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 % G T50 P 50 % YFV 17D RNA 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) 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.