Targeting the Leishmania kinome for the development of novel anti-parasitic strategies

13 Institutions from 8 countries

LSHTM

UR2CSIC UPF

CNRS

PHX

TUBS

UVIC

IPM

IPT IIT IPK

14 teams and 63 scientists / 3M€ funding with 16 positions

LSHTM

UR2CSIC UPF

CNRS

PHX

TUBS

Steering Committee

(SC)SD + 6 WP Leaders

Steering Committee

(SC)SD + 6 WP Leaders

Coordinator, IP

Scientific Director (SD)G. Spaeth, IP

Coordinator, IP

Scientific Director (SD)G. Spaeth, IP

WP7:Managt /Communication

WP Leader: G.Spaeth, IP

Structure of the LEISHDRUG consortium

WP 1 :Development of relevant host-cell-based screening methodologies

WP Leader: S. Shorte, IP

WP 1 :Development of relevant host-cell-based screening methodologies

WP Leader: S. Shorte, IP

WP2 : SMT screen of a small molecules library using recomb. Leish-mania kinases

WP Leader:L. Meijer

WP2 : SMT screen of a small molecules library using recomb. Leish-mania kinases

WP Leader:L. Meijer

WP3 :Sequence-based MAPK inhibitors and peptide carriers for drug deluvery

WP Leader:L. Rivas, CSIC

WP3 :Sequence-based MAPK inhibitors and peptide carriers for drug deluvery

WP Leader:

WP 5 : Identification of atypical Leishmaniakinases and kinasestargeted by lead compounds

WP Leader :D. Zilberstein , IIT

WP 6: SAR analysis for leads and their targets by structure analyses and bio -informatics

WP Leader:C.Notredame , CRG

WP 4: Validation of novel anti-leishmanialcompounds in models of infection

WP Leader:S. Croft , LSHTM

Cluster 1 Cluster 2 Cluster 3

screening evaluation target ident.

European CommissionScientific (or Project) OfficerEuropean CommissionScientific (or Project) Officer

Scientific Advisory Board Scientific Advisory Board

Public ScientificCommunity

LEISHDRUG concept

DifferentiationpH726°C

pH537°C

Protein kinases

Phospho-protein substrates

Stage-specific targets

LEISHDRUG objectives

to screen small molecule and peptide libraries for hit compounds with leishmanicidal activity using phenotype- and target-based strategies.

to identify anti-parasitic lead compounds and assess their pharmacokinetic profiles using cell-culture and experimental infection models.

to identify parasite-specific drug targets usingproteomics approaches and initiate lead optimization by structure-based drug design.

Hit definition

Go/No-Go Strategies Axis I

Kinase biasedlibraryGO:

WP1WP2

WP3WP4

Inhibitorlibraries

DruggabilitySynthesis

WP1WP2Evaluate

chemistry

IC50 ~ 1 µMLow toxicity

WP4

-Anti

In vivoToxicology

Activity on kinase

WP5

Targetidentification

Inhibition of Leishmania spp

Identify hit compounds

WP1WP2

WP3WP4

Leads

WP6Target

structure

StructureKinase/compound

set up in situ screen

WP1

IPP

IPK

BMM / L. amazonensis

THP1 / L. donovani

LEISHDRUG screening strategies

Kinase- biasedcompound libraries

Axis I: In situ drug screening

(Semi)-conservedLeishmania kinases

(MAPKs)

Genetic analysis

Stage-specificphosphorylation

events (phosphoproteomics)

Kinase screen

Druggable Leishmania protein kinases

Axis II: Target-based screening

WP1: Geneviève Milon and Eric Prina, Institut Pasteur, Paris

Inhibitorlibraries

Evaluatechemistry -Anti

In vivoToxicology

Targetidentification

Identify hit compounds

Leadcompound

Target structure

In situ screen (WP1)

IP chimiothèque (> 35000 compounds)

CNRS Roscoff (WP2) (1520 kinase-biased compounds)

FP6 PRO-KINASERESEARCH (960 kinase-biased compounds)

CPP-phosphopeptideMimics (WP3, WP5)

LeuOMe

High

Low

cell

surv

ival

uninfected macrophages

infected macrophages

Negative control (no compound)Positive control (amphotericin B)

-2

-1

0

1

2

-4 -3 -2 -1 0 1

Host cell survival rate

Vacuole/Cell

Compound

assaydevelopment

hitdiscovery

WP2

Recomb. kinase phosphorylatesrec. substrate

IC50 in Mrange, good

ligand efficiency

hitvalidation

WP1

hit tolead

WP4

No host cell toxicity, kills in- tracell. parasites

structuredetermination

WP6

Active sitesdistinct fromhost kinases

WP5 WP3

kinaseidentification

geneticvalidation

Kinase essential for growthGO: Active in

amastigotes

Go/No-Go Strategies Axis II

Genomics Proteomics

WP5 WP6

MPK4

MPK7

MPK10

CK1

20PKs

assaydevelopment

hitdiscovery

hitvalidation

structuredetermination

kinaseidentification

geneticvalidation

Genomics Proteomics

Genomics (WP6)Phosphoproteomics (WP5)

Morales et al. Proteomics 2008, Morales et al., PNAS 2010, hem et al., Proteomics 2010

Qualtitative and quantitative gel-based and LC-basedphosphoproteomics revealed > 600 phospho-

proteins and > 900 phosphorylation sites.

LinJ33_V3.0350 (hsp83)

Kinomics (WP5)

pH4 pH7 MW

pH4 pH7 MW

2d In-gel kinase assay

assaydevelopment

hitdiscovery

hitvalidation

structuredetermination

kinaseidentification

geneticvalidation

Genomics Proteomics

X-ray analysis (WP6)

LmaMPK10 (1.9Å)

In silico (WP6)

Using motifs for ligand prediction

7 1628

assaydevelopment

hitdiscovery

hitvalidation

structuredetermination

kinaseidentification

geneticvalidation

Genomics Proteomics

-casein

MBP

MEK1

MPK10 MPK10K/A5.5 6.5 7.5 8.0 5.5 6.5 7.5 8.0

autoauto

LmaMPK10 (WP2, WP5)

3710 compounds (NCI, Prestwick, ChemDiv)

LmaCK1 (WP2)

Workflow the LEISHDRUG consortium

Hit-to-target identification

In situ activity of target-based hits

Hit-to-target identificationHit-to-target Identification (WP2/5)

In situ activity of target-based hitsIn situ activity of target-based hits

In vitro/in vivo validation,pharmacokinetics (WP4)In vitro/in vivo validation,pharmacokinetics (WP4)

Compound library (WP1)Small molecule library (WP2)

Peptide library (WP3)

Compound library (WP1)Small molecule library (WP2)

Peptide library (WP3)

Hit compounds

CombinatorialChemistry

(WP2)

CombinatorialChemistry

(WP2)

Structure -basedDrug design (WP6)

Structure -basedDrug design (WP6)

Phenotype-basedScreen (WP1)

In situ identification of compoundsthat affect intra-

cellular Leishmaniasurvival

Phenotype-basedScreen (WP1)

In situ identification of compoundsthat affect intra-

cellular Leishmaniasurvival

Target-basedScreen (WP2)

In vitro identification of compounds

that inhibit the activityof recombinant

kinases

Target-basedScreen (WP2, WP3)

In vitro identification of compounds

that inhibit the activityof recombinant

kinases

Develop medium-highthroughput screen

Develop medium-highthroughput screen

Phospho-proteomics (WP5)Activity-based screen (WP5)Phospho-proteomics (WP5)Activity-based screen (WP5)

Bio-informatics (WP6)

P44

P47P48P49P50P51

P45P46P52P58

P53P54

P55P57

P56

Dissemination activities• 2 Web sites

(www.leishdrug.org; FP7 Leishdrug wiki page)

• 13 poster and oral presentations (+15)

• 9 reports already published or in press (PNAS, Proteomics, PLoS NTD, PLoS One, BMC Bioinformatics, NAR, TiP, … )

• 1 Book project on LEISHDRUG (Springer)

• 1 International Symposium (www.pasteur.fr/eu-conference-neprodi)

FP7 trypanosomatid initiatives

KALADRUG-R: New tools for monitoring drug resistance and treatment

TRYPOBASE : Nucleobase derivatives as drugs against trypanosomal diseases

LEISHDRUG: Targeting the Leishmania kinome for drug development

Screening Hit-to-leadHits Leads

crystallo-graphy

molecularmodelling

pharmaco-kinetics andtoxicity

combinatorialchemistry

improved anti-trypanosomatidchemotherapy

compoundlibraries

TRYPOBASE

LEISHDRUG

KALADRUG-RGuard

Currentdrugs

pharmaco-vigilance

drug resistancemarkers

epidemiologicaldynamics

effectiveness

genomediversity

TiP 2010

Towards a future TrypNet?

ChagasEpiNetComparative epidemiology

of genetic lineages of Trypanosoma cruz

LeishDNAvaxNanoTrypDevelopment of

new tools to control infections due to parasites of the

Trypanosomatidae family

RAPSODY

Diagnostics Epidemiology VaccinationDrugs

LeishDrug

KalaDrug-R

TrypBaseNucleobase derivatives

as drugs against trypanosomal diseases

Targeting the Leishmaniakinome for drug development

Development of a DNA vaccine for

visceral leishmaniasis

Pre-clinical studies of a PSA-based human

vaccine candidate targeting all forms of

Leishmaniasis New tools for monitoring drug resistance and

treatment

Prevention, treatment, and monitoring of trypanosomatid diseases

ChagasEpiNet

LeishDNAvaxNanoTryp

RAPSODY

KalaDrug-R

TrypBase

LeishDrug

Thank you

assaydevelopment

hitdiscovery

hitvalidation

structuredetermination

kinaseidentification

geneticvalidation

Genomics Proteomics

Peptide mimics (WP3) Conditional KO analysis (WP5)

WT + pXNG+MPK4

3 passages in GCV

MPK4 TK

GFPSAT

MPK4MPK4

HYGPAC

MPK4 TK

GFPSAT

3 passages in GCV

Concentration (µM)

Signal

MKKK MKK MAPK growthP

P

Signal

MKKK MKK MAPK deathP

P