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Published on: June 9, 2022
A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and selects for
Benjamin Van Loy1, Eugènia Pujol2,3, Kenichi Kamata4
1Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium.
Abstract:
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.
Insights
A novel guanidine-substituted diphenylurea compound, EPB-113, inhibits coronavirus replication by targeting the nsp15 enzyme. This discovery offers a new strategy against drug-resistant variants and future zoonotic coronavirus outbreaks.
Area of Science:
- Virology
- Drug Discovery
- Molecular Biology
Background:
- Current COVID-19 treatments like vaccines and antivirals are crucial but insufficient against emerging drug-resistant variants and novel zoonotic coronaviruses (CoVs).
- There is a critical need for novel therapeutics with distinct antiviral mechanisms to combat future coronavirus threats.
- The viral non-structural protein-15 (nsp15) with uridine-specific endoribonuclease (EndoU) activity is a potential target for antiviral development.
Purpose of the Study:
- To identify and characterize novel compounds that inhibit coronavirus replication through a distinct mechanism.
- To investigate the potential of targeting the nsp15-EndoU activity for developing new antiviral therapeutics.
- To evaluate the efficacy and mechanism of action of a novel guanidine-substituted diphenylurea compound, EPB-113.
Main Methods:
- Screening of novel guanidine-substituted diphenylurea compounds for antiviral activity against coronaviruses.
- Cell culture assays to determine the activity of EPB-113 against human coronavirus 229E (HCoV-229E) and SARS-CoV-2.
- Resistance selection studies to identify mutations conferring resistance to EPB-113.
- Biophysical and enzymatic assays using recombinant nsp15 proteins to elucidate the mechanism of action.
- Interferon (IFN) assays and co-infection studies in human macrophages and fibroblasts.
Main Results:
- A novel compound, EPB-113, was identified that suppresses CoV replication by interfering with nsp15-EndoU activity.
- EPB-113 demonstrated potent and selective activity against HCoV-229E and suppressed SARS-CoV-2 replication.
- Resistance mutations were mapped to the catalytic His250 residue of nsp15, indicating direct interaction with the compound.
- EPB-113 enhances nsp15-EndoU activity and reduces protein stability, primarily acting via an IFN-independent mechanism during viral RNA synthesis.
- EPB-113-resistant mutants induced high levels of IFN-I, were impaired in replication in macrophages, and were outcompeted by wild-type viruses.
Conclusions:
- Targeting the nsp15 enzyme with molecules like EPB-113, which induce conformational changes leading to enhanced EndoU activity, is a viable antiviral strategy.
- The nsp15-EndoU represents a promising, albeit challenging, drug target for developing new antivirals against coronaviruses.
- The distinct mechanism of EPB-113 and its resistance profile provide a foundation for future antiviral drug development against current and future coronavirus threats.
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