A Novel and Versatile Class of Coronavirus non-covalent Mpro Inhibitors based on 1,4,4-Trisubstituted Piperidines

This pilot study shows that a significant proportion of Long COVID-19 cases are positive for HERV-W ENV expression along with a subgroup of ME/CFS samples from a pre-COVID pandemia collection, raising the question of whether the presence of HERV-W ENV protein, known to induce TLR4-driven immuno- and neuro-pathogenicity, could be a common factor to their overlapping symptoms. Being this the case, HERV-W ENV could constitute a future therapeutic target, following the steps of other neurologic or autoimmune diseases such as multiple sclerosis or diabetes type I. Particularly since ongoing clinical trials assaying HERV-directional therapies based on antiretroviral agents or monoclonal antibodies are showing promising results.The COVID19 pandemia has greatly encouraged the development of vaccines and novel antivirals to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of anti-influenza H1N1 1,4,4-trisubstituted piperidines, developed in our goup, we performed a SAR analysis of these unique inhibitors that allowed to define the structural elements essential for antiHcoV-229E activity. Four of the best molecules were confirmed to be equally active against SARS-CoV-2. A TOA experiment indicated that these new CoV inhibitors interact at a post virus entry point lying at the stage of viral poly protein processing and the start of viral RNA synthesis. Enzymatic assays were performed with different CoV proteins involved in these processes. The compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was modest, the ability to bind to the catalytic site of Mpro was assessed by in silico studies. The combination of results from TOA, enzymatic assays, resistance selection and in silico molecular modeling allowed us to conclude that the 1,4,4-trisubstituted piperidines represent a structurally novel and unique class of compounds that inhibit CoV Mpro inhibitors via a non-covalent mechanism, making these inhibitors fundamentally different from other Mpro inhibitors represented by the approved drug nirmatrelvir. The five points of diversity make these N-substituted piperidinebased compounds highly versatile and amenable to further rational optimization to maximize their activity and selectivity and gain full insight their antiviral mechanism.6th Innovative Approaches for Identification of Antiviral Agents Summer School September 26th-30th 2022, Santa Margherita di Pula, Sardinia, Ital

A Versatile Class of 1,4,4-Trisubstituted Piperidines Block Coronavirus Replication In Vitro

There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure–activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2’-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.This research was funded by grants from the Spanish MICINN (Projects PID2019-104070RB- C21 and PID2019-104070RB-C22); the Spanish Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028); the European Union’s Innovative Medicines Initiative (IMI) under Grant Agreement 101005077 [Corona Accelerated R&D in Europe (CARE) project]; and Fundació La Marató de TV3, Spain (Projects No. 201832-30 and No. 202135-30). B.V.L. holds an SB-PhD fellowship from the FWO Research Foundation Flanders (project: 1S66321N)Peer reviewe

Synthesis, Structure–Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure–activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity

Carneic Acids from an Endophytic Phomopsis sp. as Dengue Virus Polymerase Inhibitors

International audienceThirteen carneic acids were isolated from the fungal endophyte Phomopsis sp. SNB-LAP1-7-32. Their structures were identified by mass spectrometry and extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Compounds 1-13 were investigated for their antipolymerase activities against DENV polymerase and Zika NS5. Five of them exhibited significant inhibition of dengue polymerase with IC50 values in the 10 to 20 mu M range without cytotoxicity. None inhibited Zika virus NS5 protein

A fluorescence-based high throughput-screening assay for the SARS-CoV RNA synthesis complex

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Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

International audienceno abstrac

Antiviral Compounds from Codiaeum peltatum Targeted by a Multi-informative Molecular Networks Approach

From a set of 292 Euphorbiaceae extracts, the use of a molecular networking (MN)-based prioritization approach highlighted three clusters (MN1-3) depicting ions from the bark extract of Codiaeum peltatum. Based on their putative antiviral potential and structural novelty, the MS-guided purification of compounds present in MN1 and MN2 afforded two new daphnane-type diterpenoid orthoesters (DDO), codiapeltines A (1) and B (2), the new actephilols B (3) and C (4), and four known 1,4-dioxane-fused phenanthrene dimers (5-8). The structures of the new compounds were elucidated by NMR spectroscopic data analysis, and the absolute configurations of compounds 1 and 2 were deduced by comparison of experimental and calculated ECD spectra. Codiapeltine B (2) is the first daphnane bearing a 9,11,13-orthoester moiety, establishing a new major structural class of DDO. Compounds 1-8 and four recently reported monoterpenyl quinolones (9-12) detected in MN3 were investigated for their selective activities against chikungunya virus replication and their antipolymerase activities against the NS5 proteins of dengue and zika viruses. Compounds 3-8 exhibited strong inhibitory activities on both dengue and zika NS5 in primary assays, but extensive biological analyses indicated that only actephilol B (3) displayed a specific interaction with the NS5 targets.status: publishe

Antiviral Compounds from Codiaeum peltatum Targeted by a Multi-informative Molecular Networks Approach

International audienc

Remdesivir and SARS-CoV-2: Structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites

International audienceThe rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

International audienceAlphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses