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
HERPESVIRIDAE PICORNAVIRIDAE CORONAVIRIDAE PARAMYXOVIRIDAE

: Adenoviruses
: Cytomegalovirus, Varicella-zoster virus : Enteroviruses (Coxsackie B, ECHO) Rhinoviruses : Coronaviruses : Parainfluenza viruses Respiratory syncytial virus SARS (Severe Acute Respiratory Disease) virus

ORTHOMYXOVIRIDAE : Influenza (A, B, C) viruses

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) Transcriptase complex (PB1, PB2 and PA)

RNPs (RNA, NP)

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 H

NH2.HCl

Amantadine
Symmetrel

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
2,3

Galactose N-acetyl-glucosamine Core sugars protein

Galactose
1,4

N-acetyl-glucosamine Core sugars 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 4-Guanidino-Neu5Ac2en Zanamivir Relenza

GS4104, Ro64-0796 Ethyl ester of GS4071 Oseltamivir Tamiflu

HO HO H3C O HO

OH H H N O OH OH O

Sialic acid N-Acetylneuraminic acid (NANA)

HO OH HO H H3C O H N HO O OH O R O

HO OH HO H H N O HO

O O R OH

influenza

H3C

neuraminidase

Sialyl -glycoside R = glycoprotein

Transition state

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

HO OH HO H H3C O H N HO O

HO OH HO H O O HO OH

H2O
H3C O

H N

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 H3C O H N HO O

DANA

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

HO OH HO H F F F O H N HO O

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

HO OH HO H H3C O H N HN NH H2N O

Zanamivir Relenza

HO P HO H3C O H3C H3C O NH2 H N O CH3

O OH

Oseltamivir phosphate Tamiflu

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

H3 C O H3 C H3 C O H2 N H N O C OH

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

H2N HN

NH O OH H N H OH CH3 CH3

H3C O

RWJ-270201
Smee et al., Antimicrob. Agents Chemother. 45: 743-748 (2001) Sidwell et al., Antimicrob. Agents Chemother. 45: 749-757 (2001)

H3C O N N

CH3 CH3 F F F O

HO O

NH2

A-192558
Wang et al., J. Med. Chem. 44: 1192-1201 (2001)

CH3

H3C O H3C

H N

H OH N H H O 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)

OH NH2 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 g/ml, while it showed no cytotoxicity at concentrations up to 1,000 g/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 H3C O O (CH2)6 CH C O C2H5 C2H5

Phenoxyalkyldiketone Arildone WIN38020

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

N H3C

O (CH2)7 O

O N

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 H3C O N N N

Pyridazinamine R61837

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

N H3C

N N (CH2)2 O

O C O C2H5

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 N O O N O

CF3

Pleconaril (VP-63843)
Romero, Exp. Opin. Invest. Drugs 10: 369-379 (2001)

BTA188

H3C N N

N O

CH2 CH3

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 S S S N N

Benzothiazole targeted at hydrophobic pocket in the core of VP1. EC50 against HRV-14 in cell culture: 0.8 M.
Tsang et al., Chem. Biol. 8: 33-45 (2001)

Ruprintrivir
O NH O N H O N O O 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)

O N H O CH2 CH3

H3 C

Pyridone
O NH O N H3 C O N H N O O 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)

O N H O CH CH3 CH3

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 Respiratory syncytial virus Parainfluenza virus Functions

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

Small envelope (M2) Nucleocapsid-associated

Transcriptional regulation; unique to pneumoviruses

Nucleoprotein (N, NP)

Phosphoprotein (P) Large polymerase complex (L)
aNot

44
37 200

58
60 250

Major RNA-binding nucleocapsid protein

Major phosphorylated protein; RNA-dependent RNA polymerase activity Large nucleocapsid-associated protein; major polymerase subunit; RNA-dependent RNA polymerase activity

bThere

present in the virus. 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 H2N N N N

HO O

HO

OH

Ribavirin Virazole

H2N O N S O O O NH N HN N HN O N H2 N O H2 N O S O N

RFI-641
NH2 O N S O NH N SO3 Na N N HN O S N NH2 O NH2 O O NH O

H2N

NaO3S

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

(A) 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)

N CH3 N NH N N CH3

NH2

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 Sendai HPIV-3

CDV PDV
RPV Measles PPRV DMV Tupaia Hendra Nipah HPIV-4b HPIV-4a Mumps SV5 HPIV-2 Mapuera NDV

Genus Respirovirus/ Paramyxovirus Genus Morbillivirus

Tentative new genus Megamyxovirus

Genus Rubulavirus

OUTBREAK-INSPIRED HUNT FOR VIRUSES

Hendra virus
(1994, Megamyxovirus)

Nipah virus
(1999, Megamyxovirus)

Australian bat lyssavirus

(1996, Rhabdoviridae)

Tioman virus
(2000, Rubulavirus)

Menangle virus
(1997, Rubulavirus)

Dolphin, porpoise and cetacean morbillivirus

(1991, Morbillivirus)

Tupaia virus
(1999, Paramyxovirus)

Salem virus
(2000, not yet classified)

DIFFERENCES BETWEEN
HENDRA & NIPAH VIRUS Multiple host species Reservoir host : fruit bats (flying foxes) ? Clinical hosts : horse, pig, man,... Experimental hosts : guinea pig, cat Not very contagious No human-to-human spread, but infection is very lethal and OTHER PARAMYXOVIRINAE Host specific : human

Highly contagious
Human-to-human spread, droplet infection

CONCLUSION APPROVED ANTIVIRAL DRUGS FOR THE TREATMENT OF THE MAJOR RESPIRATORY TRACT VIRUS INFECTIONS Adenoviruses Picornaviruses Entero Rhino Orthomyxoviruses Influenza : none
: none : none : Neuraminidase inhibitors: zanamivir, oseltamivir : Amantadine and rimantadine
(for influenza A only)

Paramyxoviruses Parainfluenza : none Respiratory syncytial virus : Ribavirin SARS virus : none