V National Conference BIFI, Zaragoza, Febrero 2011

V National Conference BIFI, Zaragoza, Febrero 2011

OLGA [email protected]

APPROCHES FOR ANTIVIRAL COMPOUND DEVELOPMENT

http://bifi.es/index.php


STEPS:

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.

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.


1º TARGET IDENTIFICATION

NS3 protein

Protease

HelicaseNTPase


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.


2º HIGH THROUGHPUT SCREENING (HTS)

Two ways for finding interesting molecules:

B/ THERMAL SHIFT SCREENING:

A/ ACTIVITY BASED-SCREENING :



A/ ACTIVITY BASED-SCREENING : A molecule inducing an effect must alter protein properties (function, behavior). Methods for detecting function or behavior are requiredOur protein target exhibits protease activity and it can be measured in vitro using a fluorescence (FRET) peptide substrate.



-500 0 500 1000 1500 2000 2500 3000 3500 4000

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Y A

xis

Titl

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X Axis Title

6B8 4B8 2A10 7C8 6A9 7E8 13C11 6A11 8B3

Example:

B8 D4 E12 G8

-600

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Slope

ResultsKinetic 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.

AssayNS3 + 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

A/ SCREENING ACTIVITY BASED:



B/ THERMAL SHIFT SCREENING: A molecule inducing an effect must bind to the protein. Methods for detecting binding are requiredIf a ligand binds preferentially to a certain protein conformational state, it stabilizes (increases the population of) such conformational stateTherefore, we can find ligands for a given protein by searching for molecules that increase the conformational stability of the proteinThere 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 case STABILIZATION OF THE UNFOLDED STATE AT pH5 is studied.


V National Conference BIFI, Zaragoza, Febrero 2011B/ SCREENING THERMAL SHIFT:

20 30 40 50 60 70

0

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Control NS3 sin Zn Control NS3 con Zn NS3 sin Zn + compX1 NS3 sin Zn + compX2 NS3 sin Zn + compX3 NS3 sin Zn + compX4 NS3 sin Zn + compX5 NS3 sin Zn + compX6

Flu

ore

sc

en

ce

(A

.U)

Temperature (ºC)

Assay: pH5, with/without EDTA


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 (-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.


4º In Vivo CELL STUDIES

Bartenschlager, Science 1999, 285(5424)110-3.


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.

0

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1 10 1000

50

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% A

cti

vid

ad

XT

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Concentracion compuesto (M)

% A

cti

vid

ad

luc

ife

ras

a Cell viability


5º EXPLORING NEW TRANSPORT AND SELECTIVE LIBERATION OF DRUGS

AuNP

AuNP1OAV

OAV

OAV

OAV

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

AuNP2

OAV

OAV

OAV

OAV

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

AuNP4A

OAV

OAV

OAV

OAV

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

AuNP4B

= Fabfragment

AuNP3

OAV

OAV

OAV

OAV

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

V

OA

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OA

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OA

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OA

V

= Internalization peptide

Selective transport

Selective recognition of infected cells

Entry in cells


FINANCIAL SUPPORTPROYECTOS NACIONALESTITULO 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: 2013CUANTÍA: 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: 2013CUANTÍA: 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: 2010CUANTÍA: 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 2010CUANTÍA: 12.700 €INVESTIGADOR PRINCIPAL: Dr. Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI)

PROYECTOS AUTONÓMICOSTITULO 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: 2011CUANTÍA: 52.000€INVESTIGADOR PRINCIPAL: Adrián Velázquez Campoy


THANK YOU FOR YOUR ATTENTION!!

V National Conference BIFI, Zaragoza, Febrero 2011

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