Cribado, identificación y ensayos in vitrode nuevos compuestos inhibidores de la                pproteasa NS3 del virus de...
HCV life cycle: targets for STAT-C                       compoundsHepatology A clinical text book (2009)Mauss, Berg, Rocks...
Genomic organization of HCV and                polyprotein processing                  l    t i          i        HCV RNA ...
NS3 protein                                                                             Helicase                          ...
Weak points in NS3 protease• Catalytic active site     Inhibiting of the enzymatic activity by competitive inhibitors bloc...
NS4A interacts with NS3                                                                    NS4A peptide (21-34 NS4A)NS3NS3...
General goal:                Identification and development of potent, selective                and adaptive inhibitors of...
pET7-NS3     *     NS3 protease      from HCV      genotype      1b J-strain         E.coli                               ...
Fluorometric activity assay HCV Protease Fl o escence Resonance Ene g T ansfe (FRET)               Fluorescence           ...
Isothermal Titration Calorimetry (ITC)                                                                       tim e (m in) ...
4                                                         OAV08         OAV06                                             ...
Inhibitors of NS3 Protease High                 Throughput Screening (HTS)Inhibitors of NS3 protease HTS
High Throughput Screening (HTS)                           with a chemical library  Materials:                      Chemic...
 Result:                Kinetic measurements: Fluorescence Signal Initial Slope (Vi )  Example:               Kinetic ex...
Analyzed data  Data processing:                                                400                                       ...
 Final test:                                           Selection of Best Compounds                                       ...
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Cribado, identificación y ensayos in vitro de nuevos compuestos inhibidores de la proteasa NS3 del virus de la Hepatitis C. Olga Abián

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Ponencia presentada en el marco de las I Jornadas Científicas del Instituto de Investigación Sanitaria Aragón (8-9 noviembre, 2010. Zaragoza)

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Cribado, identificación y ensayos in vitro de nuevos compuestos inhibidores de la proteasa NS3 del virus de la Hepatitis C. Olga Abián

  1. 1. Cribado, identificación y ensayos in vitrode nuevos compuestos inhibidores de la pproteasa NS3 del virus de la Hepatitis C OLGA ABIAN omabian.iacs@aragon.es Zaragoza, 8 de noviembre de 2010Patología digestiva
  2. 2. HCV life cycle: targets for STAT-C compoundsHepatology A clinical text book (2009)Mauss, Berg, Rockstroh, Sarrazin, WedemeyerIntroduction: Life cycle
  3. 3. Genomic organization of HCV and polyprotein processing l t i i HCV RNA p7 NS4A NS4B 5’ C E1 E2 NS2 NS3 NS5A NS5B 3’ Structural Proteins Non-Structural Proteins Polyprotein p7 NS4A NS4B C E1 E2 NS2 NS3 NS5A NS5B Cellular protease NS2-3 protease NS3 protease C E1 E2 NS2 NS3 NS5A NS5B capsid envelope p7 NS4A NS4B RNA-dependent RNA polymerase NS3 NS3 NS3 protease NTPase Cofactor helicaseIntroduction:The importance of searching drugs against Hepatitis C disease
  4. 4. NS3 protein Helicase NTPase ProteaseIntroduction: General Characteristics of the target molecule: NS3 protease
  5. 5. Weak points in NS3 protease• Catalytic active site Inhibiting of the enzymatic activity by competitive inhibitors blocking g y y y p g the active site• Allosteric binding site Inhibiting the enzymatic activity by non-competitive inhibitors hampering g y y y p p g NS3 activating conformational changes induced by pNS4A• Structural Zn+2 Ion binding site More complicated due to the strength of bindingIntroduction: Weak points
  6. 6. NS4A interacts with NS3 NS4A peptide (21-34 NS4A)NS3NS3/pNS4A Ser His Catalytic triad Asp NS3 is an allosteric proteinIntroduction: General Characteristics of the target molecule: NS3 protease
  7. 7. General goal: Identification and development of potent, selective and adaptive inhibitors of NS3 protease. Specific goals: We are interested in rational design and screening- g g based strategies for searching inhibitors. Integration of structural, genetic and thermodynamic information is needed. Steps:  Obtaining p g pure enzyme y  Protein characterization*  Screening of bioactive molecules against NS3 protease  Potential inhibitors characterization*  In vivo testing of inhibitors * (spectroscopy, calorimetry, crystallography, …)General Objectives
  8. 8. pET7-NS3 * NS3 protease from HCV genotype 1b J-strain E.coli More than 10mg protein/L culture * A kind gift from Dr. C. Steinkühler, Istituto di Ricerche Molecolare (IRBM), P.Angeletti, RomeMethods: Obtaining pure NS3 protease
  9. 9. Fluorometric activity assay HCV Protease Fl o escence Resonance Ene g T ansfe (FRET) Fluorescence Energy TransferFRET Substrate or Resonance Energy Transfer (RET) (RET S1) P1 Acceptor DonorAc - Asp - Glu - Asp(EDANS) - Glu - Glu - Abu - ψ - [COO] - Ala - Ser - Lys(DABCYL) - NH2 700000 600000 Fluorescence signal (AU) 500000 400000 300000 200000 -500 0 500 1000 1500 2000 2500 3000 3500 4000 time (s) Taliani, M. et al. Anal. Biochem. 240, 60 (1996)1- Kinetic method
  10. 10. Isothermal Titration Calorimetry (ITC) tim e (m in) 0 30 60 90 120 3.0 2.5 ML  ML dQ/d (cal/s) 2.0 1.5 Ligand 1.0 dt 0.5 0.0 10.0 8.0 Protein cal/mol) (NS3) 6.0 Ka 4.0 H Q (kc 2.0 n 0.0 0.0 00 0.5 05 1.0 10 1.5 15 2.0 20 2.5 25 3.0 30 [Ligand]T/[Macromolecule]T2- Calorimetric method
  11. 11. 4 OAV08 OAV06 0 -4 mol kcal/m -8 Δ G  Δ H  TΔS -12 -16 G H -TS G = H – TSconf – TSsolv – TStr Interaction P-L • van der Waals Entropy gain due to Entropy loss due to • hydrogen bonds release of water restricted conformational • de/protonation degrees of freedom molecules DesolvationPotential Inhibitors Characterization
  12. 12. Inhibitors of NS3 Protease High Throughput Screening (HTS)Inhibitors of NS3 protease HTS
  13. 13. High Throughput Screening (HTS) with a chemical library  Materials: Chemical Library: Thousand of potentials ligands (e.g. inhibitors)  Equipment: Plate-Reader Fluorimeter: FluoDia T70 Microbeam, S.A  Assay: NS3 + Library compound + FRET Substrate (RET S1)Inhibitors of NS3 protease HTS
  14. 14.  Result: Kinetic measurements: Fluorescence Signal Initial Slope (Vi )  Example: Kinetic example in a multi-plate experiment Raw data 900000 800000 700000 a.u) B8 ence signal (a 600000 B10 A10 500000 C8 A9 Fluoresce 400000 E8 C11 A11 300000 B3 200000 -500 0 500 1000 1500 2000 2500 3000 3500 4000 Time (s)Inhibitors of NS3 protease HTS
  15. 15. Analyzed data  Data processing: 400 u) Library compounds y p nitial rates (a.u Controls (Without ligand) Upper Limit2 200 Upper Limit1 Upper Limit2 Upper Limit1 In 0 A10 B8 C7 D5 E3 F1 G2 G12 H10 Wells  Vi (i iti l rate)= Initial slope (initial t ) I iti l l The lower the slope, the greater the inhibition  Control positive limits Average of control slopes (M) Standard deviation of control slopes (SD) Upper Limit (M+3SD) and Lower Limit (M-3SD)Inhibitors of NS3 protease HTS
  16. 16.  Final test: Selection of Best Compounds 2500 2000 tual rates(a.u) 1500 Controls 1000 Potencial Inhibitors Init 500 0 -500 Compounds  C fi i positive compounds Confirming iti d  Selecting the most promising compounds (15) OAV01-OAV02-OAV03-……-OAV15Inhibitors of NS3 protease HTS

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