TREATMENT OF VIRAL RESPIRATORY INFECTIONS

Erik DE CLERCQ Rega Institute for Medical Research, K.U.Leuven B-3000 Leuven, Belgium RESPIRATORY TRACT VIRUS INFECTIONS

ADENOVIRIDAE : Adenoviruses HERPESVIRIDAE : Cytomegalovirus, Varicella-zoster virus PICORNAVIRIDAE : Enteroviruses (Coxsackie B, ECHO) Rhinoviruses CORONAVIRIDAE : Coronaviruses ORTHOMYXOVIRIDAE : Influenza (A, B, C) viruses PARAMYXOVIRIDAE : Parainfluenza viruses Respiratory syncytial virus “SARS (Severe Acute Respiratory Disease) virus” RESPIRATORY TRACT VIRAL DISEASES

Adenoviruses: Adenoiditis, Pharyngitis, Bronchopneumonitis Cytomegalovirus: Interstitial pneumonitis Varicella-zoster virus: Pneumonitis Enteroviruses (Coxsackie B, ECHO): URTI (Upper Respiratory Tract Infections) Rhinoviruses: Common cold Coronaviruses: Common cold Influenza viruses: Influenza (upper and lower respiratory tract infections) Parainfluenza viruses: Parainfluenza (laryngitis, tracheitis) Respiratory syncytial virus: Bronchopneumonitis INFLUENZA VIRUS Influenza virus

Electron micrographs of purified influenza virions. Hemagglutinin (HA ) and neuraminidase (NA) can be seen on the envelope of viral particles. Ribonucleoproteins (RNPs) are located inside the virions.

http://www.virology.net/Big_Virology/BVRNAortho.html Influenza

Layne et al., Science 293: 1729 (2001) NA (neuraminidase)

Lipid bilayer M1 (membrane protein)

M2 (ion channel) RNPs (RNA, NP) Transcriptase complex (PB1, PB2 and PA)

HA (hemagglutinin) Simplified representation of the influenza virion showing the neuraminidase

(NA) glycoprotein, the hemagglutinin (HA) glycoprotein and the matrix M2 protein

M2

McClellan and Perry, Drugs 61: 263-283 (2001) Distribution of influenza A hemagglutinin (H) subtypes in nature

Figure adapted from Murphy & Webster. Orthomyxoviruses.

http://www.brown.edu/Courses/Bio_160/Projects1999/flu/mechanism.html Distribution of influenza A hemagglutinins in nature

http://www.brown.edu/Courses/Bio_160/Projects1999/flu/mechanism.html Distribution of influenza A neuraminidases in nature

http://www.brown.edu/Courses/Bio_160/Projects1999/flu/mechanism.html Amantadine/rimantadine: mechanism of action limited to influenza A viruses H

NH .HCl H 2

H

Amantadine Symmetrel® H

NH2.HCl

H CH3 H

Rimantadine Flumadine® The tetrameric M2 helix bundle

Sansom & Kerr, Protein Eng. 6: 65-74 (1993) Neuraminidase (NA):

Cleaves sialic acid from cell-surface glycoprotein

Glycoproteins and neuraminidase

Sialic acid Sialic acid α2,3 ! Galactose Galactose β1,4 N-acetyl-glucosamine N-acetyl-glucosamine

Core sugars Core sugars

protein protein Influenza virus neuraminidase

Functions: • removes terminal sialic acid residues • promotes release of virus particles from the cells • destroys cellular receptors recognized by hemagglutinin • prevents virus aggregation at the cell surface • prevents viral inactivation by respiratory mucus NEURAMINIDASE INHIBITORS

GG167 GS4104, Ro64-0796 4-Guanidino-Neu5Ac2en Ethyl ester of GS4071 Zanamivir Oseltamivir Relenza® Tamiflu® HO OH H O HO O H N OH H3C OH O HO

Sialic acid N-Acetylneuraminic acid (NANA) HO HO

OH OH HO HO H O H δ+ O O O H H H3C N OH influenza H3C N OH O O δ- neuraminidase O R O HO R HO

Sialyl α-glycoside Transition state R = glycoprotein

Abdel-Magid et al., Curr. Opin. Drug Discov. Dev. 4: 776-791 (2001) HO HO

OH OH HO HO H H O O Ο H O O H 2 H H C N H C N OH 3 3 ΟΟΗΗ ΟΟΗΗ O O HO HO R-O- R-OH

Transition state Sialic acid

Abdel-Magid et al., Curr. Opin. Drug Discov. Dev. 4: 776-791 (2001) HO

OH HO H O Ο H H3C N ΟΟΗΗ O HO

DANA

Abdel-Magid et al., Curr. Opin. Drug Discov. Dev. 4: 776-791 (2001) HO

OH HO H O Ο H F N ΟΟΗΗ F F O HO

FANA

Abdel-Magid et al., Curr. Opin. Drug Discov. Dev. 4: 776-791 (2001) HO

OH HO H O Ο H H3C N ΟΟΗΗ O HN NH

H2N

Zanamivir Relenza® HO O P HO OH

H3C O Ο H C 3 H N

H3CO

O NH2 CH3

Oseltamivir phosphate Tamiflu® Oseltamivir = GS4104 = oral prodrug (ethyl ester) form of GS4071

H3C O H CO 3 H N C

H3C OH

O NH2

GS4071 GS4071 bound to influenza neuraminidase

Kim et al., J. Am. Chem. Soc. 119: 681-690 (1997) GS4071 bound to influenza neuraminidase Zanamivir (Relenza®)

• active against both influenza A and B

•IC50 : 0.21-2.6 ng/ml for influenza neuraminidase • efficacy demonstrated in mouse and ferret models for influenza (upon topical administration) • has to be administered by inhalation : 10 mg bid • therapeutically effective (5 days) : significant reduction in duration of illness • prophylactically effective (4 weeks) : significant reduction in number of ill subjects • well tolerated : clinical adverse events not different from placebo • no evidence for emergence of drug-resistant virus Oseltamivir (Tamiflu®)

• active against both influenza A and B

•IC50 : < 1 ng/ml for influenza neuraminidase • efficacy demonstrated in mouse and ferret models for influenza (upon oral administration) • can be administered orally : 75 or 150 mg bid • therapeutically effective (5 days) : significant reduction in duration of illness • prophylactically effective (6 weeks) : significant reduction in number of ill subjects • well tolerated : clinical adverse events not different from placebo • no evidence for emergence of drug-resistant virus RESISTANCE MUTATIONS TO NEURAMINIDASE INHIBITORS Neuraminidase 119 Glu → Gly: • specific for zanamivir; • Glu 119 interacts with guanidinium group of zanamivir 292 Arg → Lys: • found for zanamivir; cross-resistance to oseltamivir • Arg 292 interacts with carboxylic acid group of zanamivir and oseltamivir Hemagglutinin Some mutations (i.e. 198 Thr → Ile) diminish affinity of hemagglutinin for its receptor

McKimm-Breschkin, Antiviral Res. 47: 1-17 (2000) Benefits offered by neuraminidase inhibitors

Therapeutically: • Reduction in illness duration by 1-2 days • Reduction in risk-virus transmission to household or healthcare contacts • Reduction in complications (sinusitis, bronchitis) • Reduction in use of antibiotics Prophylactically: • Seasonal prevention of infection H2NNH O HN OH H H N OH

H3C

O CH3

CH3

RWJ-270201

Smee et al., Antimicrob. Agents Chemother. 45: 743-748 (2001) Sidwell et al., Antimicrob. Agents Chemother. 45: 749-757 (2001) H3CCH3

ONCH3 FF N F O

HO NH2 O

A-192558

Wang et al., J. Med. Chem. 44: 1192-1201 (2001) CH3

H H H3C N OH N O H H O H3C O

CH3

A-315675

DeGoey et al., J. Org. Chem. 67: 5445-5453 (2002) Hanessian et al., J. Am. Chem. Soc. 124: 4716-4721 (2002) NOH

NH2 F N O

T-705

T-705 showed potent inhibitory activity against influenza A, B, and C viruses, with 50% effective inhibitory concentrations of 0.013 to 0.48 µg/ml, while it showed no cytotoxicity at concentrations up to 1,000 µg/ml. T-705 was also active against a neuraminidase inhibitor-resistant virus and amantadine-resistant viruses.

Furuta et al., Antimicrob. Agents Chemother. 46: 977-981 (2002) HUMAN RHINOVIRUS (HRV) HRV 14 Human rhinovirus type 14 Antirhinoviral targets for therapy

Agents can (a) prevent viral attachment to host-cell receptors (ICAM-1 and the low density lipoprotein receptor), (b) inhibit viral uncoating or (c) inhibit viral protein synthesis by blocking 3C viral protease.

McKinlay, Curr. Opin. Pharmacol. 1: 477-481 (2001) O

C C2H5

H3CO(CHO 2)6 CH

C C2H5 O

Phenoxyalkyldiketone Arildone WIN38020

De Clercq, In: Antiviral Agents and Human Viral Diseases. Galasso, Whitley & Merigan, eds. Lippincott-Raven Publishers, pp 1-44 (1997) O O N (CH2)7 O N H3C

Isoxazole Disoxaril WIN 51711

De Clercq, In: Antiviral Agents and Human Viral Diseases. Galasso, Whitley & Merigan, eds. Lippincott-Raven Publishers, pp 1-44 (1997) CH3 N N

H3C O NN

Pyridazinamine R61837

De Clercq, In: Antiviral Agents and Human Viral Diseases. Galasso, Whitley & Merigan, eds. Lippincott-Raven Publishers, pp 1-44 (1997) N N O

H3CCN(CH2)2 OCO2H5

Phenoxypyridazinamine Pirodavir R77975

De Clercq, In: Antiviral Agents and Human Viral Diseases. Galasso, Whitley & Merigan, eds. Lippincott-Raven Publishers, pp 1-44 (1997) Interaction of WIN 52035 with VP1 of human rhinovirus (HRV-14)

Andries, In: Antiviral Chemotherapy. Jeffries & De Clercq, eds. John Wiley & Sons, Chichester, pp 287-319 (1995) Interaction of R61837 with VP1 of human rhinovirus (HRV-14)

Andries, In: Antiviral Chemotherapy. Jeffries & De Clercq, eds. John Wiley & Sons, Chichester, pp 287-319 (1995) N CF3 N O O O N

Pleconaril (VP-63843)

Romero, Exp. Opin. Invest. Drugs 10: 369-379 (2001) BTA188

H3C N NCHO 2 CH3 NN O

BTA showed potent in vitro activity against 87 of 100 rhinovirus serotypes with a median EC50 of 10 ng/ml, superior to pleconaril. BTA188 is targeted at the hydrophobic pocket in the core of VP1.

Hayden et al., Antiviral Res. 50: A127 (2001) Benzothiazole

H3C N N S S S N

Benzothiazole targeted at hydrophobic pocket in the core of VP1.

EC50 against HRV-14 in cell culture: 0.8 µM. Tsang et al., Chem. Biol. 8: 33-45 (2001) Ruprintrivir O NH

O O

N N H3C H H N O O O CH2 CH O 3 F

Inhibitor of human rhinovirus C3 protease, currently undergoing clinical evaluation for the prevention and treatment of the common cold.

Hsyu et al., Antimicrob. Agents Chemother. 46: 392-397 (2002) Pyridone O NH

O O N N N H3C H H N O O CH3 O CH

O CH3 F F

Human rhinovirus (HRV) 3C protease inhibitor showed an average EC50 of 45 nM across 15 serotypes of HRV.

Dragovich et al., J. Med. Chem. 45: 1607-1623 (2002) RESPIRATORY SYNCYTIAL VIRUS (RSV) Respiratory Syncytial Virus (RSV) and Parainfluenza Virus (PIV)

Hall, N. Engl. J. Med. 344: 1917-1928 (2001) Characteristics of the proteins of respiratory syncytial virus and parainfluenza virus

Protein Molecular mass Functions Respiratory Parainfluenza syncytial virus virus kilodaltons Structural protein Surface Fusion (F) 68 60 Penetration; major protection antigen Attachment (G) 90 —a Viral attachment; major protective antigen Hemagglutinin neuraminidase — a 69 Viral attachment and release; major protective antigen (HN) Small hydrophobic (SH [1A]) 4.8 – 30b — a Unknown Matrix Matrix (M) 28 40 Mediates attachment of nucleocapsid to envelope 22b — a

Small envelope (M2) Transcriptional regulation; unique to pneumoviruses Nucleocapsid-associated Nucleoprotein (N, NP) 44 58 Major RNA-binding nucleocapsid protein Phosphoprotein (P) 37 60 Major phosphorylated protein; RNA-dependent RNA polymerase activity Large polymerase complex (L) 200 250 Large nucleocapsid-associated protein; major polymerase subunit; RNA-dependent RNA polymerase activity aNot present in the virus. bThere are four glycosylated and nonglycosylated forms with molecular masses of 4.8, 7.5, 13 to 15, and 21 to 30 kd.

Hall, N. Engl. J. Med. 344: 1917-1928 (2001) Epidemiologic pattern of infections with respiratory syncytial virus in relation to the occurrence of bronchiolitis from 1993 through 1998 Number of cases or isolates

1993 1994 1995 1996 1997 1998

Hall, N. Engl. J. Med. 344: 1917-1928 (2001) Proportion of samples taken from cases of influenza-like illness positive for influenza or respiratory syncytial virus in 0-5-year-olds during 1996-1997

Zambon et al., Lancet 358: 1410-1416 (2001) Proportion of samples taken from cases of influenza-like illness positive for influenza or respiratory syncytial virus in 0-5-year-olds during 1996-1997

Zambon et al., Lancet 358: 1410-1416 (2001) O

N H2N N N

HO O

HO OH

Ribavirin Virazole® H2N RFI-641 O

N H2N S NH2 O O O O NH N N O NH2 HN N NaO3S S O N O HN NH N O O S SO3Na N NH O N N H2N HN O OO O H2N S N NH2 O

NH2 RFI-641 inhibits RSV fusion mediated by F (Fusion) protein, but also interferes with the attachment (G) protein Nikitenko et al., Bioorg. Med. Chem. Lett. 11: 1041-1044 (2001) Razinkov et al., Chem. Biol. 8: 645-659 (2001) Morphology of CV-1 cells treated with RFI-641

• Noninfected, nontreated CV-1 cells; (B) RSV-infected, untreated CV-1 cells; (C) RSV-infected, RFI-641 (> 8 h)-treated CV-1 cells

Huntley et al., Antimicrob. Agents Chemother. 46: 841-847 (2002) Therapeutic efficacy of RFI-641 after intranasal administration to RSV-infected African green monkeys

RFI-164 was administered intranasally, daily, starting 1 day after virus infection

Huntley et al., Antimicrob. Agents Chemother. 46: 841-847 (2002) NNH2 CH3

N NH N

NCH3

HO

R-170591 interferes with RSV fusion

Andries et al., 40th ICAAC, Toronto, Canada, 17-20 September 2000, Abstract H-1160 Benzodithiin (RD3-0028)

S S

RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, leading to loss of infectivity

Sudo et al., Microbiol. Immunol. 45: 531-537 (2001) PARAMYXOVIRIDAE • Paramyxovirinae • Respirovirus/Paramyxovirus (Sendai virus, Parainfluenza 1 and 3) • Rubulavirus (Mumps virus, Parainfluenza 2, 4a and 4b) • Morbillivirus (Measles virus) • Megamyxovirus (Hendra and Nipah virus) • Pneumovirinae • Pneumovirus (Pneumovirus, Respiratory Syncytial Virus) • Metapneumovirus (Human metapneumovirus) PHYLOGENY OF THE PARAMYXOVIRINAE

HPIV-1 Genus Sendai Respirovirus/ HPIV-3 Paramyxovirus CDV PDV RPV Genus Measles Morbillivirus PPRV DMV Tupaia Hendra Tentative new genus Nipah Megamyxovirus HPIV-4b HPIV-4a Mumps SV5 Genus Rubulavirus HPIV-2 Mapuera NDV OUTBREAK-INSPIRED HUNT FOR VIRUSES

• Hendra virus • Nipah virus (1994, Megamyxovirus) (1999, Megamyxovirus) •Australian bat lyssavirus • Tioman virus (1996, Rhabdoviridae) (2000, Rubulavirus) •Menangle virus •Dolphin, porpoise and cetacean morbillivirus (1997, Rubulavirus) (1991, Morbillivirus) •Tupaia virus •Salem virus (1999, Paramyxovirus) (2000, not yet classified) DIFFERENCES BETWEEN

HENDRA & NIPAH VIRUS OTHER PARAMYXOVIRINAE

Multiple host species Host specific : human Reservoir host : • fruit bats (flying foxes) ? Clinical hosts : • horse, pig, man,... and Experimental hosts : • guinea pig, cat Not very contagious Highly contagious No human-to-human spread, Human-to-human spread, but infection is very lethal droplet infection CONCLUSION APPROVED ANTIVIRAL DRUGS FOR THE TREATMENT OF THE MAJOR RESPIRATORY TRACT VIRUS INFECTIONS

Adenoviruses : none Picornaviruses Entero : none Rhino : none Orthomyxoviruses Influenza : Neuraminidase inhibitors: zanamivir, oseltamivir : Amantadine and rimantadine (for influenza A only) Paramyxoviruses Parainfluenza : none Respiratory syncytial virus : Ribavirin SARS virus : none