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

Mise au point d'une méthode de screening d'inhibiteurs de la Main protéase (nsp5) du Sars-CoV-2

International audienceLe SARS-CoV-2 est l’agent infectieux responsable de la pandémie de Covid-19 apparu en 2019. Afin d’endiguer la pandémie, le développement de nouvelles thérapie représente un enjeux crucial. Le génome du SARS-CoV-2 est traduit en 2 polyprotéines pp1a et pp1ab contenant les 16 enzymes nsp (non structural protein) nécessaires au cycle viral. Les 2 polyprotéines pp1a et pp1ab sont clivées en protéines fonctionnelles par les protéases virales nsp3 et nsp5. Le clivage des polyprotéines est réalisé a des sites de clivages spécifiques des 2 protéases virales nsp3 et nsp5. L’inhibition de l’activité protéolytique des protéases virale empêche la formation du complexe de réplication et bloque donc le cycle vira

N-arylsulfonamide-based adenosine analogues to target RNA cap N7-methyltransferase nsp14 of SARS-CoV-2

International audienceRNA cap methylations have been shown to be crucial for the life cycle, replication, and infection of ssRNA viruses, as well as for evading the host's innate immune system. Viral methyltransferases (MTases) therefore represent an attractive target for the development of compounds as tools and inhibitors. In coronaviruses, N7methyltransferase function is localized in nsp14, which has become an increasingly important therapeutic target with the COVID-19 pandemic. In recent years, we have been developing SAH-derived bisubstrates with adenosine and an N-arylsulfonamide moiety targeting both SAM and RNA binding sites in nsp14. We report here the synthesis of 31 SAH analogues with the N-arylsulfonamide attached to the 5'-position of adenosine via different linkers such as N-ethylthioether, N-ethylsulfone, N-ethylamino or N-methyltriazole. The compounds were obtained efficiently by amine sulfonylation or click chemistry. Their ability to inhibit SARS-CoV-2 N7-MTase was evaluated and the best inhibitors showed a submicromolar inhibitory activity against N7-MTase nsp14

Facile access to 4′-( N -acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N 7-guanine-methyltransferase nsp14

International audienceN-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4′-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14

5′-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2′-O-methyltransferases

International audienceViral RNA cap 2'-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2'-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme's catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2'-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5'-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2'-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2'-O-MTases

Structure-guided optimization of adenosine mimetics as selective and potent inhibitors of coronavirus nsp14 N7-methyltransferases

International audienceThe COVID-19 pandemic reveals the urgent need to develop new therapeutics targeting the SARS-CoV-2 replication machinery. The first antiviral drugs were nucleoside analogues targeting RdRp and protease inhibitors active on nsp5 Mpro. In addition to these common antiviral targets, SARS-CoV-2 codes for the highly conserved protein nsp14 harbouring N7methyltransferase (MTase) activity. Nsp14 is involved in cap N7-methylation of viral RNA and its inhibition impairs viral RNA translation and immune evasion, making it an attractive new antiviral target. In this work, we followed a structure-guided drug design approach to design bisubstrates mimicking the S-adenosylmethionine methyl donor and RNA cap. We developed adenosine mimetics with an N-arylsulfonamide moiety in the 5'-position, recently described as a guanine mimicking the cap structure in a potent adenosine-derived nsp14 inhibitor. Here, the adenine moiety was replaced by hypoxanthine, N 6-methyladenine, or C7-substituted 7-deazaadenine. 26 novel adenosine mimetics were synthesized, one of which selectively inhibits nsp14 N7-MTase activity with a subnanomolar IC50 (and seven with a single-digit nanomolar IC50). In the most potent inhibitors, adenine was replaced by two different 7-deaza-adenines bearing either a phenyl or a 3-quinoline group at the C7-position via an ethynyl linker. These more complex compounds are barely active on the cognate human N7-MTase and docking experiments reveal that their selectivity of inhibition might result from the positioning of their C7 substitution in a SAM entry tunnel present in the nsp14 structure and absent in the hN7-MTase. These compounds show moderate antiviral activity against SARS-CoV-2 replication in cell culture, suggesting delivery or stability issue

Potent Inhibition of SARS-CoV-2 nsp14 N 7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues

International audienceEnzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-L-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure−activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔT m ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors

A naturally inspired chemical library of 25 molecules was synthesised guided by 3-D dimensionality and natural product likeness proved to have antiviral activity against SARS-CoV-2