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Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

ABSTRACT: To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC₅₀: 0.6 μM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

SUBMITTER: Stevaert A

PROVIDER: S-EPMC8084268 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

Stevaert Annelies A Krasniqi Besir B Van Loy Benjamin B Nguyen Tien T Thomas Joice J Vandeput Julie J Jochmans Dirk D Thiel Volker V Dijkman Ronald R Dehaen Wim W Voet Arnout A Naesens Lieve L

Journal of medicinal chemistry 20210420 9

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives ...[more]

PMID: 33877845

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Project description:Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Project description:The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target.

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Dataset Information

Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

Publications

Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

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