Development of protease inhibitors against norovirus and other emerging

viruses

Ladislau C. Kovari, Ph.D

HIV DART and Emerging Viruses,Miami, FL, USA; November 27-29, 2018

Dept. of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA

Center for AIDS Research, Dept. of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA

Cocrystal Pharma, Inc.Tucker, GA, USA and Bothell, WA, USA

Background• NoVs are the leading cause of gastroenteritis in humans

worldwide:• Hallmarks of the disease: watery diarrhea, vomiting, abdominal

cramps, nausea, and sometimes fever• 700 million infections (Aron et al., Expert Review of Vaccines, 2016)• 200,000 deaths (Bartsch et al., PLoS One, 2016)

• Each year in the United States:• 21 million infections• 71,000 hospitalizations• 800 deaths

• On average, people will experience 4 to 5 episodes of NoV in their life (CDC, 2016).

• Children, immunocompromised individuals, and the elderly are at higher risk for long-term or fatal infections.

• Currently there is no antiviral therapy or vaccine to treat or prevent NoV infections.

Norovirus strikes shelters for Californiawildfire evacuees

Features of NoV epidemiology

http://www.cdc.gov/ncidod/dvrd/revb/gastro/norovirus.htmhttp://rivn.nl/en/aboutrivm/projects.index

Clinical course of NoV disease

Various treatment protocols tested in normal and immunocompromised patients with NoV gastroenteritis

Steinhoff et al., Gastroenterology, 1980; Rossingnol et al. Aliment. Pharm., 2006; Siddiqet al., J. Infect., 2011; Florescu et al. Pediatr. Transplant, 2011; Wingfield et al. J. Clin.Virol., 2010; Boillat Blanco et al. Transplant Infect. Dis., 2011; Kaufman et al., J. Pediatr.Gastroenterol. Nutr., 2005; Roos-Weil, Transplantation, 2011; Schorn et al. Clin. Infect.Dis, 2010.

Pathology of NoV infection in the jejunumA. Normal jejunal tissue

from biopsy of a volunteer

B. Broadened and flattened villi in jejunal tissue from the same volunteer during illness with NoVinduced gastroenteritis.

C. Scanning electron micrograph (EM) showing normal jejunaltissue from biopsy of pig.

D. Scanning EM showing shortening, blunting, fusion, or absence of villiin jejunal biopsy from the same pig following challenge with NoV.A, B. Agus et al. Ann. Intern. Med, 1973

C, D. Flynn et al. Am. J. Vet. Res., 1988

Clinical and public health intervention scenarios for the use of new anti-NoV agents

• Treating immunocompromised patients with chronic NoV infection (e.g.,HIV infection or AIDS, cancer, solid organ transplantation, stem cell transplantation, asplenia and congenital immune deficiencies).

• Preventing/reducing NoV shedding from acute infection in patients with an intact immune system -- limiting the spread of the virus in the hospital, nursing home, school, or military settings.

• Use as a prophylactic anti-NoV agent in uninfected persons during NoVoutbreaks to prevent/contain the spread of NoV infection (less than 20 virus particles cause disease, difficult to disinfect NoV contamination due to the relative resistance of particles to disinfectants and high environmental stability).

Genetic classification of noroviruses

in 9 genogroups

GIHuman

GII.4

GII.20

GII.3GII.11

GII.18

GII.19

GII.8GII.9

GII.14

GII.6GII.22

GII.10

GII.2GII.5

GII.12GII.1

GII.17GII.21

GII.13

GII.7

GII.16

GVI.1

GVI.2

GIV.1 GIV.2

GII.15

GVII

0.2

GV.2 GV.1

GIII.1GIII.3

GIII.2

GIIHuman, Porcine

GIVHuman, Canine

GVIcanine

GVMurine

GIIIBovine, ovine

GVIIcanine

Chhabra & Vinjé, 2016

GI.6

GI.1

GI.2GI.4

GI.5

GI.3

GI.7

GI.8GI.9

GVIIIhuman

Chiba

Yuzawa

GI: 5-10%GII: 90-95%GIV: < 0.1%GVIII: ?

Norwalk

Single-cell replication cycle of noroviruses

Adapted from Thorne and Goodfellow (2014). Adapted from Green, Virology, 2013.

Department of Biochemistry, Microbiology and Immunology

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2915564/

Lei et al. 2016 Science.

Kuiper et al. 2018 Infectious Disorders-Drug Targets. Submitted.

Norovirusrus

Zika virus

Chikungunya virus

The viral protease converts the viral polyprotein intofunctional proteins

NoV drug discovery diagram:Activities, the decision making process, and benchmarks for the project

Determining the structure of the drug target:Crystal structure of the hNoV GII.4 protease

Kuiper et al., Infectious Disorders – Drug Targets, 2018 Muzzarelli et al., Biochemistry, in press.

Cleavage junctions• Proteolytic processing by NoV Pro occurs at 5 sites

• Q-G, E-G, E-A serve as the cleavage junctions

• Q-G sites are preferentially processed first

• Neighboring residues implicated in substrate recognition

Muhaxhiri, Z., et al. J. Virol. (2013).

Norovirus cleavage product: a starting point for inhibitor design

Compound 2905

Anti-norovirus activity:EC50 = 0.09 μM (replicon)EC90 = 0.5 μM (replicon)IC50 = 0.204 μM (enzyme)IC90 = 1.59 μM (enzyme)

Toxicity (CC50) in:Huh7 >100 μMPBM >100 μMCEM = 36 μMVero > 100 μM

Inhibition of NoV PR

Incubation time: 90 min (time required to form the covalent bond between the enzyme and the inhibitor)

Compound 3150

IC50 (nM)

74 +/- 4

Antiviral drug design strategy targeting the NoV PR

Efficacy of NoV protease inhibitors Amblard et al, Bioorg. & Med. Chem. Letters, 2018

Mechanism of action studies of compound 2905

Inhibition of norovirus replication by 2905 at 10 μM (5-day time-course)

Western blot analysis of NoVprotease (19 kDa) production in the absence and in the presence of the inhibitor 2905

Compound 2905 activity over time

We have identified four new norovirus PIs (2905, 2906, 2897, 3150)for further characterization and optimization.

• EC50 is in the low nanomolar range (EC50 = 90 nM).• Aldehyde based compounds are the most potent warheads.• Mechanism of action: covalent inhibition of the NoV PR• Favorable toxicity profile in 4 different cell types ( Huh7, PBM, CEM, Vero)• Therapeutic index: CC50/EC50 = 1,111 for compound 2905• Favorable ADME/Tox prediction (QikProp 1 to 4 star rating for compounds

2905, 2906, 2897, and 3150) • Specificity studies: the lead compound (2905) is specific for NoV PR.

NEXT STEP ?Proof of concept In Vivo - Animal Studies in collaboration with the Christiane Wobus lab at the University of Michigan

Animal models to study human NoV disease

Each animal model has strengths and weaknesses

• Immunocompromised mice (low cost, used for antiviral development)

Balb/c Rag/gamma chain-deficient (Rag-γc-/-) miceTaube et al. mBIO (2013); Karst el al. Cell Host &Microbe (2014)

• Gnotobiotic pigs (diarrhea, high cost, used forantiviral and vaccine development)Cheetham et al. J. Virol. (2006); Souza et al. J. Virol. (2007)

• Chimpanzees (intravenous route, asymptomatic infection, no more NIH funding for the chimpanzee model)

Bok et al. PNAS (2011)• Gnotobiotic calves (diarrhea, high cost)

Souza et al. J. Virol. (2008)• Rhesus macaques (asymptomatic infection, high cost, Biomedical

Primate Research Center, Netherlands)Rockx et al. J. Med. Virol. (2005)

Wayne State University

Kendall Muzzarelli

Benjamin Kuiper

Joshua Holcomb

Nicholas Spellmon

Zhe Yang

Iulia Kovari

Ladislau Kovari

U. of Michigan

Christiane Wobus

Virginia Tech

Lijuan Yuan

Emory UniversityFranck AmblardPeng LiuShaoman ZhouJack YoonLeda BassitBryan CoxJames KohlerRaymond Schinazi

Argonne National Laboratory, LS-CATJoseph Brunzelle