V National Conference BIFI, Zaragoza, Febrero 2011

1. V National Conference BIFI, Zaragoza, Febrero 2011 APPROCHES FOR ANTIVIRAL COMPOUND DEVELOPMENT OLGA ABIAN oabifra@unizar.es

2. V National Conference BIFI, Zaragoza, Febrero 2011 STEPS: 1º TARGET IDENTIFICATION: A certain protein with biomedical therapy interest which can be inhibited, activated or stabilized by compound binding. 2º ACTIVE COMPOUND SEARCHING:A chemical library with thousands of compounds is used. In order to be efficient, the detection methods must be characterized by High Throughput Screening (HTS). Then, we need fast experimental procedures or multiple simultaneous measurements, or both. From thousands of molecules we must identify a few interesting molecules (hits, lead compounds). 3º CHARACTERIZATION OF THE SELECTED COMPOUNDS:The compounds that have been identified as promising drug candidates are studied in detail using the different biophysical (calorimetric, spectroscopic or crystallographic) techniques available in the facilities of our institute. 4º In Vivo CELL STUDIES:In some cases, the efficiency of the compound can be measured in vivo using a cell system. In other cases in which this is not possible, the toxicity of the compound can be determined using cell cultures. 5º EXPLORING NEW TRANSPORT AND SELECTIVE LIBERATION OF DRUGS: We can also try increasing the bioavailability of the compounds we have discovered: Selectively recognition of cells where drug should be preferably released, so adverse effects due to the systemic distribution will be reduced; and an easy way to ensure cell internalization, so compounds active in vitro but not in vivo are rescue.

3. V National Conference BIFI, Zaragoza, Febrero 2011 1º TARGET IDENTIFICATION Helicase NTPase NS3 protein Protease

4. V National Conference BIFI, Zaragoza, Febrero 2011 TARGET CHARACTERIZATION A/ Structure: Spectroscopic study By far and near Circular Dichroism, Spectrophotometry and Fluorescence B/ Zn interaction: Calorimetry (ITC and DSC) When Zn is removed at pH5 (where no aggregation nor precipitation occurs) the protein unfolds. The binding heat capacity value associated with the binding of Zn and other spectroscopic evidences suggest that this is a “folding by binding” process (Abian et al. Proteins 2009). C/ Allosteric activation mechanism : Fluorescence The activity of NS3 protease was measured in the presence and absence of its cofactor pNS4A at different substrate concentration. The cooperativity parameters for the binding of pNS4A and substrate were determined.

5. V National Conference BIFI, Zaragoza, Febrero 2011 2º HIGH THROUGHPUT SCREENING (HTS) Two ways for finding interesting molecules: A/ ACTIVITY BASED-SCREENING : B/ THERMAL SHIFT SCREENING:

6. V National Conference BIFI, Zaragoza, Febrero 2011 2º HIGH THROUGHPUT SCREENING (HTS) Two ways for finding interesting molecules: A/ ACTIVITY BASED-SCREENING : A molecule inducing an effect must alter protein properties (function, behavior). Methods for detecting function or behavior are required Our protein target exhibits protease activity and it can be measured in vitro using a fluorescence (FRET) peptide substrate.

7. V National Conference BIFI, Zaragoza, Febrero 2011 • Assay NS3 + Compound from the chemical library + FRET Substrate (RET S1) from Anaspec Ac - Asp - Glu - Asp(EDANS) - Glu - Glu - Abu - ψ - [COO] - Ala - Ser - Lys(DABCYL) - NH2 Donor Acceptor • Example: • Results Kinetic measurements: Vi (initial slope) The ratio of the control positive slopes (X) defines the lower limit(X-2SD). Below this limit the compounds are considered as possible inhibitors. The more lower positive value the best inhibitor compound. A/ SCREENING ACTIVITY BASED:

8. V National Conference BIFI, Zaragoza, Febrero 2011 2º HIGH THROUGHPUT SCREENING (HTS) Two ways for finding interesting molecules: B/ THERMAL SHIFT SCREENING: A molecule inducing an effect must bind to the protein. Methods for detecting binding are required If a ligand binds preferentially to a certain protein conformational state, it stabilizes (increases the population of) such conformational state Therefore, we can find ligands for a given protein by searching for molecules that increase the conformational stability of the protein There are several compounds (ANS, Sypro-Orange, …) which bind the hydrophobic parts of the protein that are exposed during the thermal unfolding and are used as probes. In our caseSTABILIZATION OF THE UNFOLDED STATE AT pH5 is studied.

9. V National Conference BIFI, Zaragoza, Febrero 2011 B/ SCREENING THERMAL SHIFT: • Assay: pH5, with/without EDTA

10. V National Conference BIFI, Zaragoza, Febrero 2011 3º CHARACTERIZATION OF THE SELECTED COMPOUNDS A/ Activity: The activity of the protein was measured at different compound concentrations and so, the inhibition constants (Ki) were determined. B/ Selectivity: The activity of human protease (a-chymotrypsin) in the presence of the selected compounds was determined. C/ Binding affinity: Affinity constants and thermodynamic parameters determination using ITC calorimetry D/ Crystallographic Structure: Obtaining the crystallographic structures of the protease with the compound is needed.

11. V National Conference BIFI, Zaragoza, Febrero 2011 4º In Vivo CELL STUDIES Bartenschlager, Science 1999, 285(5424)110-3.

12. V National Conference BIFI, Zaragoza, Febrero 2011 A/ Efficacy in vivo: The luciferase activity from cell culture is measure after 3 days of incubation with the compounds. B/ Toxicity in vivo: The viability of the cells in the presence of the compounds is measured using XTT. In this cells the replicon system is not present.

13. V National Conference BIFI, Zaragoza, Febrero 2011 OAV OAV OAV OAV OAV OAV OAV OAV Entry in cells Selective transport OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV AuNP OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV OAV AuNP1 AuNP2 = Internalization peptide OAV OAV OAV OAV = Fabfragment AuNP3 Selective recognition of infected cells AuNP4A AuNP4B 5º EXPLORING NEW TRANSPORT AND SELECTIVE LIBERATION OF DRUGS

14. V National Conference BIFI, Zaragoza, Febrero 2011 FINANCIAL SUPPORT PROYECTOS NACIONALES TITULO DEL PROYECTO: " Nanopartículas multifuncionales para el transporte y liberación selectiva de fármacos frente al virus de la hepatitis C (VHC)” " ENTIDAD FINANCIADORA: Instituto Carlos III DURACION DESDE: 2011 HASTA: 2013 CUANTÍA: 83.000€ INVESTIGADOR PRINCIPAL: Olga Abian Franco TITULO DEL PROYECTO: "Proteasa NS3 del virus de la hepatitis C: Identificación y caracterización de inhibidores competitivos y alostéricos" ENTIDAD FINANCIADORA: Ministerio de Ciencia e Innovación DURACION DESDE: 2010 HASTA: 2013 CUANTÍA: 90.000€ INVESTIGADOR PRINCIPAL: Adrián Velázquez Campoy TITULO DEL PROYECTO: "Implementación de estudios "in vivo" e "in vitro" de compuestos antiinfecciosos efectivos frente a Helicobacter pylori y el Virus de la Hepatitis C (VHC)" ENTIDAD FINANCIADORA: Instituto Carlos III DURACION DESDE: 2008 HASTA: 2010 CUANTÍA: 40.000€ INVESTIGADOR PRINCIPAL: Olga Abian Franco TITULO DEL PROYECTO: “Proteasa NS3 del virus de la hepatitis C: Inhibidores competitivos, alostéricos y bases moleculares de la resistencia a fármacos” ENTIDAD FINANCIADORA: Universidad de Zaragoza DURACION 2010 CUANTÍA: 12.700 € INVESTIGADOR PRINCIPAL: Dr. Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI) PROYECTOS AUTONÓMICOS TITULO DEL PROYECTO: “Equilibrio Conformacional de la proteasa NS3 en virus de la hepatitis C: estado no nativo e identificación de un nuevo tipo de inhibidores alostéricos” ENTIDAD FINANCIADORA: DGA DURACION DESDE: 2009 HASTA: 2011 CUANTÍA: 52.000€ INVESTIGADOR PRINCIPAL: Adrián Velázquez Campoy

15. V National Conference BIFI, Zaragoza, Febrero 2011 THANK YOU FOR YOUR ATTENTION!!