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SILVER WP1

SILVER WP1. Overview Of Screening Capabilities Current Status and Planning Result Highlights. Overview of Screening Capabilities. Compound Library Screening (WP1). ScreeningCampaign. Hit Confirmation/ Selection. Hit Explosion. Hit-to- Lead. Chemical Validation.

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SILVER WP1

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  1. SILVER WP1 Overview Of Screening Capabilities Current Status and Planning Result Highlights

  2. Overview of Screening Capabilities

  3. Compound Library Screening (WP1) ScreeningCampaign HitConfirmation/ Selection Hit Explosion Hit-to-Lead Chemical Validation

  4. Screening Facility at KULeuven (P3) State-of-the-art cell based BSL-2 screening platform at KULeuven (P3) Efficient data management (barcode tracked)/database. BSL-2 & BSL-3 animal facilities

  5. Compound Libraries • ChemBridge Diversity: • 30 k Compounds received from Univ Med (P1) • Selection of druglike compounds with maximum pharmacophorediversity. • CISTIM Library: • 33 K Compounds receivedfrom CISTIM (P24) • Special compound selection based on different parameters (e.g., Lipinski’s rule of five, solubility, toxicophores, frequent hitters, unstable fraction)

  6. Screening Campaigns at KULeuven (P3) Cell-culture based screening -> cover all viral targets Automated plate processing and data capture Antiviral activity determined by CPE quantification or reporter gene assays 4 concentrations assay starting at 50 µg/mL 30 k compounds -> 2500 test plates Additional microscopic scoring of antiviral activity and toxicity(Quality control)

  7. Screening Campaigns for Rabies and Toscana Virus Test plates (ChemBridge) prepared at KULeuven (P3)and shipped to Inst. Pasteur (P6) and Univ. Med. (P1) 1 concentration assay 30 k compounds -> 750 test plates Read-out: CPE or ELISA Additional microscopic scoring of antiviral activity and toxicity (Qualtity control)

  8. HIT CONFIRMATION / SELECTION Dengue Virus Plaque Assay Starts at ~10 k compounds screened Purchase of fresh powder 8-points screen Determining antiviral spectrum(ex Enteroviruses) Viral Yield Assays Chemical validation, Literature Check, …

  9. HIT Explosion Chemical validation • Acquisition of commercial analogues (~20): variation on R, ideally one position at a time • Evaluation of analogues in biological assays • Establishing Structure-Activity –Relationship (SAR) • Total synthesis (from scratch) to compare analytical data of in house synthesised and commercial compounds. • Evaluation of in house synthesised and commercial compounds in biological assays) Commercial hit R1 CC50 > 50 μg/ml; EC50 = 0.3 μg/ml R3 Scaffold Synthesised hit CC50 > 50 μg/ml; EC50 = 0.24 μg/ml R2 Rn

  10. ChemInformatics at KULeuven (P3) • Selection of potential hits from commercially available libraries • Predictive QSAR modelingwith graph kernels • Efficient enumeration of subgraphswith careful normalization and large-margin classification. • State-of-the-art predictive power • Active optimization • Iterative: acquire, test, model, repeat. • Escapes from local optima by exclicit uncertainty modeling • Suggestions for synthesis

  11. Status and planning

  12. Compound Library Screening Status (WP1) Hit Confirm. Assay Optimisation Screening HitExplosion Hit-to-Lead ALPHAVIRUS Chikungunya Virus Coxsackievirus B4 (CVB4) ENTEROVIRUS NOROVIRUS Murine Norovirus Metapneumovirus P5 EMC PARAMYXOVIRUS Dengue / Yellow Fever Virus DENGUEVIRUS CORONAVIRUS Coronavirus P5 EMC RHABDOVIRUS Rabies virusP6Inst. Pasteur BUNYAVIRUS Toscana virusP1 UnivMed

  13. Compound Library Screening Planning (WP1) 2010 2011 2012 2013 Today

  14. Result Highlights

  15. R110 (AAF)2-R110 Coronavirus Assay Optimisation Released Proteases Virus Cell CPE Collaboration KULeuven and EMC (P3 and P5) Coronavirus NL63 produces limited CPE-> Quantification? A new methodology to quantify CPE wassuccessfully developed (reported at ICAR2011)

  16. Alphavirus-Norovirus-EnterovirusscreensCollaborationKULeuvenand CISTIM (P3 and P24) ALPHAVIRUS (CHIKV) (screen and hit selection) NOROVIRUS (partial screen and hit selection) ENTEROVIRUS (CVB4) (screen and hit selection) Screening CISTIM library ~33000 compounds Hits - resupply Verification and profiling Confirmed Hits • ALPHAVIRUS (CHIKV) • NOROVIRUS Commercial Analogues • In collaboration with UIBK (P17) • ALPHAVIRUS (CHIKV):first family selected Selected families Hit-to-Lead

  17. 1-Alphavirus – First Hit Selected for Hit-to-Lead Analogue acquisition Compound with confirmed selective antiviral activity on the replication of Chikungunya virus (899 strain) in Vero cells (subtype A) Example of an active commercial analogue Family transferred to UIBK (P17) for Hit-to-Lead Next steps for P24: acquisition of analogues for confirmed hits

  18. 2-Murine Norovirus – First Hit Selected for MoAStudies Analogue acquisition Compound with confirmed selective antiviral activity on the replication of murinenorovirus Example of an active commercial analogue MOA studies (TOA, resistance studies) are ongoing

  19. 3-Enterovirus: Hit Confirmation Ongoing Screening Results Confirmation Compound 1 Compound 2 Hit Explosion by Q3 2011 – Hit Optimization by Q4 2011

  20. Inhibitors identified in collaborations

  21. KRICT compounds CPE Reduction Assay CPE score [%VC] Control Virus Yield Reduction Infectious Virus Titer[Log CCID50] Concentration KR22809 [µM] Luciferase activity ≡ RNA replication 35S methionine pulse labeling experiments KR22809 (50µM) KR22809 (50µM) Luciferase activity (x10exp4 RLU) Concentration KR22809 [µM] Time hours post transfection • NOT affected: • Rate of protein synthesis • Processing of polyprotein

  22. KRICT compounds: characterization Resistance pattern of CVB3 Highest concentration with still sufficient virus replication ([µM] Passage number

  23. Ivermectine as a potent inhibitor of YFV replication

  24. 3’5’ di-O-trityluridine: an inhibitor of flavivirus RNA dependent RNA polymerase

  25. ...and see WP3 & WAVES for • GPC-N against entero RdRp • GPV entero capsid binder • XP126 against entero • SG class against entero

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