A broad inhibitor of coronavirus replication acting via the nsp15 endoribonuclease Benjamin Van Loy 1 , Annelies Stevaert 1 , Gökçe Cihan-Üstündag 2 , Julie Vandeput 1 , Dirk Jochmans 1 , Ronald Dijkman 3 , Volker Thiel 3 , Gültaze Çapan 2 and Lieve Naesens 1 1 Rega Institute, KU Leuven, Belgium; 2 Istanbul University, Turkey; 3 Bern University, Switzerland arise from zoonotic reservoirs, imply that antiviral drugs against are urgently needed. Introduction ZA5 shows robust and broad anti-coronavirus activity Conclusion References ZA5 acts via nsp15 ZA5 executes prolonged inhibition of CoV replication Uninfected 25 µM ZA5 12.5 µM ZA5 6.25 µM ZA5 No compound 20 µM ZA5 ZA5 is active against SARS-CoV-2 and several other CoVs and therefore qualifies as a pan-CoV inhibitor. Resistance studies showed that ZA5 acts via nsp15, revealing an unexplored druggable target. Structure-aided design with the nsp15 crystal structure is ongoing to further improve the activity of this lead compound. The H250A nsp15 catalytic site mutant form of HCoV-229E [2] is 7-fold less sensitive to ZA5, but equally sensitive to the polymerase inhibitor GS-441524. This supports that ZA5 acts via nsp15. Time-of-addition profile of ZA5, an entry inhibitor and the RNA synthesis inhibitor K22. ZA5 gradually loses its activity when added after virus infection, suggesting a prolonged action mechanism. The SARS-CoV-2 pandemic proves that pan-coronavirus (CoV) inhibitors are urgently needed, since it is very likely that highly pathogenic CoVs will continue to arise from zoonotic reservoirs. We here report the discovery of a small molecule inhibitor of coronavirus replication, designated ZA5, possessing potent and broad anti-coronavirus activity. ZA5 acts via nsp15, a hexameric endoribonuclease that is part of the replication-transcription complex. Since nsp15 is considered to have a role in CoV replication [1] and immune evasion [2], inhibition of nsp15 may have a dual outcome: a direct suppressive effect on CoV replication and an indirect effect via reversal of viral immune evasion and boosting of host antiviral immunity. This dual pharmacological effect appears an obvious asset to treat CoV infections. [email protected] [1] Guarino LA et al. J Mol Biol 2005, 353(5): 1106. [2] Kindler E et al. PLoS Pathog 2017, 13(2): e1006195. [3] Lundin A et al. PLoS Pathog 2014, 10(5): e1004166. CPE reduction assay with HCoV-229E in human embryonic lung (HEL) cells. ZA5 dose-dependently inhibits the virus, giving full inhibition at 25 µM without any cytotoxicity. ZA5 prevents dsRNA formation in CoV-infected human airway 16HBE cells. ZA5 inhibits all five CoV strains tested thus far. ZA5 produces up to 4-log10 reduction in virus yield, similarly as the reference compound K22 [3]. Resistance selection by serial virus (HCoV-229E) passaging indicated that ZA5-resistance is associated with substitution F230L, located near the CoV nsp15 EndoU domain. This mutation resulted in a 14-fold increase in EC 50 value. Virus Cells Cytotoxicity (µM) Antiviral activity (µM) EC50 EC90 HCoV-229E HEL ≥100 9.0 4.1 HCoV-NL63 Vero118 >50 ND 7.0 MHV-A59 L2 >100 ND 5.6 FIPV CRFK >100 13 ND SARS-CoV-2 VeroE6-GFP >100 9.6 ND Superposition of nsp15 crystal structures (right) and alignment of c- terminal part of nsp15 (below) showing ZA5-resistance mutation (pink triangle) and catalytic triad (blue triangles). Colored in darker colors are fully conserved residues. The fact that residue 230-F/Y is conserved in all human and animal CoVs aligns with the finding that ZA5 has broad CoV activity. HCoV-229E SARS-CoV-1 MHV-A59 H234 K289 H249 Y/F230

A broad inhibitor of coronavirus replication acting via the nsp15 … · 2020. 10. 29. · ZA5 shows robust and broad anti -coronavirus activity. Conclusion. References. ZA5 acts

Download PDF Report

Upload
others
View
2
Download
0

Embed Size (px)

Citation preview

Page 1: A broad inhibitor of coronavirus replication acting via the nsp15 … · 2020. 10. 29. · ZA5 shows robust and broad anti -coronavirus activity. Conclusion. References. ZA5 acts

1Rega Institute, KU Leuven, Belgium; 2Istanbul University, Turkey; 3Bern University, Switzerland

arise from zoonotic reservoirs, imply that antiviral drugs against are urgently needed.

Introduction

ZA5 shows robust and broad anti-coronavirus activity

Conclusion

References

ZA5 acts via nsp15 ZA5 executes prolonged inhibition of CoV replication

Uninfected 25 µM ZA5 12.5 µM ZA5 6.25 µM ZA5 No compound 20 µM ZA5

ZA5 is active against SARS-CoV-2 and severalother CoVs and therefore qualifies as a pan-CoVinhibitor. Resistance studies showed that ZA5 actsvia nsp15, revealing an unexplored druggabletarget. Structure-aided design with the nsp15 crystalstructure is ongoing to further improve the activity ofthis lead compound.

The H250Ansp15 catalytic site mutant form of HCoV-229E [2] is 7-fold lesssensitive to ZA5, but equally sensitive to the polymerase inhibitor GS-441524.This supports that ZA5 acts via nsp15.

Time-of-addition profile of ZA5, an entry inhibitor andthe RNA synthesis inhibitor K22. ZA5 gradually losesits activity when added after virus infection,suggesting a prolonged action mechanism.

The SARS-CoV-2 pandemic proves that pan-coronavirus (CoV) inhibitors are urgentlyneeded, since it is very likely that highly pathogenic CoVs will continue to arise from zoonoticreservoirs. We here report the discovery of a small molecule inhibitor of coronavirusreplication, designated ZA5, possessing potent and broad anti-coronavirus activity. ZA5 actsvia nsp15, a hexameric endoribonuclease that is part of the replication-transcription complex.Since nsp15 is considered to have a role in CoV replication [1] and immune evasion [2],inhibition of nsp15 may have a dual outcome: a direct suppressive effect on CoV replicationand an indirect effect via reversal of viral immune evasion and boosting of host antiviralimmunity. This dual pharmacological effect appears an obvious asset to treat CoV infections.

[email protected]

[1] Guarino LA et al. J Mol Biol 2005, 353(5): 1106.[2] Kindler E et al. PLoS Pathog 2017, 13(2): e1006195.[3] Lundin A et al. PLoS Pathog 2014, 10(5): e1004166.

CPE reduction assay with HCoV-229E in human embryoniclung (HEL) cells. ZA5 dose-dependently inhibits the virus,giving full inhibition at 25 µM without any cytotoxicity.

ZA5 prevents dsRNAformation in CoV-infectedhuman airway 16HBE cells.

ZA5 inhibits all five CoV strains tested thus far. ZA5 produces up to 4-log10reduction in virus yield,similarly as the referencecompound K22 [3].

Resistance selection by serial virus (HCoV-229E)passaging indicated that ZA5-resistance isassociated with substitution F230L, located near theCoV nsp15 EndoU domain. This mutation resulted ina 14-fold increase in EC50 value.

Virus CellsCytotoxicity(µM)

Antiviral activity (µM)EC50 EC90

HCoV-229E HEL ≥100 9.0 4.1

HCoV-NL63 Vero118 >50 ND 7.0

MHV-A59 L2 >100 ND 5.6

FIPV CRFK >100 13 ND

SARS-CoV-2 VeroE6-GFP >100 9.6 ND

Superposition of nsp15 crystalstructures (right) and alignment of c-terminal part of nsp15 (below)showing ZA5-resistance mutation(pink triangle) and catalytic triad (bluetriangles). Colored in darker colorsare fully conserved residues. The factthat residue 230-F/Y is conserved inall human and animal CoVs alignswith the finding that ZA5 has broadCoV activity.

HCoV-229ESARS-CoV-1MHV-A59

H234

K289

H249

Y/F230