Cribado, identificación y ensayos in vitro de nuevos compuestos inhibidores de la pproteasa NS3 del virus de la Hepatitis C

OLGA ABIANomabian.iacs@aragon.es

Zaragoza, 8 de noviembre de 2010Patología digestiva

HCV life cycle: targets for STAT-C compounds

Hepatology A clinical text book (2009)Mauss, Berg, Rockstroh, Sarrazin, Wedemeyer

Introduction: Life cycle

Genomic organization of HCV and l t i ipolyprotein processing

HCV RNA NS4BNS4Ap7

5’ 3’C E1 E2 NS3 NS5A NS5BNS2

Structural Proteins Non-Structural Proteins

Polyprotein NS4BNS4Ap7

C E1 E2 NS3 NS5A NS5BNS2

NS2-3 protease

C E1 E2 NS3 NS5A NS5BNS2

Cellular protease NS3 protease

NS2C E1 E2 NS3 NS5A NS5B

p7capsid envelope RNA-dependentRNA polymerase

NS3

NS4BNS4A

NS3protease

NS3NTPasehelicase

protease Cofactor

Introduction: The importance of searching drugs against Hepatitis C disease

NS3 proteinNS3 protein

HelicaseHelicaseNTPase

Protease

Introduction: General Characteristics of the target molecule: NS3 protease

Weak points in NS3 protease

• Catalytic active siteInhibiting of the enzymatic activity by competitive inhibitors blockingg y y y p gthe active site

• Allosteric binding siteInhibiting the enzymatic activity by non-competitive inhibitors hamperingg y y y p p gNS3 activating conformational changes induced by pNS4A

• Structural Zn+2 Ion binding siteMore complicated due to the strength of binding

Introduction: Weak points

NS4A interacts with NS3

NS4A peptide (21-34 NS4A)

NS3NS3/pNS4A

Ser

His

AspCatalytic triad

NS3 is an allosteric protein

Introduction: General Characteristics of the target molecule: NS3 protease

General goal:

Identification and development of potent, selectiveand adaptive inhibitors of NS3 protease.

Specific goals:

We are interested in rational design and screening-g gbased strategies for searching inhibitors.Integration of structural, genetic and thermodynamicinformation is needed.

Obtaining pure enzyme

Steps:

g p y Protein characterization* Screening of bioactive molecules against NS3 protease Potential inhibitors characterization* In vivo testing of inhibitors

* (spectroscopy, calorimetry, crystallography, …)

General Objectives

pET7-NS3*

NS3 protease from HCV genotype

1b J-strain

E.coliE.coli

* A kind gift from Dr. C. Steinkühler, Istituto di Ricerche Molecolare (IRBM), P.Angeletti, Rome

More than 10mg protein/L culture

Methods: Obtaining pure NS3 protease

Fl o escence Resonance Ene g T ansfe (FRET)

Fluorometric activity assayHCV Protease

FRET Substrate (RET S1)

Fluorescence Resonance Energy Transfer (FRET)or Resonance Energy Transfer (RET)

P1Donor Acceptor

700000

Ac - Asp - Glu - Asp(EDANS) - Glu - Glu - Abu - ψ - [COO] - Ala - Ser - Lys(DABCYL) - NH2

500000

600000

700000

sign

al (A

U)

200000

300000

400000

Fluo

resc

ence

Taliani, M. et al. Anal. Biochem. 240, 60 (1996)

-500 0 500 1000 1500 2000 2500 3000 3500 4000

200000

time (s)

1- Kinetic method

Isothermal Titration Calorimetry (ITC)

3.00 30 60 90 120

tim e (m in)

MLLM

1.0

1.5

2.0

2.5

dt (

cal/s

)

MLLM

Ligand

10.00.0

0.5

1.0

dQ/d

4.0

6.0

8.0

cal/m

ol)

H Ka

Protein(NS3)

0 0 0 5 1 0 1 5 2 0 2 5 3 0

0.0

2.0

Q (k

c

n

0.0 0.5 1.0 1.5 2.0 2.5 3.0

[Ligand]T/[Macromolecule]T

2- Calorimetric method

4 OAV08 OAV06

-4

0

mol

STΔHΔGΔ -12

-8

kcal

/m

-16 G H -TS

G = H – TSconf – TSsolv – TStr

Interaction P-L• van der Waals• hydrogen bonds

Entropy gain due to release of water

Entropy loss due torestricted conformational• de/protonation

Desolvation moleculesrestricted conformational degrees of freedom

Potential Inhibitors Characterization

Inhibitors of NS3 Protease HighInhibitors of NS3 Protease High Throughput Screening (HTS)

Inhibitors of NS3 protease HTS

High Throughput Screening (HTS)

Materials:

with a chemical library Materials:

Chemical Library: Thousand of potentials ligands (e.g. inhibitors)

Equipment: Equipment:

Plate-Reader Fluorimeter:

FluoDia T70 Microbeam, S.A

Assay: Assay:

NS3 + Library compound + FRET Substrate (RET S1)

Inhibitors of NS3 protease HTS

Result: Kinetic measurements: Fluorescence Signal Initial Slope (Vi ) Result: Kinetic measurements: Fluorescence Signal Initial Slope (Vi )

900000

Example:

Raw data

Kinetic example in a multi-plate experiment

700000

800000a.

u)

B8

Raw data

500000

600000

ence

sig

nal (

a B8 B10 A10 C8 A9E8

200000

300000

400000

Fluo

resc

e E8 C11 A11 B3

-500 0 500 1000 1500 2000 2500 3000 3500 4000

200000

Time (s)

Inhibitors of NS3 protease HTS

Data processing:

Analyzed data

processing:400

Library compoundsu)

200

y p Controls (Without ligand) Upper Limit2 Upper Limit1 Upper Limit2 Upper Limit1

nitia

l rat

es (a

.u

0

In

Vi (i iti l t ) I iti l l

A10 B8 C7 D5 E3 F1 G2 G12 H10

Wells

Vi (initial rate)= Initial slopeThe lower the slope, the greater the inhibition

Control positive limitsAverage of control slopes (M)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

Final test: Selection of Best Compounds

2000

2500

1000

1500

tual

rate

s(a.

u)

Controls Potencial Inhibitors

0

500Init

C fi i iti d

-500

Compounds

Confirming positive compounds

Selecting the most promising compounds (15)

OAV01-OAV02-OAV03-……-OAV15

Inhibitors of NS3 protease HTS