RSV HCV - escmid.org

Design of small molecule inhibitors of RNA virus replication

Towards antivirals against VHF viruses

Johan NeytsRega Institute, University of Leuven, Belgium

N

N

NH

N

O

NH2

OOH

Gertrude Elion, 1918 - 1999


Virus Target Drugs on the market

Herpes Polymerase Acyclovir, valacyclovir, penciclovir, famiclovir, brivudin, foscarnet, cidofovir

HBV RT/polymerase Lamivudine, adefovir, entecavir, telbuvidine

HIV Reverse transcriptase

NRTI: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir

NNRTI: nevirapine, delavirdine, efavirenz

Protease Saquinavir, ritonavir, indinavir, nelfinafir, amprenavir, lopinavir

Fusion- CCR5 T20 - Maraviroc

Integrase Raltegravir

Influenza M2 Amantadine, rimantadine

Neuraminidase Oseltamivir, zanamivir

RSV Ribavirin

HCV Ribavirin + peg interferon


N

NH

O

O

O

Br

OH

OH

Herpes HBV HIV And more

CMV retinitis

HPMPC

BVDU-TP inhibits the viral polymerase

PMEA-TP is chain terminator

PMPA-TP is chain terminator HIV

TIBO derivatives

T-tropic HIV strains

CXCR4 antagonists

Brivudin

BVDU

Adefovir dipivoxil

Bis(POM)-PMEA

Tenofovir disoproxyl fumarate

Bis(POC)-PMPA-fumarateRNA viruses?

Antivirals from the Rega Institute

N

NH

O

O

O

Br

OH

OH


CMV retinitis

HPMPC


PMEA-TP is a chain terminator

PMPA-TP is chain terminator HIV

TIBO derivatives


CXCR4 antagonists

Brivudin

BVDU

Adefovir dipivoxil

Bis(POM)-PMEA



N

N

N

N

NH2

OPPP


N

NH

O

O

O

Br

OH

OH


CMV retinitis

HPMPC



PMPA-TP is a chain terminator HIV

TIBO derivatives


CXCR4 antagonists

Brivudin

BVDU

Adefovir dipivoxil

Bis(POM)-PMEA



N

N

N

N

NH2

OPPP

N

N

N

N

NH2

OP

O

O OO

OO

O

OO

+ emtricitabine

efavirenz +


Ribavirin and VHFRibavirin and VHFActive Not (or limited) active

Arenaviruses Filoviruses

Bunyaviruses Flaviviruses

Treatment of suspected or confirmed clinical cases of VHF

[10 days]

Post exposure prophylaxis [7 days]

Intravenous : initial dose of 2g followed by 1 g every 6 hr for 4 days followed by 0.5 g every 8 hr for 6 days

Intravenous : Initial dose of 30 mg/kg followed by 15 mg/kg every 6 hr for 4 days, followed by 7.5 mg/kg every 8 hr.

Per os : 2 g as loading dose followed by 4 g/day in 4 doses for 4 days followed by 2 g/day for 6 days

Per os : 2 g / day in 4 doses

Bossi et al., Eurosurveillance (2004)

Task Force on Biological & Chemical Agent Threats, European Commission


Can we design more potent ribavirin analogues?

Ribavirin Ribavirin

Mycophenolic acid

Ribavirin Ribavirin

Mycophenolic acid

Ribavirin Ribavirin

Mycophenolic acid

GTP

1. Inhibition of IMP-dehydrogenase

Mechanism of action of ribavirin?N

N

N

H2N

O

OHO

HO OH


2. 2. Inhibition of the Inhibition of the viral polymeraseviral polymerase by ribavirinby ribavirin--TP (reovirus, VSV, influenza… )TP (reovirus, VSV, influenza… )

3. Inhibition of 3. Inhibition of guanylyltransferaseguanylyltransferase activity and thus capping (sindbis)activity and thus capping (sindbis)

4. 4. Induction of an Induction of an error catastropheerror catastrophe (polio… .)(polio… .)

5. 5. ImmunomodulationImmunomodulation

Crotty et al. (2001) Proc. Natl. Acad. Sci. USA 98, 6895-6900


O

N

N

C

H2N

O

HO

HO OH

HC

MORE POTENT ANALOGUES OF RIBAVIRIN...

EICAR EC50 µg/ml Ribavirin EICAR

YFV-17D 27 1

DENV 25 2

Junin 12 0.2

RSV 4 0.2

Measles 8 0.5

De Clercq et al., Antimicrob. Agents & Chemother. 35:679-84

Leyssen et al., J. Virol. 80:149-60.

but ....EICAR is at least 10-fold more cytostatic in cell culture

than ribavirin

5- ethynyl 1 beta-D ribofuranosyl imidazole

carboxamide


Ribavirin EICARMPA

0

25

50

75

100

125

100 25 10 2.5 1 0.25 0.1 0.025 0.01

[ ] compound (µg/m l)

% G

TP

0

25

50

75

100

125

100 25 10 2.5 1 0.25 0.1 0.025 0.01

[ ] compound (µg/m l)

% Y

FV

17D

RN

A

N

N

N

H2N

O

OHO

HO OH

O

N

N

C

H2N

O

HO

HO OH

HC

HOOC

H3COO

CH3 OH

O

CH3


GTP depletion Anti-YFV activity

Antiviral activity of ribavirin correlates with GTP depletionRibavirinEICARMPA

R2 = 0.998

0.001

0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100

EC50 for inhibition of YFV 17D RNA replication (µg/ml)

EC

50 f

or

GT

P d

eple

tio

n (

µg

/ml)

DENV: R² = 0.991

MODV: R² = 0.999

MMLV: R² = 0.987

Leyssen et al., J. Virol. 80:149-60


Ribavirin

EICAR MPA

Correlation between reduction of viral RNA and infectious virus for YFV

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08

R² = 0.901

R² = 0.889

R² = 0.938

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08

R² = 0.880


No increased mutation frequency in pre-extinction populationRibavirin EICAR MPA

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1 2 3 4 5

VC 1.25 0.25 0.05 0.01 [µg/ml]

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1 2 3 4 5

VC 12.5 2.5 0.5 0.1 [µg/ml]

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1 2 3 4 5

VC 300 240 180 120 60 12 [ µg/ml]


1.22 1.74 1.49 1.40 1.32

MPAEICARRibavirinVirus controlReference MPAEICARRibavirinVirus controlReference

Unique mutations/1000 NT

Leyssen et al., (2006) Mol. Pharm

Merimepodib

Mycophenolic acid (CellCept)


Genus Hepacivirushepatitis C virus

FlaviridaeFlaviridae

Genus Flavivirus

Genus Pestiviruse.g. bovine viral diarrhea virus (surrogate)


Evaluation of the anti-BVDV activity of novel classes of compounds

Collaboration with ~20 medicinal chemists world-wide

Identification of a selective inhibitor of BVDV replication

N

N

N

F

FF

F

Hit with EC50 = 16 µM

Analogue with increased activity:

EC50 = 1.5 µM

N

NN

F

FF

F

Analogue with decreased activity:

EC50 >100 µM

N

NN

FF

F

F

Purstinger et al., Bioorg. Med. Chem. Letters. (2006)

þ

ý


Building a structure-activity relationship : Hit to lead

N

NN

Br

Purstinger et al., Bioorg. Med. Chem. Letters. (2006)

þ ý


Study of the mechanism of action

Generation and characterisation of resistant virus

Study of the molecular mechanism of action

F224S mutation in polymerase is responsible for resistance

A B

BPIP

Phe224Ala 222

Ala 221

C

A B

BPIP

Phe224Ala 222

Ala 221

C

A B

BPIP

Phe224Ala 222

Ala 221

C

A B

BPIP

Phe224Ala 222

Ala 221

C

Paeshuyse et al., J. Virol.

(2006)


Study of the mechanism of action of other BVDV inhibitors

AG110 is cross-resistant with BPIP

E291G mutation only 7Å away from BPIP-induced mutation

Identification of a hot spot for inhibition of viral replication Paeshuyse et

al., J. Virol (2007)


From pestivirus to HCV inhibitors ....

N

NN

FF

F

F

BVDV : 1.5 µM

HCV : >50 µM

O

N

N

NN

F

Cl

F

BVDV : 0.7 µM

HCV : 0.004 µM

GS-9190

~1000 analogs ~700 analogs

Pürstinger et al., Bioorg. Med. Chem. Lett. (2007)

þ ýþ ý


GS-9190 in clinical trial

-2.15

-2.00

-1.85

-1.70

-1.55

-1.40

-1.25

-1.10

-0.95

-0.80

-0.65

-0.50

-0.35

-0.20

-0.05

0.10

0 1 2 3 4 5 6 7 8 9 10

Time (days)

Cha

nge

from

Bas

elin

e H

CV

RN

A Lo

g 10 co

pies

/mL

Cohort 1 (40mg)

Cohort 2 (120mg)

Placebo

Dosing period

þ ý


DEBIO-025 is a non-immunosuppressive cyclosporin analog

Name Pos3 N4 Pos4

CsA H Me LeuDEBIO-025 Ala Et Val

O

NH

O

N

O

NHN

O

N

HN

HN

O

O

O

O

N

O

NNO

O

N

HO

Cyclophilin binding domain

Calcineurin binding domain

Paeshuyse et al., (2006) Hepatology


Debio-025 is in advanced Phase 2 clinical trial

-28

1

2

3

4

5

6

7

8

9

DEBIO-025

Placebo

0 5 10 15 20 25 30 35 40 45 50

Treatment

Time (Days)

Log 1

0 c

opie

s/m

l

DEBIO-025 1200 mg BID (n = 16) Placebo (n = 3)

Mean: -3.6 Log10

(p = 0.0018)

Flisiak R, Horban A, Kierkus J, Stanczak J, Cielnak I, Stanczak GP, Wiercinska-Drapalo A, et al. The cyclophilininhibitor Debio-025 has a potent dual anti-HIV and anti-HCV activity in treatment-naive HIV/HCV co-infectedsubjects. Hepatology 2006; 44: 609A. (AASLD meeting Boston 2006)Flisiak et al, Hepatology, accepted

þ ý


HCV NS3 Protease Product Based HCV NS3 Protease Product Based InhibitorsInhibitors

BILN-2061


Lamarre et al., Nature. (2003) 426:186-9.

Effect of BILN-2061 on HCV titersDesign of small molecule inhibitors of RNA virus replication

Erbel et al., (2006) Nature Structural & Molecular Biology

Interaction of NS3pro with part of NS2b and a

substrate based inhibitor


NS5bRNA dependent RNA polymerase inhibitors

1. NUCLEOSIDE ANALOGUES2. NON NUCLEOSIDE ANALOGUES


O NHO

HO OH

CH3

N

NH2

O

1Inhibits HCV and other +ssRNA viruses including flaviviruses

Mononegavirales

Segmented viruses

Plus strand viruses

Design of small molecule inhibitors of RNA virus replicationCourtesy : S. Gunther

Furuta et al, Antimicrob. Agents & Chemother. (2005) 49:. 981–986

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide)



Lethal PTV hamster model


Small molecule inhibitor of New World arenaviruse entry

Bolken et al., Antiviral Research 2006

Tacaribe lethal mouse model


Small molecule inhibitors of arenavirus entry

Lee et al., JBC (2008)


3-Deazaneplanocin A induces massively increased interferon production in Ebola virus-infected mice

Bray et al., Antiviral Research (2002)

S-adenosyl homocysteine hydrolase inhibitors block methylation of the cap

High speed, high accuracy, high reproducibility

Full assay setup –maximal autonomy –minimal user interventions

Programming of wide variety of protocols: combination exps, resistance generation,...

Continuous innovation depending on current and future needs


Carefully select library of small drugable molecules.

Screen against target or surrogate

Hit identification followed by lead optimization

0,81

1,2

0,1 1 10 100 1000

Concentration (µg/ml)

Green = uninfected, untreated Red = infected, untreatedBrown staining = live cells Yellow staining = dead cells

Evaluation of the toxicity Evaluation of antiviral effect



Models for screening

Infectious virus (BSL-4 pathogens)

Surrogate viruses ?Filo: noneArena :mopeia .... (old world), Tacaribe...(New World)Bunya : Dugbe , Punta Toro....Flavi : can use dengue, YFV-17D

MinigenomeFilo, Arena ....

Virus-like particles

CONCLUSION

Today only ribavirin (a more or less a-specific drug) is available

Few RNA viruses attract attention from the pharma industryhepatitis C virus, respiratory syncytial virus, influenza virus ...

Develop antivirals with broad-spectrum activitybroad spectrum (-) ss RNA virus inhibitorsbroad spectrum (+) ss RNA virus inhibitors

Off label use of drugs approved for commercially interesting viruses (influenza, HCV) could be an option.

Acknowledgements

Academic research groupHCV team

Jan Paeshuyse, Inge Vliegen, Lotte Coelmont, Leen Delang, Susan Obeid, Katrien Geerts

Picornavirus teamArmando De Palma, Hendrik Jan Thibaut, Miette Stuyck

Flavivirus teamSuzanne Kaptein, Tine De Burghgraeve

Animal modelsCarolien De Keyzer

��

Academic drug discovery facility : Headed by Pieter LeyssenStijn Delmotte, Tom Bellon

Flaviviridae, Picornaviridae, Caliciviridae, Rhabdoviridae, Togaviridae,...

RSV HCV - escmid.org

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