Design of small molecule inhibitors of RNA virus replication
Towards antivirals against VHF viruses
Johan Neyts Rega Institute, University of Leuven, Belgium Design of small molecule inhibitors of RNA virus replication
Gertrude Elion, 1918 - 1999 O N HN
H N N N 2
HO O Design of small molecule inhibitors of RNA virus replication
Virus Target Drugs on the market Herpes Polymerase Acyclovir, valacyclovir, penciclovir, famiclovir, brivudin, foscarnet, cidofovir HBV RT/polymerase Lamivudine, adefovir, entecavir, telbuvidine
HIV Reverse NRTI: zidovudine, didanosine, zalcitabine, stavudine, transcriptase 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 Antivirals from the Rega Institute
Herpes HBV HIV And more
O CMV retinitis Br HN
O N HPMPC HO O
HO BVDU-TP inhibits the PMEA-TP is chain PMPA-TP is chain viral polymerase terminator terminator HIV
TIBO derivatives
T-tropic HIV strains
CXCR4 antagonists
Brivudin Adefovir dipivoxil Tenofovir disoproxyl fumarate RNA viruses? BVDU Bis(POM)-PMEA Bis(POC)-PMPA-fumarate Antivirals from the Rega Institute
Herpes HBV HIV And more CMV retinitis O NH Br 2 HN N N O N HPMPC HO N N O P P P O
HO BVDU-TP inhibits the PMEA-TP is a chain PMPA-TP is chain viral polymerase terminator terminator HIV
TIBO derivatives
T-tropic HIV strains
CXCR4 antagonists
Brivudin Adefovir dipivoxil Tenofovir disoproxyl fumarate RNA viruses? BVDU Bis(POM)-PMEA Bis(POC)-PMPA-fumarate Antivirals from the Rega Institute
Herpes HBV HIV And more
O NH CMV retinitis NH 2 2 Br N N HN N O O N N N HPMPC O N O O O HO N N P O O O O P P P O O O HO BVDU-TP inhibits the PMEA-TP is a chain PMPA-TP is a chain viral polymerase terminator terminator HIV
TIBO derivatives
+ emtricitabine efavirenz + T-tropic HIV strains
CXCR4 antagonists
Brivudin Adefovir dipivoxil Tenofovir disoproxyl fumarate RNA viruses? BVDU Bis(POM)-PMEA Bis(POC)-PMPA-fumarate Design of small molecule inhibitors of RNA virus replication
Herpes HBV HIV And more
O NH CMV retinitis NH 2 2 Br N N HN N O O N N N HPMPC O N O O O HO N N P O O O O P P P O O O HO BVDU-TP inhibits the PMEA-TP is a chain PMPA-TP is a chain viral polymerase terminator terminator HIV
TIBO derivatives
+ emtricitabine efavirenz + T-tropic HIV strains
CXCR4 antagonists
Brivudin Adefovir dipivoxil Tenofovir disoproxyl fumarate RNA viruses? BVDU Bis(POM)-PMEA Bis(POC)-PMPA-fumarate Design of small molecule inhibitors of RNA virus replication Ribavirin and VHF
Active Not (or limited) active Arenaviruses Filoviruses Bunyaviruses Flaviviruses
Treatment of suspected or Post exposure prophylaxis [7 confirmed clinical cases of VHF days] [10 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 Per os : 2 g / day in 4 doses followed by 4 g/day in 4 doses for 4 days followed by 2 g/day for 6 days
Bossi et al., Eurosurveillance (2004) Task Force on Biological & Chemical Agent Threats, European Commission Design of small molecule inhibitors of RNA virus replication Can we design more potent ribavirin analogues?
O
N H2N N Mechanism of action of ribavirin? N
HO O
Mycophenolic acid
HO OH 1. Inhibition of IMP-dehydrogenase
GTP
Ribavirin Ribavirin Design of small molecule inhibitors of RNA virus replication
2. Inhibition of the viral polymerase by ribavirin-TP (reovirus, VSV, influenza… ) 3. Inhibition of guanylyltransferase activity and thus capping (sindbis)
4. Induction of an error catastrophe (polio… .) 5. Immunomodulation
Crotty et al. (2001) Proc. Natl. Acad. Sci. USA 98, 6895-6900 Design of small molecule inhibitors of RNA virus replication MORE POTENT ANALOGUES OF RIBAVIRIN...
O
EICAR N EC µg/ml H2N 50 Ribavirin EICAR
HC C N YFV-17D 27 1 DENV 25 2 HO O Junin 12 0.2 5- ethynyl 1 beta-D ribofuranosyl imidazole RSV 4 0.2 carboxamide HO OH Measles 8 0.5
but ....EICAR is at least 10-fold more cytostatic in cell culture than ribavirin
De Clercq et al., Antimicrob. Agents & Chemother. 35:679-84 Leyssen et al., J. Virol. 80:149-60. Design of small molecule inhibitors of RNA virus replication
GTP depletion Anti-YFV activity
125 125
100 100
75 75
GTP T G % 50 50 YFV RNA A N R D 7 1 V F Y %
25 25
0 0 100 25 10 2.5 1 0.25 0.1 0.025 0.01 100 25 10 2.5 1 0.25 0.1 0.025 0.01 [ ] compound (µg/m l) [ ] compound (µg/m l)
O O Ribavirin EICAR MPA N N H2N H2N CH3 N N N H3CO HC C O HO HOOC HO O O O
CH3 OH
HO OH HO OH Design of small molecule inhibitors of RNA virus replication
Antiviral activity of ribavirin correlates with GTP depletion
MPA EICAR Ribavirin
100 R2 = 0.998 DENV: R² = 0.991 MODV: R² = 0.999 MMLV: R² = 0.987 10
1
0.1
0.01 EC50 for GTP depletion (µg/ml) depletion GTP for EC50
0.001 0.001 0.01 0.1 1 10 100 EC50 for inhibition of YFV 17D RNA re plication (µg/ml)
Leyssen et al., J. Virol. 80:149-60 Design of small molecule inhibitors of RNA virus replication
Correlation between reduction of viral RNA and infectious virus for YFV
1.0E+09 1.0E+09
1.0E+08 Ribavirin 1.0E+08
1.0E+07 1.0E+07
1.0E+06 1.0E+06
1.0E+05 1.0E+05
1.0E+04 1.0E+04
1.0E+03 1.0E+03
1.0E+02 1.0E+02
1.0E+01 R² = 0.901 1.0E+01 R² = 0.938
1.0E+00 1.0E+00 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+09 EICAR MPA 1.0E+08 1.0E+08
1.0E+07 1.0E+07
1.0E+06 1.0E+06
1.0E+05 1.0E+05
1.0E+04 1.0E+04
1.0E+03 1.0E+03
1.0E+02 1.0E+02 R² = 0.880 R² = 0.889 1.0E+01 1.0E+01
1.0E+00 1.0E+00 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 Design of small molecule inhibitors of RNA virus replication No increased mutation frequency in pre-extinction population
Ribavirin EICAR MPA
1.0E+09 1.0E+09 1.0E+09
1.0E+08 1.0E+08 1.0E+08
1.0E+07 1.0E+07 1.0E+07
1.0E+06 1.0E+06 1.0E+06
1.0E+05 1.0E+05 1.0E+05
1.0E+04 1.0E+04 1.0E+04
1.0E+03 1.0E+03 1.0E+03
1.0E+02 1.0E+02 1.0E+02
1.0E+01 1.0E+01 1.0E+01
1.0E+00 1.0E+00 1.0E+00 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
VC 300 240 180 120 60 12 [ µg/ml] VC 12.5 2.5 0.5 0.1 [µg/ml] VC 1.25 0.25 0.05 0.01 [µg/ml]
Unique mutations/1000 NT
Reference Virus control Ribavirin EICAR MPA
1.22 1.74 1.49 1.40 1.32
Leyssen et al., (2006) Mol. Pharm Design of small molecule inhibitors of RNA virus replication Design of small molecule inhibitors of RNA virus replication
Merimepodib
Mycophenolic acid (CellCept) Design of small molecule inhibitors of RNA virus replication
Flaviridae
Genus Hepacivirus Genus Flavivirus hepatitis C virus
Genus Pestivirus e.g. bovine viral diarrhea virus (surrogate) Design of small molecule inhibitors of RNA virus replication
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
Analogue with F increased activity: N EC = 1.5 µM N 50 N F F F F N N þ N F F F ý N F
Analogue with F N N Hit with decreased activity: F EC50 = 16 µM EC50 >100 µM F Purstinger et al., Bioorg. Med. Chem. Letters. (2006) Design of small molecule inhibitors of RNA virus replication Building a structure-activity relationship : Hit to lead
N
N N
þ ý Br
Purstinger et al., Bioorg. Med. Chem. Letters. (2006) Design of small molecule inhibitors of RNA virus replication
Study of the mechanism of action Paeshuyse et al., J. Virol. Generation and characterisation of resistant virus (2006) Study of the molecular mechanism of action A B
BPIP
Phe224 Ala 221 Ala 222
C F224S mutation in polymerase is responsible for resistance Design of small molecule inhibitors of RNA virus replication
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
Paeshuyse et Identification of a hot spot for inhibition of viral replication al., J. Virol (2007) Design of small molecule inhibitors of RNA virus replication
From pestivirus to HCV inhibitors ....
F F F N N
N N N N F F F þ ý þ ý O GS-9190 N ~1000 analogs ~700 analogs BVDV : 1.5 µM BVDV : 0.7 µM HCV : >50 µM Cl HCV : 0.004 µM
Pürstinger et al., Bioorg. Med. Chem. Lett. (2007) Design of small molecule inhibitors of RNA virus replication
GS-9190 in clinical trial þ ý
0.10 -0.05 -0.20 -0.35 -0.50 -0.65 -0.80 copies/mL
10 -0.95 -1.10 -1.25 -1.40 Change from Baseline -1.55 HCV RNALog -1.70 -1.85 Cohort 1 (40mg) Cohort 2 (120mg) -2.00 Placebo -2.15 0 1 2 3 4 5 6 7 8 9 10 Time (days) Dosing period Design of small molecule inhibitors of RNA virus replication
DEBIO-025 is a non-immunosuppressive cyclosporin analog
Cyclophilin binding domain
HO O
N N N N N O H O O O O N H O H O N O N N H N N O O
Calcineurin binding domain
Name Pos3 N4 Pos4 Paeshuyse et CsA H Me Leu al., (2006) Hepatology DEBIO-025 Ala Et Val Design of small molecule inhibitors of RNA virus replication
Debio-025 is in advanced Phase 2 clinical trial þ ý
9
8
7 Mean: -3.6 Log 6 10 (p = 0.0018) 5
copies/ml 4 10 Log 3 DEBIO-025 1200 mg BID (n = 16) Placebo (n = 3) 2 DEBIO-025 1 Treatment Placebo
-28 0 5 10 15 20 25 30 35 40 45 50 Time (Days)
Flisiak R, Horban A, Kierkus J, Stanczak J, Cielnak I, Stanczak GP, Wiercinska-Drapalo A, et al. The cyclophilin inhibitor Debio-025 has a potent dual anti-HIV and anti-HCV activity in treatment-naive HIV/HCV co-infected subjects. Hepatology 2006; 44: 609A. (AASLD meeting Boston 2006) Flisiak et al, Hepatology, accepted Design of small molecule inhibitors of RNA virus replication HCV NS3 Protease Product Based Inhibitors
BILN-2061 Design of small molecule inhibitors of RNA virus replication Effect of BILN-2061 on HCV titers
Lamarre et al., Nature. (2003) 426:186-9. Design of small molecule inhibitors of RNA virus replication
Interaction of NS3pro with part of NS2b and a substrate based inhibitor
Erbel et al., (2006) Nature Structural & Molecular Biology Design of small molecule inhibitors of RNA virus replication NS5b RNA dependent RNA polymerase inhibitors
1. NUCLEOSIDE ANALOGUES 2. NON NUCLEOSIDE ANALOGUES
NH2
N HO O N O CH3 HO OH Inhibits HCV and other +ssRNA 1 viruses including flaviviruses Design of small molecule inhibitors of RNA virus replication Courtesy : S. Gunther
Mononegavirales
Segmented viruses
Plus strand viruses Design of small molecule inhibitors of RNA virus replication
T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide)
Furuta et al, Antimicrob. Agents & Chemother. (2005) 49:. 981–986 Design of small molecule inhibitors of RNA virus replication
Lethal PTV hamster model Design of small molecule inhibitors of RNA virus replication
Small molecule inhibitor of New World arenaviruse entry
Tacaribe lethal mouse model
Bolken et al., Antiviral Research 2006 Design of small molecule inhibitors of RNA virus replication
Small molecule inhibitors of arenavirus entry
Lee et al., JBC (2008) Design of small molecule inhibitors of RNA virus replication
S-adenosyl homocysteine hydrolase inhibitors block methylation of the cap
3-Deazaneplanocin A induces massively increased interferon production in Ebola virus-infected mice Bray et al., Antiviral Research (2002) Design of small molecule inhibitors of RNA virus replication Design of small molecule inhibitors of RNA virus replication
Carefully select library of small drugable molecules. Screen against target or surrogate Hit identification followed by lead optimization
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 Design of small molecule inhibitors of RNA virus replication
Green = uninfected, untreated Red = infected, untreated Brown staining = live cells Yellow staining = dead cells Evaluation of the toxicity Evaluation of antiviral effect
1,2 1 0,8 0,1 1 10 100 1000 Concentration (µg/ml) Design of small molecule inhibitors of RNA virus replication
Models for screening
Infectious virus (BSL-4 pathogens)
Surrogate viruses ? Filo: none Arena :mopeia .... (old world), Tacaribe...(New World) Bunya : Dugbe , Punta Toro.... Flavi : can use dengue, YFV-17D
Minigenome Filo, 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 industry hepatitis C virus, respiratory syncytial virus, influenza virus ...
Develop antivirals with broad-spectrum activity broad spectrum (-) ss RNA virus inhibitors broad spectrum (+) ss RNA virus inhibitors
Off label use of drugs approved for commercially interesting viruses (influenza, HCV) could be an option. Acknowledgements
Academic research group HCV team Jan Paeshuyse, Inge Vliegen, Lotte Coelmont, Leen Delang, Susan Obeid, Katrien Geerts Picornavirus team Armando De Palma, Hendrik Jan Thibaut, Miette Stuyck Flavivirus team Suzanne Kaptein, Tine De Burghgraeve Animal models Carolien De Keyzer
˜™ Academic drug discovery facility : Headed by Pieter Leyssen Stijn Delmotte, Tom Bellon
Flaviviridae, Picornaviridae, Caliciviridae, Rhabdoviridae, Togaviridae,...