TREATMENT OF VIRAL RESPIRATORY INFECTIONS Erik DE CLERCQ Rega Institute for Medical Research,...

TREATMENT OF VIRAL RESPIRATORYINFECTIONS

Erik DE CLERCQRega 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

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

Influenza

NA (neuraminidase)

M1 (membrane protein)

M2 (ion channel)

Transcriptase complex(PB1, PB2 and PA)

Lipid bilayer

RNPs (RNA, NP)

HA (hemagglutinin)

McClellan and Perry, Drugs 61: 263-283 (2001)

Simplified representation of the influenza virion showing the neuraminidase(NA) glycoprotein, the hemagglutinin (HA) glycoprotein and the matrix M2

protein

M2

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

H

NH2.HCl

Amantadine

Symmetrel®

H

H

H

CH3

NH2.HCl

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

Galactose

N-acetyl-glucosamine

Core sugars

protein

Galactose

N-acetyl-glucosamine

Core sugars

protein

Sialic acid

2,3

1,4

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®

Sialic acidN-Acetylneuraminic acid (NANA)

OHN

HO

HO OH

H

HO

H3C

O

OH

O

OH

influenza

neuraminidase

Sialyl -glycosideR = glycoprotein

Transition state

+

-

O

HO

OH

HO

HN

O

OHO

R

HOH

H3C

O

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

O

HO

OH

HO

HN

O

OHO

R

HOH

H3C

O

H2O

Transition state Sialic acid

R-O- R-OH

O

HO

OH

HO

HN

O

OH

HOH

H3C

O

O

HO

OH

HO

HN

HOH

H3C

O

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

DANA

O

HO

OH

HO

HN

HOH

H3C

O

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

FANA

O

HO

OH

HO

HN

HOH

FF

F

O

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

ZanamivirRelenza®

O

HO

OH

HN

HN

HOH

H2N

NH

H3C

O

Oseltamivir phosphateTamiflu®

NO

HN

H3C O

O

H3C

H3C

CH3H2

PHO O

HO OH

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

GS4071

NO

HN

H3C

C

OH

O

H3C

H3C O

H2

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

Neuraminidase119 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 oseltamivirHemagglutininSome 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

RWJ-270201

OH

O

HN

H3C

CH3

CH3

HN

H2N NHO

OH

H

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

A-192558

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

NH2

N

HO

O

O

F

F FO N CH3

H3C CH3

A-315675

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

NH

O

OHHN

H3C O

CH3

H

CH3

H3C

HO

T-705

Furuta et al., Antimicrob. Agents Chemother. 46: 977-981 (2002)

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.

N

N OH

NH2F

O

HUMAN RHINOVIRUS (HRV)

HRV 14Human 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)

PhenoxyalkyldiketoneArildone

WIN38020

OH3C O (CH2)6 CH

C

C

C2H5

C2H5

O

O

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

IsoxazoleDisoxaril

WIN 51711

N

O

O(CH2)7

ON

H3C

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

PyridazinamineR61837

NN

N NOH3C

CH3

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

PhenoxypyridazinaminePirodavirR77975

NN

N (CH2)2 O C OH3C C2H5

O

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)

Pleconaril (VP-63843)

NO

O

NO

N CF3

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

BTA188

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

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

H3C

N N

N

O

ON CH2 CH3

Benzothiazole

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)

N

S

S S

N

N

H3C

Ruprintrivir

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

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

N

NH

O

NH

O

O

F

O

NH

O

H3C

O

OCH2

CH3

Pyridone

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)

N

NH

O

NNH

O

O

F

O

NH

O

H3C

F

O

OCH

CH3

CH3

RESPIRATORY SYNCYTIAL VIRUS (RSV)

Hall, N. Engl. J. Med. 344: 1917-1928 (2001)

Respiratory Syncytial Virus (RSV) and Parainfluenza Virus (PIV)

Characteristics of the proteins of respiratory syncytial virus and parainfluenza virus

Protein Molecular mass Functions

Respiratory syncytial virus

Parainfluenza virus

kilodaltons

Structural protein

Surface

Fusion (F) 68 60 Penetration; major protection antigen

Attachment (G) 90 —a Viral attachment; major protective antigen

Hemagglutinin neuraminidase (HN)

— a 69 Viral attachment and release; major protective antigen

Small hydrophobic (SH [1A]) 4.8 – 30b — a Unknown

Matrix

Matrix (M) 28

22b

40

— a

Mediates attachment of nucleocapsid to envelope

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)

Hall, N. Engl. J. Med. 344: 1917-1928 (2001)

Nu

mb

er o

f ca

ses

or

iso

late

s

1993 1994 1995 1996 1997 1998

Epidemiologic pattern of infections with respiratory syncytial virusin relation to the occurrence of bronchiolitis from 1993 through 1998

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

Proportion of samples taken from cases of influenza-like illness positive forinfluenza 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 forinfluenza or respiratory syncytial virus in 0-5-year-olds during 1996-1997

Ribavirin

Virazole®

OHO

HO OH

H2N

N

N

N

O

RFI-641

Nikitenko et al., Bioorg. Med. Chem. Lett. 11: 1041-1044 (2001)Razinkov et al., Chem. Biol. 8: 645-659 (2001)

RFI-641 inhibits RSV fusion mediated by F (Fusion) protein, but also interfereswith the attachment (G) protein

SO

O

N

O

NH2

O

NH2

NH

N

N

N

NH

HN

HN

NaO3S

SO3Na

N

N

N

NH

HN

SN

H2N

O

O

H2N

O

O

SN

O

H2N

O

H2N

OO

SN

NH2

O

O

NH2

O O

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

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

R-170591 interferes with RSV fusion

Andries et al., 40th ICAAC, Toronto, Canada, 17-20 September 2000, Abstract H-1160

N

N

NH

N NH2CH3

N CH3

HO

Benzodithiin (RD3-0028)

Sudo et al., Microbiol. Immunol. 45: 531-537 (2001)

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

S

S

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

Genus Respirovirus/

Paramyxovirus

Genus Morbillivirus

Tentative new genus Megamyxovirus

Genus Rubulavirus

HPIV-1Sendai

HPIV-3

CDVPDV

RPVMeasles

PPRVDMV

Tupaia

HendraNipah

HPIV-4b

HPIV-4a

Mumps

SV5HPIV-2

MapueraNDV

OUTBREAK-INSPIRED HUNT FOR VIRUSES

• Hendra virus(1994, Megamyxovirus)

•Australian bat lyssavirus (1996, Rhabdoviridae)

•Menangle virus (1997, Rubulavirus)

•Tupaia virus (1999, Paramyxovirus)

• Nipah virus (1999, Megamyxovirus)

• Tioman virus (2000, Rubulavirus)

•Dolphin, porpoise and cetacean morbillivirus (1991, Morbillivirus)

•Salem virus (2000, not yet classified)

DIFFERENCES BETWEEN

HENDRA & NIPAH VIRUS OTHER PARAMYXOVIRINAE

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

Host specific : human

and

Highly contagious

Human-to-human spread, droplet infection

CONCLUSIONAPPROVED ANTIVIRAL DRUGS FOR THE TREATMENT OF

THE MAJOR RESPIRATORY TRACT VIRUS INFECTIONS

Adenoviruses : nonePicornaviruses

Entero : noneRhino : none

OrthomyxovirusesInfluenza : Neuraminidase inhibitors: zanamivir, oseltamivir

: Amantadine and rimantadine (for influenza A only)

ParamyxovirusesParainfluenza : noneRespiratory syncytial virus : RibavirinSARS virus : none