tetano
Editor, Senior Moderator
PLoS Pathog
. 2025 Feb 11;21(2):e1012571.
doi: 10.1371/journal.ppat.1012571. Online ahead of print. A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and selects for interferon-susceptible mutant viruses
Benjamin Van Loy[SUP] 1 [/SUP], Eugènia Pujol[SUP] 2 3 [/SUP], Kenichi Kamata[SUP] 4 [/SUP], Xiao Yin Lee[SUP] 4 [/SUP], Nikolai Bakirtzoglou[SUP] 1 [/SUP], Ria Van Berwaer[SUP] 1 [/SUP], Julie Vandeput[SUP] 1 [/SUP], Cato Mestdagh[SUP] 1 [/SUP], Leentje Persoons[SUP] 1 [/SUP], Brent De Wijngaert[SUP] 1 [/SUP], Quinten Goovaerts[SUP] 1 [/SUP], Sam Noppen[SUP] 1 [/SUP], Maarten Jacquemyn[SUP] 1 [/SUP], Kourosh Ahmadzadeh[SUP] 1 [/SUP], Eline Bernaerts[SUP] 1 [/SUP], Juan Martín-López[SUP] 2 3 [/SUP], Celia Escriche[SUP] 2 3 [/SUP], Bert Vanmechelen[SUP] 1 [/SUP], Besir Krasniqi[SUP] 5 [/SUP], Abhimanyu K Singh[SUP] 1 [/SUP], Dirk Daelemans[SUP] 1 [/SUP], Piet Maes[SUP] 1 [/SUP], Patrick Matthys[SUP] 1 [/SUP], Wim Dehaen[SUP] 5 [/SUP], Jef Rozenski[SUP] 6 [/SUP], Kalyan Das[SUP] 1 [/SUP], Arnout Voet[SUP] 4 [/SUP], Santiago Vázquez[SUP] 2 3 [/SUP], Lieve Naesens[SUP] 1 [/SUP], Annelies Stevaert[SUP] 1 [/SUP]
Affiliations
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.
. 2025 Feb 11;21(2):e1012571.
doi: 10.1371/journal.ppat.1012571. Online ahead of print. A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and selects for interferon-susceptible mutant viruses
Benjamin Van Loy[SUP] 1 [/SUP], Eugènia Pujol[SUP] 2 3 [/SUP], Kenichi Kamata[SUP] 4 [/SUP], Xiao Yin Lee[SUP] 4 [/SUP], Nikolai Bakirtzoglou[SUP] 1 [/SUP], Ria Van Berwaer[SUP] 1 [/SUP], Julie Vandeput[SUP] 1 [/SUP], Cato Mestdagh[SUP] 1 [/SUP], Leentje Persoons[SUP] 1 [/SUP], Brent De Wijngaert[SUP] 1 [/SUP], Quinten Goovaerts[SUP] 1 [/SUP], Sam Noppen[SUP] 1 [/SUP], Maarten Jacquemyn[SUP] 1 [/SUP], Kourosh Ahmadzadeh[SUP] 1 [/SUP], Eline Bernaerts[SUP] 1 [/SUP], Juan Martín-López[SUP] 2 3 [/SUP], Celia Escriche[SUP] 2 3 [/SUP], Bert Vanmechelen[SUP] 1 [/SUP], Besir Krasniqi[SUP] 5 [/SUP], Abhimanyu K Singh[SUP] 1 [/SUP], Dirk Daelemans[SUP] 1 [/SUP], Piet Maes[SUP] 1 [/SUP], Patrick Matthys[SUP] 1 [/SUP], Wim Dehaen[SUP] 5 [/SUP], Jef Rozenski[SUP] 6 [/SUP], Kalyan Das[SUP] 1 [/SUP], Arnout Voet[SUP] 4 [/SUP], Santiago Vázquez[SUP] 2 3 [/SUP], Lieve Naesens[SUP] 1 [/SUP], Annelies Stevaert[SUP] 1 [/SUP]
Affiliations
- PMID: 39932973
- DOI: 10.1371/journal.ppat.1012571
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.