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

replication. Agnihothram et al. Mbio 2014

1990 20202000 2010

MHV1986

Rapid Evolution of Pardes Protease Inhibitors

Pardes Biosciences - Confidential 9

Metabolic ClPathways

8 PardesScaffolds

Pardes Warhead Platform

Patents

Literature

De novo Design

Previous SARS, MERS, EV71,

RHV, NoV Efforts

FBDD

Xtal StructuresSBDD

April2020

Dec. 18th2020

Series 1

Series 4

PBI-0451 Development Candidate

>500 analogs

>300 analogs

Metabolic ClPathways

5 Warhead Variants

® Potency ® Pan-CoV ® Solubility, ® Metabolic Stability ® Oral PK ®Permeability

Pardes Biosciences oral 3CL-protease inhibitor PBI-0451 reversibly targets the active site cysteine

Cys145

Covalent bond to Cys145 observed in Xray co-crystal structure at 2.15Å resolution

PBI-0451 is broadly active against beta- and alpha-CoVsDuvinacovirus

Sarbecovirus

Embecovirus

Merbecovirus

Rhinacovirus

George & Pruijssers

Virus Biochemical 3CLproIC50 (µM)

Viral cell cultureEC50 (µM)

SARS-CoV-2 0.02-0.03 0.006-0.06SARS-CoV 0.05-0.08* n.d.MERS-CoV 0.4-0.6 0.47MHV 0.28 0.08MHV/HCoV-OC43 0.15-0.2 0.43-0.57**MHV/HCoV-HKU1 0.07-0.13 0.67**CoV-229E 0.12-0.17 0.1-0.12CoV-NL63 0.24-0.37 n.d.

* Ki = 2.70 nM (Morrison), 5.18 +/- 0.8 nM (Global fit)

CoV Strain% Homology to

SARS-CoV2 3CLpro

SARS-CoV2 -SARS-CoV 96%MERS 53%HKU1 49%OC43 49%NL63 44%229E 41%

Adapted from Cui et al. 2018 Nat. Rev. Microbiol.

Stobart et al. 2013 J. Virol.

Virus Biochemical 3CLproIC50 (µM)

Viral cell cultureEC50 (µM)

SARS-CoV-2 0.02-0.03 0.006-0.06SARS-CoV 0.05-0.08* n.d.MERS-CoV 0.4-0.6 0.47MHV 0.28 0.08MHV/HCoV-OC43 0.15-0.2 0.43-0.57**MHV/HCoV-HKU1 0.07-0.13 0.67**CoV-229E 0.12-0.17 0.1-0.12CoV-NL63 0.24-0.37 n.d.

* Ki = 2.70 nM (Morrison), 5.18 +/- 0.8 nM (Global fit)** CPE data from MHV/OC43 nsp5 or MHV/HKU1 nsp5 chimera

CoV Strain% Homology to

SARS-CoV2 3CLpro

SARS-CoV2 -SARS-CoV 96%MERS 53%HKU1 49%OC43 49%NL63 44%229E 41%

PBI-0451 is broadly active against beta- and alpha-CoVsDuvinacovirus

Sarbecovirus

Embecovirus

Merbecovirus

Rhinacovirus Adapted from Cui et al. 2018 Nat. Rev. Microbiol.

Analysis of PBI-0451 inhibition of SARS-CoV-2 replication in continuous and induced cell lines

PBI-0451 is potently active against SARS-CoV-2 WA-1 in A549-hACE2 human lung cells

0.1 1 10 100 1000-25

0

25

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125

-50

0

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100

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[PBI-0451] (nM)

% In

hibi

tion %

Toxicity

EC50= 27.60 nM

0 200 400 600 800 10001

2

3

4

5

6

7

[PBI-0451] (nM)

Lum

ines

cenc

e (L

og10

)

NanoluciferasePBI-0451

RNA genome copiesPBI-0451

0 500 1000 1500 2000 2500 30003

4

5

6

7

8

9

[PBI-0451] (nM)

SARS

-CoV

-2 R

NA (L

og10

Cop

ies/

mL)

Experiment 1 qPCR

0.1 1 10 100 1000-25

0

25

50

75

100

125

[PBI-0451] (nM)

% In

hibi

tion

EC50 = 13.54 nM

Experiment 1 qPCR

Infectious viral titerPBI-0451

0 500 1000 1500 2000 2500 30001

2

3

4

5

6

[PBI-0451] (nM)

SARS

-CoV

-2 T

iter

(Log

10 P

FU/m

L)

Experiment 1 plaque assay

LOD

0.1 1 10 100 1000 10000-25

0

25

50

75

100

125

[PBI-0451] (nM)

% In

hibi

tion

EC50 = 6.08 nM

Experiment 1 plaque assay

Pruijssers and Hughes

PBI-0451 is potently active against SARS-CoV-2 WA-1 in iPS-derived human alveolar type II (iAT2) pneumocyte cultures

0 1 2 3 4-1

0

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6

Time post-infection (d)

SAR

S-C

oV-2

RN

A v

s in

put

(Log

10 c

opie

s/w

ell)

MOI 0.04 PFU/cellMOI 0.004 PFU/cellMOI 0.0004 PFU/cell

0 100 200 300 400 500 6003

4

5

6

7

8

9

[PBI-0451] (nM)

SARS

-CoV

-2 R

NA (L

og10

Cop

ies/

mL)

AT2 Experiment 1 qPCR

0 100 200 300 400 500 6001

2

3

4

5

6

[PBI-0451] (nM)

SARS

-CoV

-2 T

iter

(Log

10 P

FU/m

L)

AT2 Experiment 1 plaque assay

LOD

1 10 100 1000 10000-25

0

25

50

75

100

125

-25

0

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100

125

[PBI-0451] (nM)

% In

hibi

tion %

ToxicityEC50 = 8.06 nMExperiment 1 plaque assay

Ave. EC50 = 26.02 nM Ave. EC50 = 36.21 nMRNA genome copiesInfectious viral titer

Pruijssers, Kook, and Hughes

1 10 100 1000 10000-25

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125

-25

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[PBI-0451] (nM)

% In

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tion %

Toxicity

EC50 = 34.14 nMExperiment 1 qPCR

• SynergyFinder 2.0 software predicts the interaction between the PBI-0451 and either RDV or molnupiravir is neither antagonistic or synergistic.

• It is likely additive.

Combined activity of PBI-0451 and polymerase inhibitors against SARS-CoV-2

0.0001 0.01 1-25

0

25

50

75

100

125

[PBI-0451] (µM)

% In

hibi

tion

+ 2.5 µM NHC EC50 = 83 nM]+ 1.25 µM NHC EC50 = 84 nM]

PBI-0451 alone [EC50 = 53 nM]+ 0.31 uM NHC [EC50 = 69 nM]+ 0.63 uM NHC [EC50 = 64 nM]

0.0001 0.01 1-25

0

25

50

75

100

125

[PBI-0451] (µM)

% In

hibi

tion

PBI-0451 alone [EC50 = 65 nM]+0.16 µM RDV [EC50 = 86 nM]+0.31 µM RDV [EC50 = ~101 nM]+0.63 µM RDV [EC50 = 78 nM]+1.25 µM RDV [EC50 = 93 nM]

PBI-0451 x

molnupiravir (NHC)

PBI-0451 x

Remdesivir (RDV)

ZIP Synergy score: -1.26

ZIP Synergy score: 0.29

Pruijssers and Hughes

Phenotypic resistance of SARS-CoV-2 to PBI0451 required extended passage

Ongoing analysis• RNA seq of nsp5 and complete genomes• Comparison with GISAID databases • Reverse Genetic analysis of any candidate

mutations • Phenotypes for replication, competitive fitness

Stevens & Pruijssers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

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Passage number

% C

PE

[PBI0451] nM

*

PBI-0451: Orally Bioavailable, Favorable PK Profile

• Favorable physicochemical and non-clinical ADME properties• Orally bioavailable: mice (39%) & dogs (79%)• Achieves high exposure in lung tissue• Allometric PK scaling to humans indicates potential for BID to QD dosing clinically

0 6 12 18 241

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50 mpk100 mpk

Mean ± SD

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PO administration to DogsPO administration to CD1 mice

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Coadministerd w/ ritonavir 20 mpkMean ± SD

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Protease Inhibitors and Coronaviruses• The nsp5 (3CLpro) protease is highly conserved structurally and

functionally across all coronaviruses

• Nsp5 activity is required for viral replication

• nsp5 protease inhibitors abort protein processing and viral RNA synthesis

• PBI-0451 is an orally bioavailable protease inhibitor with broad-spectrum CoV activity, favorable PK/PD, and a high barrier to resistance

• PBI-0451 has no antagonism and likely additivity with remdesivir or molnupiravir in vitro

AcknowledgementsDenison lab, VUMCAndrea Pruijssers PhDLaura Stevens, MSTia Hughes, MSAmelia George, MSJennifer Gribble-BowserXiaotao Lu, MS

Guttentag lab, VUMCSeughyi KookSusan Guttentag MD

Pardes BiosciencesFundingNIH NIAID 1U19AI142759Dolly Parton COVID Relief fund

Special thanks to Dr. Daryl Kotton, Boston University Medical Center for providing the iAT2 cells