A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and combines a direct antiviral effect with subtle boosting of interferon

Abstract

AbstractThe approval of COVID-19 vaccines and two classes of antiviral drugs has been crucial in addressing 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. HCoV-229E viruses selected for resistance to EPB-113 carried mutations at or near the catalytic residue His250 in the nsp15 EndoU domain. EndoU is known to reduce the levels of viral dsRNA and avoid induction of type I interferon (IFN-I). Accordingly, the mutant viruses selected under EPB-113 were unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. This highlights the critical role of EndoU in maintaining CoV replication. By targeting nsp15, EPB-113 combines a direct antiviral effect with subtle boosting of IFN-I. 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 reduces the thermal stability of hexameric nsp15, while enhancing its EndoU cleavage activity. This EndoU-stimulating mechanism offers an explanation why the virus escapes EPB-113 by becoming EndoU-deficient. Our findings suggest that targeting nsp15 as an antiviral strategy may require a mechanism that alters the conformation of this protein and disrupts its EndoU activity and other functions related to immune evasion and viral replication. Based on the appealing pharmacological profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target.Author summaryDespite significant progress in controlling the global COVID-19 crisis through vaccines and antiviral drugs, SARS-CoV-2 infection remains a serious health threat for vulnerable individuals, such as elderly and immunocompromised patients. These populations could benefit from innovative treatments that target the virus from multiple angles. In this study, we focus on the nsp15 endoribonuclease (EndoU), a poorly understood coronavirus protein that aids in viral evasion from the host innate immune response and is assumed to regulate viral RNA synthesis. Our research centers around a newly identified, first-in-class coronavirus inhibitor, designated EPB-113, which specifically targets nsp15. This compound has the unique property to suppress the virus in two complementary ways via subtle induction of interferon combined with a direct impact on viral RNA synthesis. EPB-113 selects for escape mutants that carry substitutions in the core of EndoU, leading to a severe replication defect. When incubated with nsp15 protein, EPB-113 induces a structural change and enhances its EndoU enzyme activity. Hence, our study introduces a novel drug concept that disrupts the multifunctional nsp15 protein and suppresses coronavirus replication on multiple fronts.