Novel Protease Inhibitors as Potent Broad-Spectrum Coronavirus Antivirals Pruijssers, A. J.; Stevens, L. J.; George, A. S.; Hughes, T. M.; Lu, X.; Kook, S.; Arnold, L. D.; Kearney, B. P.; Fenaux, M.; Lopatin, U.; Guttentag S. H.; and Denison, M. R.. Vanderbilt University Medical Center, Nashville, TN, USA Pardes Biosciences, Inc., Carlsbad, CA, USA Funding NIH NIAID 1U19AI142759 Dolly Parton COVID Relief fund
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Novel Protease Inhibitors as Potent Broad-Spectrum Coronavirus Antivirals
Pruijssers, A. J.; Stevens, L. J.; George, A. S.; Hughes, T. M.; Lu, X.; Kook, S.; Arnold, L. D.; Kearney, B. P. ; Fenaux, M.; Lopatin, U.; Guttentag S. H.; and Denison, M. R..
Vanderbilt University Medical Center, Nashville, TN, USAPardes Biosciences, Inc., Carlsbad, CA, USA
FundingNIH NIAID 1U19AI142759
Dolly Parton COVID Relief fund
RemdesivirMolnupiravir
nsp3 – PLpronsp5- 3CLpro, Mpro
Coronaviruses translate and proteolytically process replicase proteins into 16 nonstructural proteins (nsp1-16)
ORFs 1a, 1b non-structural proteins (nsp 1-16)
1 2 3 4 5 67-11 12 13 14 1516
2S
3a
M6 7b
E N
4 5 9 10
Translation
Polyprotein 1ab
Processing by virus-encoded proteases nsp3 -PLpro and nsp5 3CLpro (Mpro)
nsp3
nsp5- 3CLpro
RibosomalFrameshift
PLproLOGO plot nsp5 cleavage sites across 17 CoVS
Kiemer et al BMC bioinformatics 2004
Polyprotein 1a
Protease inhibitors block and abort coronavirus RNA synthesis
1 2 3 4 5 67-11 12 13 14 1516
Translation
nsp3
nsp53CLproMpro
RibosomalFrameshift
PLpronsp3
ORFs 1a, 1b non-structural proteins (nsp 1-16)
Processing by virus-encoded proteases nsp3 -PLpro and nsp5 3CLpro (Mpro)
• nsp5 activity absolutely required for replication
• Protease inhibition at any point stops RNA synthesis
E64d
E64d
E64d
Untreated
E64d 1h pi
E64d 3h pi
E64d 6 h pi
10 2 3 4 5 6 7 8 9 10Time (hrs post infection)
103
104
105
[3 H] c
pm
Kim et al . . . Denison, J Virol 1995
Polyprotein 1aPolyprotein 1ab
Ullrich and Nitsche 2020 Bioorg. Med. Chem. Lett.
The nsp5 protease (3CLpro, Mpro) is conserved across coronaviruses
• Active as a dimer• Cleaves after a glutamine• Specific for viral substrates• Inhibition by targeting catalytic cysteine
A brief history of CoV protease discovery and inhibitionPF-00835231 inhibits SARS-CoV-2 replicationDe Vries et al. J. Virol. 2021
SARS-CoV SARS-CoV-2MERS-CoV
Development of PF-00835231 for SARS-CoVHoffmann et al. J. Med. Chem 2020
GC-376 inhibited SARS-CoV-2 replicationVuong et al. Nature Comm. 2020
GC-376 treats FIPV infectionPedersen et al. J. Feline Med. Sur. 2018
Translation and Processing of Mouse Hepatitis Virus Virion RNA in a Cell-Free System. Denison and Perlman J Virol 1986
Identification of a serine-like proteinase in MHVLu et al. J. Virol. 1995
3CLPro inhibitors block SARS-CoV replication. Gosh et al. Bioorg Med Chem Lett. 2008
GRL0617 inhibits PLPro of SARS-CoVRatia et al. PNAS 2008 GRL-001 inhibits HKU-1 and MERS-CoV