluation of candidate drugs against zoonotic porcine deltacoronavirus (PDCoV)

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. PDCoV spillovers were recently detected in Haitian children with acute undifferentiated febrile illness, underscoring the urgent need to develop anti-PDCoV therapeutics. Coronavirus 3C-like protease (CoV 3CLpro) is essential for viral replication, and therefore provides an attractive target for drugs directed against CoV. Here, we initially evaluated the anti-PDCoV effect of Nirmatrelvir (PF-07321332), an FDA-approved anti-SARS-CoV-2 drug targeting viral 3CLpro. Regrettably, a very limited anti-PDCoV effect was achieved. By analyzing the binding modes of Nirmatrelvir with PDCoV 3CLpro and SARS-CoV-2 3CLpro, we demonstrated that the S2 pocket of 3CLpro is the primary factor underlying the differential inhibitory potency of Nirmatrelvir against different CoV 3CLpros. Based on the specific characteristics of the S2 pocket of PDCoV 3CLpro, four derivatives of Nirmatrelvir (compounds T1–T4) with substituted P2 moieties were synthesized. Compound T1, with an isobutyl at the P2 site, displayed improved anti-PDCoV activity invitro (cell infection model) and invivo (embryonated chicken egg infection model), and therefore is a potential candidate drug to combat PDCoV. Together, our results identify the substrate-binding mode and substrate specificity of PDCoV 3CLpro, providing insight into the optimization of Nirmatrelvir as an antiviral therapeutic agent against PDCoV.

The S2 Pocket Governs the Genus-Specific Substrate Selectivity of Coronavirus 3C-Like Protease.

Coronavirus 3C-like protease (CoV 3CLpro) is essential for viral replication, providing an attractive target for monitoring the evolution of CoV and developing anti-CoV drugs. Here, the substrate-binding modes of 3CLpros from four CoV genera are analyzed and found that the S2 pocket in 3CLpro is highly conserved within each genus but differs between genera. Functionally, the S2 pocket, in conjunction with S4 and S1' pockets, governs the genus-specific substrate selectivity of 3CLpro. Resurrected ancestral 3CLpros from four CoV genera validate the genus-specific divergence of S2 pocket. Drawing upon the genus-specific S2 pocket as evolutionary marker, eight newly identified 3CLpros uncover the ancestral state of modern 3CLpro and elucidate the possible evolutionary process for CoV. It is also demonstrated that the S2 pocket is highly correlated with the genus-specific inhibitory potency of PF-07321332 (an FDA-approved drug against COVID-19) on different CoV 3CLpros. This study on 3CLpro provides novel insights to inform evolutionary mechanisms for CoV and develop genera-specific or broad-spectrum drugs against CoVs.

A Structural Investigation of the Interaction between a GC-376-Based Peptidomimetic PROTAC and Its Precursor with the Viral Main Protease of Coxsackievirus B3

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CLPro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3CPro protease of enteroviruses, specifically characterizing its interaction with CVB3 3CPro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3CPro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3CPro and SARS-CoV-2 3CLPro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3CPro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3CPro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3CPro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent.

Deciphering the cleavage sites of 3C-like protease in Gammacoronaviruses and Deltacoronaviruses

Coronaviruses replicate by using the 3C-like protease (3CLpro) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CLpro cleavage sites are limited, particularly in Gammacoronaviruses (GammaCoV) and Deltacoronaviruses (DeltaCoV). This study aims to fill this gap by identifying 3CLpro cleavage sites in these viruses to provide deeper insights into their pathogenic mechanisms. By integrating sequence alignments and structural model comparisons, we developed a position-specific scoring matrix (PSSM) based on self-cleavage motifs, revealing specific preferences for each residue. Utilizing AlphaFold2's predicted alignment error (PAE) and predicted local distance difference test (pLDDT), we found that most cleavage sequences are located in regions with high PAE and low pLDDT values. KEGG pathway analysis showed that potential host protein cleavage targets are mainly concentrated in pathways related to nucleo-cytoplasmic transport and endocytosis. Through in vitro cleavage experiments and mutational analysis, we identified and validated three high-scoring proteins—nucleoporin 58 (NUP58), cell division cycle 73 (CDC73), and signal transducing adaptor molecule 2 (STAM2). These findings suggest that 3CLpro not only plays a vital role in viral replication but may also influence host cell functions by cleaving host proteins. This study provides an effective tool for identifying 3CLpro cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.

Phytoconstituents of Citrus limon (Lemon) as Potential Inhibitors Against Multi Targets of SARS-CoV-2 by Use of Molecular Modelling and In Vitro Determination Approaches.

In the present work, phytoconstituents from Citrus limon are computationally tested against SARS-CoV-2 target protein such as Mpro - (5R82.pdb), Spike - (6YZ5.pdb) &RdRp - (7BTF.pdb) for COVID-19. Docking was done by glide model, QikProp was performed by in silico ADMET screening & Prime MM-GB/SA modules were used to define binding energy. When compared with approved COVID-19 drugs such as Remdesivir, Ritonavir, Lopinavir, and Hydroxychloroquine, plant-based constituents such as Quercetin, Rutoside, Naringin, Eriocitrin, and Hesperidin. bind with significant G-scores to the active SARS-CoV-2 place. The constituents Rutoside and Eriocitrin were studied in each MD simulation in 100 ns against 3 proteins 5R82.pdb, 6YZ5.pdb and 7BTF.pdb.We performed an assay with significant natural compounds from contacts and in silico results (Rutin, Eriocitrin, Naringin, Hesperidin) using 3CL protease assay kit (B.11529 Omicron variant). This kit contained 3CL inhibitor GC376 as Control. The IC50 value of the test compound was found to be Rutin -17.50 μM, Eriocitrin-37.91 μM, Naringin-39.58 μM, Hesperidine-140.20 μM, the standard inhibitory concentration of GC376 was 38.64 μM. The phytoconstituents showed important interactions with SARS-CoV-2 targets, and potential modifications could be beneficial for future development.

NLRP12 inhibits PRRSV-2 replication by promoting GP2a degradation via MARCH8

NLRP12, a member of the NLR family, has been shown to exert a vital function in orchestrating immune responses. Here, using the immunosuppressive porcine reproductive and respiratory syndrome virus (PRRSV) as a model, the role of NLRP12 in virus infection was deciphered. We demonstrated that overexpression of NLRP12 significantly restrained PRRSV replication, while NLRP12 silencing resulted in increased viral titer. Mechanistically, NLRP12 interacts with glycoprotein 2a (GP2a) through its LRR domain and recruits the membrane-associated RING-CH E3 ubiquitin ligase 8 (MARCH8) via the PYD domain. NLRP12 facilitates the lysine-48 (K48)–linked polyubiquitination of GP2a at K128 and induces its lysosome degradation via the MARCH8-NDP52 (nuclear dot protein 52 kDa) pathway. To counteract this, PRRSV Nsp2 effectively prevented the polyubiquitination of GP2a induced by NLRP12 by its deubiquitinating activity. Meanwhile, the overexpression of Nsp4 decreased the mRNA of endogenous NLRP12 and cleaved NLRP12 in a 3C-like protease activity-dependent manner, which collaboratively counteracts the antiviral function of NLRP12. Collectively, this study revealed the mechanisms of the NLRP12-MARCH8-NDP52 axis in the host defense against PRRSV, which might be harnessed for the development of anti-PRRSV therapies.

19F qNMR based pharmacokinetics, metabolism and mass balance studies of SARS-CoV-2-3CL protease inhibitor simnotrelvir (SIM0417) in humans.

Simnotrelvir (SIM0417), an inhibitor of the 3CL protease of SARS-CoV-2, has been identified as a CYP3A sensitive substrate. This study investigated the pharmacokinetics, metabolism, and mass balance of simnotrelvir following a single oral dose of 750 mg in six healthy Chinese male subjects, co-administered with four doses of 100 mg ritonavir. Analysis using 19F qNMR combined with LC-MS/MS showed that the parent drug M0 constituted over 90% of the drug-related components in plasma. Of the administered dose, 55.4% (54.3% of M0) was recovered in urine, while 36.7% (4.57% of M0) was excreted in feces. UPLC/Q-TOF MS was used to identify metabolites in human plasma, urine and feces. Notably, oxidative metabolites catalyzed by CYP3A were scarcely detected in these matrixes. The amide hydrolyzed metabolite M9 and the cyano hydrolyzed metabolite M10 were recognized as the predominant metabolites, with the main excretion being through feces (19.0% and 12.7% of the administered dose, respectively). In vitro experiments indicated that M10 is primarily formed in the duodenum and jejunum, with further metabolism to M9 by microbiota in the large intestine. Overall, the co-administration of simnotrelvir with ritonavir led to predominant metabolism by intestinal enzymes or microbiota, resulting in hydrolyzed metabolites. These findings highlight the critical role of intestinal metabolism in the pharmacokinetics of simnotrelvir and emphasize the need to consider interactions with antibiotics and individual differences of intestinal microbiota.

Mechanism of non-competitive inhibition of the SARS-CoV-2 3CL protease dimerization: Therapeutic and clinical promise of the lichen secondary metabolite perlatolinic acid

In response to the COVID-19 pandemic, identifying effective treatments against SARS-CoV-2 has become of utmost importance. This study elucidates the mechanism by which perlatolinic acid, a lichen-derived secondary metabolite, non-competitively inhibits the dimerization of the SARS-CoV-2 3CL protease, a pivotal enzyme in the virus lifecycle. Utilising a combination of kinetic parameter determination, inhibition assays, and molecular docking studies, we demonstrate that perlatolinic acid effectively disrupts the enzymatic function by binding at the dimer interface with a measured Ki value of 0.67 μM, thereby impeding the protease catalytic activity essential for viral replication. Molecular docking studies further corroborate the binding specificity of perlatolinic acid to the dimer interface, which is attributed to the loss of key interactions essential for dimerization, consequently impairing catalytic activity, highlighting its potential as a scaffold for developing broad-spectrum antiviral drugs.Despite a dose-dependent cytotoxicity of perlatolinic acid, its TC50 is approximately 43 times higher than the Ki value.Our findings suggest that perlatolinic acid holds significant promise as a lead compound for the development of new therapeutics against COVID-19, warranting further investigation and clinical evaluation.In conclusion, the study sheds light on the therapeutic potential of natural compounds in combating SARS-CoV-2, paving the way for the exploration of lichen secondary metabolites as a reservoir of potential antiviral agents.

β-Tocotrienol and δ-Tocotrienol as Additional Inhibitors of the Main Protease of Feline Infectious Peritonitis Virus: An In Silico Analysis.

Feline infectious peritonitis (FIP) is a severe and invariably fatal disease affecting both domestic and wild felines with limited effective therapeutic options available. By considering the significant immunomodulatory effects of vitamin E observed in both animal and human models under physiological and pathological conditions, we have provided a full in silico investigation of vitamin E and related compounds and their effect on the crystal structure of feline infectious peritonitis virus 3C-like protease (FIPV-3CLpro). This work revealed the β-tocotrienol and δ-tocotrienol analogs as inhibitor candidates for this protein, suggesting their potential as possible drug compounds against FIP or their supplementary use with current medicines against this disease.

In vitro study on efficacy of SKF7®, a Malaysian medicinal plant product against SARS-CoV-2

BackgroundIn early 2020, COVID-19 pandemic has mobilized researchers in finding new remedies including repurposing of medicinal plant products focusing on direct-acting antiviral and host-directed therapies. In this study, we performed an in vitro investigation on the standardized Marantodes pumilum extract (SKF7®) focusing on anti-SARS-CoV-2 and anti-inflammatory activities.MethodsAnti-SARS-CoV-2 potential of the SKF7® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays.ResultsThe SKF7® inhibited wild-type Wuhan (EC50 of 21.99 µg/mL) and omicron (EC50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC50 value of 6.31 µg/mL). The SKF7® prominently inhibited 3CL protease activity. The SKF7® inhibited the LPS induced-TLR4 response with the EC50 of 16.19 µg/mL.ConclusionsIn conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7®. Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7® in affecting the COVID-19 pathogenesis are warranted.

Anti-coronavirus and anti-pulmonary inflammation effects of iridoids, the common component from Chinese herbal medicines for the treatment of COVID-19.

The practice of Chinese herbal medicines for the treatment of COVID-19 in China played an essential role for the control of mortality rate and reduction of recovery time. The iridoids is one of the main constituents of many heat-clearing and detoxifying Chinese medicines that were largely planted and frequently used in clinical practice. Twenty-three representative high content iridoids from several staple Chinese medicines were obtained and tested by a SARS-CoV-2 pseudo-virus entry-inhibition assay on HEK-293T/ACE2 cells, a live HCoV-OC43 virus infection assay on HRT-18 cells, and a SARS-CoV-2 3CL protease inhibitory FRET assay followed by molecular docking simulation. The anti-pulmonary inflammation activities were further evaluated on a TNF-α induced inflammation model in A549 cells and preliminary SARs were concluded. The results showed that specnuezhenide (7), cornuside (12), neonuezhenide (15), and picroside III (21) exhibited promising antiviral activities, and neonuezhenide (15) could inhibit 3CL protease with an IC50 of 14.3μM. Docking computation showed that compound 15 could bind to 3CL protease through a variety of hydrogen bonding and hydrophobic interactions. In the anti-pulmonary inflammation test, cornuside (12), aucubin (16), monotropein (17), and shanzhiside methyl ester (18) could strongly decrease the content of IL-1β and IL-8 at 10μM. Compound 17 could also upregulate the expression of the anti-inflammatory cytokine IL-10 significantly. The iridoids exhibited both anti-coronavirus and anti-pulmonary inflammation activities for their significance of existence in Chinese herbal medicines, which also provided a theoretical basis for their potential utilization in the pharmaceutical and food industries.

TDFFM: Transformer and Deep Forest Fusion Model for Predicting Coronavirus 3C-Like Protease Cleavage Sites.

COVID-19, caused by the highly contagious SARS-CoV-2 virus, is distinguished by its positive-sense, single-stranded RNA genome. A thorough understanding of SARS-CoV-2 pathogenesis is crucial for halting its proliferation. Notably, the 3C-like protease of the coronavirus (denoted as 3CLpro) is instrumental in the viral replication process. Precise delineation of 3CLpro cleavage sites is imperative for elucidating the transmission dynamics of SARS-CoV-2. While machine learning tools have been deployed to identify potential 3CLpro cleavage sites, these existing methods often fall short in terms of accuracy. To improve the performances of these predictions, we propose a novel analytical framework, the Transformer and Deep Forest Fusion Model (TDFFM). Within TDFFM, we utilize the AAindex and the BLOSUM62 matrix to encode protein sequences. These encoded features are subsequently input into two distinct components: a Deep Forest, which is an effective decision tree ensemble methodology, and a Transformer equipped with a Multi-Level Attention Model (TMLAM). The integration of the attention mechanism allows our model to more accurately identify positive samples, thus enhancing the overall predictive performance. Evaluation on a test set demonstrates that our TDFFM achieves an accuracy of 0.955, an AUC of 0.980, and an F1-score of 0.367, substantiating the model's superior prediction capabilities.

Discovery of the potent covalent inhibitor with an acrylate warhead for SARS-CoV-2 3CL protease

COVID-19 has caused severe consequences in terms of public health and economy worldwide since its outbreak in December 2019. SARS-CoV-2 3C-like protease (3CLpro), crucial for the viral replications, is an attractive target for the development of antiviral drugs. In this study, several kinds of Michael acceptor warheads were utilized to hunt for potent covalent inhibitors against 3CLpro. Meanwhile, novel 3CLpro inhibitors with the P3-3,5-dichloro-4-(2-(dimethylamino)ethoxy)phenyl moiety were designed and synthesized which may form salt bridge with residue Glu166. Among them, two compounds 12b and 12c exhibited high inhibitory activities against SARS-CoV-2 3CLpro. Further investigations suggested that 12b with an acrylate warhead displayed potent activity against HCoV-OC43 (EC50 = 97 nM) and SARS-CoV-2 replicon (EC50 = 45 nM) and low cytotoxicity (CC50 > 10 μM) in Huh7 cells. Taken together, this study devised two series of 3CLpro inhibitors and provided the potent SARS-CoV-2 3CLpro inhibitor (12b) which may be used for treating coronavirus infections.

SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.

An automated positive selection screen in yeast provides support for boron-containing compounds as inhibitors of SARS-CoV-2 main protease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to cause severe disease and deaths in many parts of the world, despite massive vaccination efforts. Antiviral drugs to curb an ongoing infection remain a priority. The virus-encoded 3C-like main protease (MPro; nsp5) is seen as a promising target. Here, with a positive selection genetic system engineered in Saccharomyces cerevisiae using cleavage and release of MazF toxin as an indicator, we screened in a robotized setup small molecule libraries comprising ~2,500 compounds for MPro inhibitors. We detected eight compounds as effective against MPro expressed in yeast, five of which are characterized proteasome inhibitors. Molecular docking indicates that most of these bind covalently to the MPro catalytically active cysteine. Compounds were confirmed as MPro inhibitors in an in vitro enzymatic assay. Among those were three previously only predicted in silico; the boron-containing proteasome inhibitors bortezomib, delanzomib, and ixazomib. Importantly, we establish reaction conditions in vitro preserving the MPro-inhibitory activity of the boron-containing drugs. These differ from the standard conditions, which may explain why boron compounds have gone undetected in screens based on enzymatic in vitro assays. Our screening system is robust and can find inhibitors of a specific protease that are biostable, able to penetrate a cell membrane, and are not generally toxic. As a cellular assay, it can detect inhibitors that fail in a screen based on an in vitro enzymatic assay using standardized conditions, and now give support for boron compounds as MPro inhibitors. This method can also be adapted for other viral proteases.IMPORTANCEThe coronavirus disease 2019 (COVID-19) pandemic triggered the realization that we need flexible approaches to find treatments for emerging viral threats. We implemented a genetically engineered platform in yeast to detect inhibitors of the virus's main protease (MPro), a promising target to curb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Screening molecule libraries, we identified candidate inhibitors and verified them in a biochemical assay. Moreover, the system detected boron-containing molecules as MPro inhibitors. Those were previously predicted computationally but never shown effective in a biochemical assay. Here, we demonstrate that they require a non-standard reaction buffer to function as MPro inhibitors. Hence, our cell-based method detects protease inhibitors missed by other approaches and provides support for the boron-containing molecules. We have thus demonstrated that our platform can screen large numbers of chemicals to find potential inhibitors of a viral protease. Importantly, the platform can be modified to detect protease targets from other emerging viruses.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

The problem of SARS-CoV-2 virus resistance to direct-acting antivirals

INTRODUCTION. The high prevalence of mutations in the SARS-CoV-2 genome raises particular concerns about the resistance of the virus to current antiviral therapy, including inhibitors of the main protease, or 3C-like protease (3CLpro), and RNA-dependent RNA polymerase (RdRp).AIM. This study aimed to analyse the prevalence, spectrum, and causes of SARS-CoV-2 mutations conferring resistance to approved and pipeline RdRp and 3CLpro inhibitors on the basis of clinical, virological, and genotypic data.DISCUSSION. The authors have analysed the prevalence of SARS-CoV-2 mutations conferring resistance to antivirals (RdRp inhibitors, including remdesivir and molnupiravir, and 3CLpro inhibitors, including paxlovid) in 2021–2024. The results suggest that certain mutations existed prior to the use of these antivirals. The prevalence of resistance-conferring mutations does not exceed 0.5% of the global population. However, the results of clinical and experimental studies demonstrate the possibility of a more than 200-fold reduction in susceptibility to medicinal products and, in particular, the emergence of multidrug-resistant variants. This is especially important for immunocompromised patients. SARS-CoV-2 can persist in such patients for many months, during which spontaneous or selection-driven mutations can render antiviral therapy ineffective. This would create a risk of spreading drug-resistant variants and/or a risk of adverse outcomes for patients.CONCLUSIONS. As COVID-19 treatment coverage increases, there may be a rise in drug-resistant variants of the virus. The presented data indicate the need for genomic epidemiological surveillance, including an analysis of potential targets for medicinal products based on clinical observations. In the future, surveillance data may determine the treatment strategy and the need to develop new antivirals (RdRp and protease inhibitors) adjusted to resistant SARS-CoV-2 variants.

Persistence of an infectious form of SARS-CoV-2 post protease inhibitor treatment of permissive cells in vitro.

Reports have described SARS-CoV-2 rebound in COVID-19 patients treated with nirmatrelvir, a 3CL protease inhibitor. The cause remains a mystery, although drug resistance, re-infection, and lack of adequate immune responses have been excluded. We now present virologic findings that provide a clue to the cause of viral rebound, which occurs in ∼20% of the treated cases. Persistence of infectious SARS-CoV-2 was experimentally documented in vitro after treatment with nirmatrelvir or another 3CL protease inhibitor, but not with a polymerase inhibitor, remdesivir. This infectious form decayed slowly with a half-life of ∼1 day, suggesting that its persistence could outlive the treatment course to re-ignite SARS-CoV-2 infection as the drug is eliminated. Notably, extending nirmatrelvir treatment beyond 8 days abolished viral rebound in vitro. Our findings point in a particular direction for future investigation of virus persistence and offer a specific treatment recommendation that should be tested clinically.