Plasmodium falciparum protein kinases:

from database mining to drug discovery

Inserm team (U609)

Wellcome Centre for Molecular ParasitologyUniversity of Glasgow

U609

Outline

1. Protein kinases

2. Atypical features of malarial protein kinases and signalling pathways:

- CDKs - cyclins - MAPK pathways- Kinome

3. Malarial protein kinases as drug targets

- target validation / functional studies- screening against recombinant targets- perspectives

outline

Protein kinases:

125 in yeast (ser/thr)520 in human (ser/thr + tyr) (approx 2% of genes)

Dysregulation associated with a number of pathologies (cancer, neurodegenerative diseases…)

Kinase inhibitors on the market (Gleevec) or in clinical trials

targets in parasitic diseases???

Introduction

Protein phosphorylation:

- involved in most cellular regulatory processes

- 30% of proteins are phosphoproteins

- major mechanism of signal transduction

I II IVIII V VIA VIB VII IXVIII X XI

GxGxxG K E HRDxxxxN RAPEDFG D

Human CDK2

N-ter lobe

C-ter lobe

ATP

Structure of Ser/Thr and Tyr protein kinases

ATPPP ADPP

PProtein kinase

Kinase assay

Introduction

Introduction

Baldauf, Science 300, 1703 (June 2003)

“…from yeast to man”

Plasmodium

Introduction

Outline

1. Protein kinases

2. Atypical features of malarial protein kinases and signalling pathways:

- CDKs - cyclins- MAPK pathways- Kinome

3. Malarial protein kinases as drug targets

- target validation / functional studies- screening against recombinant targets- perspectives

outline

CDK2

CDK2

CDK1CDK4

CDK6

CDK3

CDK1Cyclin E

Cyclin ?

Cyclin D

Cyclin D

Cyclin B

Cyclin A

Cyclin A

CDK7

Cyclin H

Eukaryotic cell cycle

receptor

pheromoneHeterotrimeric G-proteins

Ste20

Ste5 Ste11 (MAPKKK)Ste7 (MAPKK)Fus3 (MAPK)KSS1

CDKs

CDKs of P. falciparum

...and four potential cyclins

PfPK5 (Cdk1)T161

Pfcrk-1 (p58)T161AMTSLRE

Pfmrk (CDK7)T161NFVLLRE

PfPK6 (Cdk1/MAPK)T161KCILRE

Pfcrk-3 (Cdk1)T161AKTYIRE

Pfcrk-4 (Cdk1/MAPK)T161EEFAVNE

PSTTIRE

PSTAIRE T161 Cdk1

Cell cycle control elements

S G2

M

G1

CDK6 CDK4

CDK3

CDK2

CDK1CDK2

CDK1

CDK7

Cyclin H

Cyclin E

Cyclin ?

Cyclin D Cyclin D

Cyclin B

Cyclin A Cyclin A

CDKs

Pfmrkhistone H1

hcycH PfPK5

Activation of PfPK5 (CDK1) by human cyclin H

Pfmrk + hcycH

Pfmrk

Le Roch et al. (2000), JBC 275, 8952; Merckx et al., JBC (2003)

Relaxed cyclin specifity for PfPK5?

CAK-independent activation

Autophosphorylation of PfPK5

Human cyclin HPfcyc-1, -3p25RINGO (A. Nebreda)

Human cyclin A (J. Endicott)

PfPK5 + hcycH

PfPK5 + Pfmrk +hcycH

Histone H1

CDKs

sw|Q07785|CC2H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E... 592 e-169sw|P34117|CC2H_DICDI CDC2-LIKE SERINE/THREONINE-PROTEIN KINASE ... 411 e-115sw|P29618|CC21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 382 e-106sw|Q38772|CC2A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 379 e-105sw|P23111|CC2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 379 e-105sw|P24100|CC2A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 374 e-103sw|P52389|CC2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 372 e-103sw|P29619|CC22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 370 e-102sw|P43450|CDK2_CARAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 369 e-102sw|Q63699|CDK2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 367 e-101sw|O55076|CDK2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 367 e-101sw|P24941|CDK2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 366 e-101sw|P48963|CDK2_MESAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 366 e-101sw|Q05006|CC22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 365 e-101sw|P23437|CDK2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 360 2e-99sw|Q00526|CDK3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2.7.1.-). 356 2e-98sw|P24923|CC21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 356 4e-98sw|Q00535|CDK5_HUMAN CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-... 355 5e-98sw|Q02399|CDK5_BOVIN CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-... 355 7e-98sw|P51166|CDK5_XENLA CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-... 354 1e-97sw|P49615|CDK5_MOUSE CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-... 354 1e-97sw|P43063|CC28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 353 3e-97sw|Q38773|CC2B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B ... 353 3e-97sw|Q03114|CDK5_RAT CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-) ... 352 6e-97sw|P06493|CC2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 351 1e-96sw|P39951|CC2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2... 351 1e-96sw|P11440|CC2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 350 2e-96sw|P48734|CC2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 350 2e-96sw|P51958|CC2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 348 7e-96sw|P97377|CDK2_MOUSE CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 344 2e-94sw|P13863|CC2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 343 3e-94sw|P35567|CC21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 341 1e-93sw|P24033|CC22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 340 1e-93sw|P48609|CDK5_DROME CELL DIVISION PROTEIN KINASE 5 HOMOLOG (EC... 340 3e-93sw|P00546|CC28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 340 3e-93sw|Q06309|CRK1_LEIME CELL DIVISION PROTEIN KINASE 2 HOMOLOG CRK... 339 3e-93sw|P23572|CC2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 339 4e-93sw|P54664|CC21_TRYCO CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 338 1e-92sw|P34112|CC2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 335 5e-92sw|P04551|CC2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-... 335 9e-92sw|P54119|CC2_AJECA CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-... 333 3e-91sw|Q00646|CC2_EMENI CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-... 332 6e-91sw|P38973|CC21_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 332 6e-91sw|P17157|PH85_YEAST NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2... 328 6e-90sw|O35831|KPT2_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2 (E... 324 2e-88sw|P23573|CC2C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE (E... 324 2e-88sw|Q00537|KPT2_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2 ... 323 4e-88sw|Q92241|PH85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2... 320 2e-87sw|Q04899|KPT3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 ... 315 6e-86sw|O35832|KPT3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 (E... 314 2e-85sw|Q00536|KPT1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1 ... 312 7e-85sw|Q04735|KPT1_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1 ... 310 2e-84sw|Q07002|KPT3_HUMAN SERINE/THREONINE PROTEIN KINASE PCTAIRE-3 ... 307 1e-83sw|P34556|CC2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 305 5e-83sw|P54666|CC23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 3 ... 300 2e-81sw|P25859|CC2B_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B ... 293 2e-79sw|Q38774|CC2C_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG C ... 286 4e-77sw|Q38775|CC2D_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG D ... 284 1e-76sw|P54665|CC22_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 284 2e-76

PfPK5: CDK

sw|Q11179|YPC2_CAEEL PUTATIVE SERINE/THREONINE-PROTEIN KINASE C... 328 1e-89sw|P28869|ERK1_CANAL EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E... 254 2e-67sw|P27638|SPK1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE SPK1 (EC ... 254 2e-67sw|P42525|ERK1_DICDI EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E... 253 5e-67sw|Q39024|MPK4_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4... 251 2e-66sw|Q39025|MPK5_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5... 248 1e-65sw|Q07176|MMK1_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 247 2e-65sw|Q40353|MMK2_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 247 3e-65sw|P27361|MK03_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2.7... 247 3e-65sw|Q40531|NTF6_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 246 5e-65sw|P47812|MK14_XENLA MITOGEN-ACTIVATED PROTEIN KINASE 2 (EC 2.7... 246 5e-65sw|P21708|MK03_RAT MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2.7.1... 246 6e-65sw|Q06060|MAPK_PEA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D5 ... 245 8e-65sw|P26696|MK01_XENLA MITOGEN-ACTIVATED PROTEIN KINASE (EC 2.7.1... 245 1e-64sw|Q39026|MPK6_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6... 245 1e-64sw|Q39021|MPK1_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 243 3e-64sw|Q40532|NTF4_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 243 4e-64sw|Q39027|MPK7_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7... 243 4e-64sw|P39745|SUR1_CAEEL MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC... 243 5e-64sw|Q40517|NTF3_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 242 7e-64sw|P27703|MK01_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64sw|P28482|MK01_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64sw|P46196|MK01_BOVIN MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64sw|Q39022|MPK2_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2... 242 9e-64sw|Q40884|MAPK_PETHY MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 242 1e-63sw|Q63844|MK03_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2.7... 240 3e-63sw|Q00772|SLT2_YEAST MITOGEN-ACTIVATED PROTEIN KINASE SLT2/MPK1... 240 5e-63sw|P70618|MK14_RAT MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.7.... 239 8e-63sw|P47811|MK14_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63sw|Q16539|MK14_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63sw|Q90336|MK14_CYPCA MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63sw|O02812|MK14_CANFA MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 238 1e-62sw|Q39023|MPK3_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3... 238 2e-62sw|Q13164|MK07_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 7 (EC 2.7... 238 2e-62sw|O08666|MK11_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2.... 236 5e-62sw|P40417|ERKA_DROME MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC... 232 8e-61sw|Q92398|SPM1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE SPM1 (EC ... 231 2e-60sw|P43068|MKC1_CANAL MITOGEN-ACTIVATED PROTEIN KINASE MKC1 (EC ... 229 9e-60sw|P14681|KSS1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE KSS1 (EC ... 229 9e-60sw|Q63538|MK12_RAT MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.7.... 228 1e-59sw|P16892|FUS3_YEAST MITOGEN-ACTIVATED PROTEIN KINASE FUS3 (EC ... 228 1e-59sw|Q09892|STY1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE STY1 (EC ... 228 2e-59sw|Q15759|MK11_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2.... 228 2e-59sw|O08911|MK12_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.... 227 3e-59sw|P53778|MK12_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.... 225 1e-58sw|O15264|MK13_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 13 (EC 2.... 222 1e-57sw|Q92207|HOG1_CANAL MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 220 5e-57sw|P32485|HOG1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 217 4e-56sw|P36005|KKQ1_YEAST PROBABLE SERINE/THREONINE-PROTEIN KINASE Y... 213 4e-55sw|P45983|MK08_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2.7... 205 2e-52sw|P49187|MK10_RAT MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2.7.... 204 3e-52sw|P49185|MK08_RAT MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2.7.1... 204 3e-52sw|P53779|MK10_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2.... 201 1e-51sw|Q61831|MK10_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2.... 200 3e-51sw|P45984|MK09_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2.7... 199 6e-51sw|P49186|MK09_RAT MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2.7.1... 199 7e-51sw|P41808|SMK1_YEAST SPORULATION-SPECIFIC MITOGEN-ACTIVATED PRO... 199 1e-50sw|P29619|CC22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 194 3e-49

Pfmap-1: MAPK

sw|P43063|CC28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 194 3e-49sw|P23437|CDK2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 192 8e-49sw|Q39026|MPK6_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6... 190 2e-48sw|Q63699|CDK2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 190 2e-48sw|P23573|CC2C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE (E... 190 2e-48sw|P24941|CDK2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 190 3e-48sw|O55076|CDK2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 190 4e-48sw|P48963|CDK2_MESAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 188 1e-47sw|P43450|CDK2_CARAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 188 1e-47sw|Q39023|MPK3_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3... 187 3e-47sw|Q07176|MMK1_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 187 3e-47sw|Q40532|NTF4_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 186 6e-47sw|P54666|CC23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 3 ... 185 1e-46sw|Q06060|MAPK_PEA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D5 ... 185 1e-46sw|P24100|CC2A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 184 2e-46sw|P23111|CC2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 184 2e-46sw|P29618|CC21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 183 4e-46sw|P39951|CC2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2... 182 7e-46sw|Q38772|CC2A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 182 7e-46sw|P06493|CC2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 182 9e-46sw|P48734|CC2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 181 1e-45sw|P34117|CC2H_DICDI CDC2-LIKE SERINE/THREONINE-PROTEIN KINASE ... 181 2e-45sw|P52389|CC2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45sw|P11440|CC2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45sw|P34556|CC2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45sw|Q04770|CDK2_ENTHI CELL DIVISION PROTEIN KINASE 2 HOMOLOG (EC... 179 6e-45sw|Q05006|CC22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 179 7e-45sw|P29619|CC22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 178 1e-44sw|Q40517|NTF3_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 178 2e-44sw|Q39025|MPK5_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5... 178 2e-44sw|Q40884|MAPK_PETHY MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 178 2e-44sw|P13863|CC2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 178 2e-44sw|Q07785|CC2H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E... 177 2e-44sw|P24923|CC21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 177 3e-44sw|P24033|CC22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 176 5e-44sw|Q39027|MPK7_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7... 176 6e-44sw|P23572|CC2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 176 6e-44sw|P35567|CC21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 176 6e-44sw|Q39021|MPK1_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 175 8e-44sw|P51958|CC2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 175 8e-44sw|P00546|CC28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 175 1e-43sw|P04551|CC2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-... 174 2e-43sw|Q39022|MPK2_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2... 174 2e-43sw|P34112|CC2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 174 2e-43sw|Q00526|CDK3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2.7.1.-). 173 3e-43sw|Q40353|MMK2_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 173 4e-43sw|Q00532|KKIA_HUMAN SERINE/THREONINE-PROTEIN KINASE KKIALRE (E... 173 6e-43sw|P32485|HOG1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 172 7e-43sw|Q15131|CDKA_HUMAN CELL DIVISION PROTEIN KINASE 10 (EC 2.7.1.... 172 7e-43sw|Q39024|MPK4_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4... 171 1e-42sw|Q04899|KPT3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 ... 171 1e-42sw|P24788|KP58_MOUSE GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K... 171 2e-42sw|P39745|SUR1_CAEEL MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC... 171 2e-42sw|Q92241|PH85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2... 171 2e-42sw|Q40531|NTF6_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 171 2e-42sw|Q38773|CC2B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B ... 171 2e-42sw|O35832|KPT3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 (E... 170 3e-42sw|P21127|KP58_HUMAN GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K... 170 3e-42sw|Q00536|KPT1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1 ... 170 4e-42sw|P40417|ERKA_DROME MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC... 170 4e-42

PfPK6: CDK-MAPK?

CDKs

0

702.2

125102050100200500

HsCDK1SpCdc2PfPK5PfCRK4PfPK6MmErk2ScFus3PfMAP1PfcPKA

MAPKs

CDKs

PfCRK4 PfPK6

CDKs

MAP kinase

MAP kinase kinase

MAP kinase kinase kinase

Cell cycle control elements

S G2

M

G1

CDK6 CDK4

CDK3

CDK2

CDK1CDK2

CDK1

CDK7

Cyclin H

Cyclin E

Cyclin ?

Cyclin D Cyclin D

Cyclin B

Cyclin A Cyclin A

MAP kinase pathway

ras

RTK

PfPK6

Pfcrk-4 ?

?

CDKs

Aim : To identify components of MAPK modules.

receptor

pheromoneG-proteins

Ste20

Ste5 Ste11 (MAPKKK)Ste7 (MAPKK)Fus3 (MAPK)KSS1

F Y L K N C TXY Mammalian/yeast MAPK

(KKYVDGSN)12(IKEQ)5

F Y L K N C TXYPfmap-1

F Y L K N C TSHPfmap-2

Two MAP kinase homologues

MAPK pathway

Lane 1 2 3 4-5 6-7

100

600

200

400%

act

ivity

Activation of GST-Pfmap-2 by gametocyte extracts

Dorin et al. (1999), JBC 274, 29912.

Activity of recombinant Pfmap-2

Pfmap-2

MBP

MAPK pathway

TEY MAPK(ERK1/2)

SMANS MEK1/2

MEKK

Typical MAPK module

TSH

TDY

Pfmap-2

Pfmap-1

?

P. falciparum

Atypical MAPK pathway in Plasmodium

MAPK pathway

?

Can we use genomic databases to solve black boxes ?

?

MAPK pathway

From Hagai Ginsburg’s web site on Plasmodium metabolic pathways

(http://sites.huji.ac.il/malaria)

« Metabolic databases have been used to predict the pathways of several organisms from their genomes. These concepts were at the base of the construction of this site ».

…it works well for reconstruction ofmetabolic pathways...

MAPK pathway

BLAST analysis using whole-length MEK1 as a query No hit in PlasmoDB

BLAST analysis using 23 residues around the SMANS activation site 1 Hit!

30% Mm MEK1 IKLCDFGVS--GQLIDSMANSFVGTRSYMSPERLQGTHYSV--QSDIWSM : ::: : :.:::.: ::: : ::: : : :: : 100% Pfnek-1 AKIGDFGLSK-NIGIESMAHSCVGTPYYWSPELLLHETKSYDDKSDMWAL : ::::::: : :::: : ::: : :: ::42% NIMA VKLGDFGLSKLMHSH-DF.STYVGTPFYMSPEIC--AAEKSTLRSDIWAV

Query: 1 GVSGQL-IDSMANSFVGTRSYMSP 23 G+S + I+SMA+S VGT Y SPSubject: 591 GLSKNIGIESMAHSCVGTPYYWSP 662

SMAHS Pfnek-1

SMANS MEK1/2( )

MAPK pathway

TEY MAPK(ERK1/2)

SMANS MEK1/2

MEKK

Typical MAPK module

TSH

TDY

Pfmap-2

Pfmap-1

P. falciparum

SMAHS Pfnek-1

?

MAPK pathway

Pfmap-2

mERK2

Pfmap-2

mERK2Coomassie

Autoradiogram

Pfnek-1: + - + - +ERK2 Pfmap-2

Phosphorylation of Pfmap-2 by Pfnek-1

Dorin et al. (2001), EJB 268:2600-2608

MBP

map-2 nek-1nek-1 +map-2

Synergistic activities of Pfnek-1 and Pfmap-2

Atypical MAPK pathway in Plasmodium

Pfnek-1: - closely related to NIMA kinases; MAPKK–like activation site.

SMAHS Pfnek-1 (100%)

SMANS MEK1/2 (30%)

DxxT NIMA/Nek (42%)

MAPK pathway

Aim : To identify components of MAPK modules.

receptor

pheromoneG-proteins

Ste20

Ste5 Ste11 (MAPKKK)Ste7 (MAPKK)Fus3 (MAPK)KSS1

- Experimental (PCR) approaches- Targeted database mining (BLASTP)

No typical MAPKK homologues!

PlasmoDB database mining using a variety of tools

(HMM, BLAST, Gene Ontology prediction..)

Alignment, manual edition

Construction of a tree including members of defined families of eukaryotic protein kinases

- Genome-wide “kinome” characterisation(with P. Ward, WCMP, and J. Packer, Abbott laboratories)

Kinome

Features of the P. falciparum kinome:

• Only 65 „typical“ S/T protein kinases (124 in yeast)

• Many can be attributed to distinct families (35-60% identity) :

- 2 MAPKs, 6 CDK-related kinases, PKA, PKG, PKB...- at least 5 CDPKs (found also in plants and Ciliates, but not in

animal cells)

• Many do not cluster with previously defined kinase groups

• Several show features from distinct kinase families

• No TyrK, no PKC, no STE

Kinome

csnk1g1Pf110377 (PfCK1)

csnk1dpfb0150c

map2k1map3k3

map4k1pak1

pf140320pf110220

mal6p1191pf130258

tgfbr1Pfb0520w (PfRaf)

zaklimk1

raf1src

insregfr

pfi1280cpfi1290w

Mal6p1146 (PfPK4)pfa0380w

pf140423pfl0080c

mal6p156Pfl1370w (Pfnek-1)

nek7Pfe1290w

nek1Mal7p1100 (Pfnek-4)

Mal7p191 (PfEST)pf110488

mal13p1196Pfc0755c (Pfcrk-4)

Mal6p1271 (Pfcrk-5)Pf130206 (PfPK6)

Pfd0740w (Pfcrk-3)Mal13p1279 (PfPK5)

cdk2Pf100141 (Pfmrk)

Pfd0865c (Pfcrk-1)Pf110147 (Pfmap-2)

Pf140294 (Pfmap-1)mapk1

Pf110096 (PfCK2)Pf080044 (PfPK1)

mal13p184Pfc0525c (PfGSK3)

gsk3bpf110156

Pf140431(Pflammer)clk1

pfc0105wpf140408

pf140392mal7p173

Pfl1885c (PfPK2)pf110242

Pfb0815w (PfCDPK1)pf070072

Pf130211 Mal6p1108 (PfCDPK2)

Pfc0420w (PfCDPK3)mapkapk2

camk2acamk1

dapk1pf110239

pf140227pfc0485w

pfb0665wmal7p118

pf110060pf110464

pfc0385cmal13p1278

pf110227Pf140346 (PfPKG)

rock1Pfl2250c

prkcaakt1

prkacaPfi1685w (PfPKA)

Pf140516 (PfKIN)Pf140476

pf130085pfi1415w

Pfb0605w (PfPK7) Pfl2280w

AGC

Camk

CMGC

NimA

Tyrosine kinase

Ste7/11/20

Casein kinase 1

Tyrosine kinase-like

Kinome

I II IVIII V VIA VIB VII IXVIII X XI

GxGxxG K E HRDxxxxN RAPEDFG D

A novel, Apicomplexa-specific family of PK-like proteins

W FIKKxxxxxWGExxxxxxNxxMExxxxxxL

LHxxGxxHLDxxxxNxL

CD[FL][GA]xxxxxPxY

PxPEYxxVxxxDxxM

WP LxxxxxR PW

• 20 genes in P. falciparum

• 1 gene in P. vivax and P. berghei• 1 gene in Toxoplasma gondii and Cryptosporidium• Not found so far in any non-apicomplexan organism

• Closest relatives (by BLASTP !): MAPKs

Kinome

PfEMP1 (var) rifin FIKK

Kinome

the organisation of Plasmodium regulatory pathways cannot be predicted from sequence databases by analogy to those in higher Eukaryotes.

Kinome

Enzyme A

Enzyme B

Enzyme C

Reconstructing metabolic pathways

metabolitesModel eukaryote Plasmodium

Kinome

not recognisable

recognisable

Problems in reconstructing regulatory pathways:

Kinome

Holton et al., Structure (2003)

Kinome

Outline

1. Protein kinases

2. Atypical features of malarial protein kinases and signalling pathways:

- CDKs- cyclins- MAPK pathways- Kinome

3. Malarial protein kinases as drug targets

- target validation / functional studies- screening against recombinant targets- perspectives

outline

Malaria parasites: - Extremely low transfection rate- No inducible expression system yet

PyrR

targeted gene

PyrS

Pyr selection

PyrR

Check for the presence of the desired genotype (PCR)

Clone parasites

Analyse phenotype

Kinases as drug targets

Validation by gene knock-out

Pst I

MscIMscI

MscI

hDHFR

MscI MscI

1734

202 1027

Pbcrk-1

Kinases as drug targets

episome

WT

Pbcrk-1 cannot be knocked-out…

Rangajaran et al., submitted

MscI

Homologous recombination

802XhoI

hDHFR

hDHFR

1734

1734

NdeINdeI

NdeI

NdeINdeI

Kinases as drug targets

episome

WT

recombinant

Pbcrk-1

…but the Pbcrk-1 locus is amenable to genetic modification.

Rangajaran et al., submitted

Homologous recombination

Problem: detailed studies of the phenotype are impossible using KO approach.

a possible solution: chemical genetics ?

Wild-type kinase

ATP

Inhibitor

ATP

Inhibitor

Large gatekeeper: F, L... Small gatekeeper: G, A...

Mutated kinase

INACTIVEACTIVE

Validation by chemical genetics

Kinases as drug targets

338

V-Src 332-EPIYIVIEYMSK--GSLLDFLKGEM-C-Fyn 333-EPIYIVTEYMNK--GSLLDFLKDGE-C-Abl 309-PPFYIITEFMTY--GNLLDYLRECN-Cdk2 83-NKLYLVFEFLHQ---DLKKFMDASA-CDC28 92-HKLYLVFEFLDL---DLKRYMEGIP-CAMKII 84-GHHYLIFDLVTG--GELFEDIVARE-FUS3 87-NEVYIIQELMQT---DLHRVISTQM-

Pfmap-2 171-DELYIVLEIADS---DLKKLFKTPI-Pfmap-1 107-NDIYLIFDFMET---DLHEVIKADL-PfPK5 73-KRLVLVFEHLDQ---DLKKLLDVCE-PfPK6 109-SCLYLAFEYCDI---DLFNLIKKHN-PfGSK-3 151-IFLNVVMEYIPQ---TVHKYMKYYS-Etc...

Bishop et al., Nature 407:395-401

3. Parasite PKs as targets: validation

0 1 10 1001000

10000

Histone H1

nM 1 N-M-PP1

His-PfPK5 WT His-PfPK5 F79G

100%

50

100%

50

0 1 10 1001000

10000

Selective inhibition of mutagenised PfPK5-Pfcyc-1 by N-M-PP1

Validation by chemical genetics

Kinases as drug targets

Next: allele replacement in the parasite

next: - selectivity studies- cellular effects of the hits

Kinases as drug targets

0%10%20%30%40%50%60%70%80%90%

100%

0,01 0,1 1 10 100Concentration (µM)

Paullone KenpaulloneGwennpaullone Alsterpaullone

A

Preliminary inhibition assay (PfGSK3).

Plate 38, 10 µM

0102030405060708090

100110120130140150

0 10 20 30 40 50 60 70 80 90

Well number

Medium-throughput screening

Chemical library screening

Perspectives --Rational drug design

Nu6102 binding to PfPK5 and Cdk2• Mode of binding highly conserved from Cdk2

• Subtle differences in ribose binding site may afford greater potency for PfPK5 selective inhibitors

•IC50: PfPK5, 215 nM CDK2, 5 nM

Holton et al., Structure (2003)Kinases as drug targets

In brief:

1. Parasite protein kinases display many divergences relative to their mammalian homologues.

2. Peculiarities of the malarial kinome:

- absence of three-component MAPK modules- a novel family of kinase-related molecules- several „orphan“ kinases/groups (incl. „hybrid“ kinases)

3. Antimalarial drug discovery based on protein kinase inhibitors:

- Screening on recombinant kinases is ongoing- Rational drug design in progress- Chemical genetics as a possible approach for functional

studies

AcknowledgementsJane Endicott, Univ. of OxfordKavita Shah, Novartis, San DiegoLaurent Meijer, CNRS, Roscoff/RockefellerDebopam Chakrabarti, UCF, OrlandoRobert Menard, Institut PasteurAli Sultan, Harvard School of Public

HealthJeremy Packer, Abbott Laboratories

WCMP-INSERM U609:Pauline WardDominique DorinKarine Le RochAnais MerckxLeila EquinetMarie-Paule NivezLuc ReinigerCaroline Doerig