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

1. Cribado, identificación y ensayos in vitro
de nuevos compuestos inhibidores de la
p
proteasa NS3 del virus de la Hepatitis C
OLGA ABIAN
omabian.iacs@aragon.es
Zaragoza, 8 de noviembre de 2010
Patología digestiva

2. HCV life cycle: targets for STAT-C
compounds
Hepatology A clinical text book (2009)
Mauss, Berg, Rockstroh, Sarrazin, Wedemeyer
Introduction: Life cycle

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
helicase
Introduction:
The importance of searching drugs against Hepatitis C disease

4. NS3 protein
Helicase
NTPase
Protease
Introduction: General Characteristics of the target molecule: NS3 protease

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 binding
Introduction: Weak points

6. NS4A interacts with NS3
NS4A peptide (21-34 NS4A)
NS3
NS3/pNS4A
Ser
His Catalytic triad
Asp
NS3 is an allosteric protein
Introduction: General Characteristics of the target molecule: NS3 protease

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. 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, Rome
Methods: Obtaining pure NS3 protease

9. Fluorometric activity assay
HCV Protease Fl o escence Resonance Ene g T ansfe (FRET)
Fluorescence Energy Transfer
FRET Substrate or Resonance Energy Transfer (RET)
(RET S1)
P1 Acceptor
Donor
Ac - 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. 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]T
2- Calorimetric method

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
Desolvation
Potential Inhibitors Characterization

12. Inhibitors of NS3 Protease High
Throughput Screening (HTS)
Inhibitors of NS3 protease HTS

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.  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. 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.  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-……-OAV15
Inhibitors of NS3 protease HTS