Plasmodium falciparum protein kinases:
from database mining to drug discovery
Inserm team (U609)
Wellcome Centre for Molecular Parasitology
University 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 phosphorylation:
- involved in most cellular regulatory processes
- 30% of proteins are phosphoproteins
- major mechanism of signal transduction
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
Structure of Ser/Thr and Tyr protein kinases
GxGxxG
K
E
I
II
III
HRDxxxxN
IV
V
VIA
VIB
DFG
APE
D
VII
VIII
IX
R
X
XI
Kinase assay
N-ter lobe
Protein kinase
ATP
P
C-ter lobe
ATPPP
Human CDK2
ADPP
Introduction
Introduction
“…from yeast to man”
Plasmodium
Baldauf, Science 300, 1703 (June 2003)
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
Eukaryotic cell cycle
pheromone
Heterotrimeric G-proteins
receptor
CDK7
Cyclin H
Ste20
Ste5
KSS1
Ste11
Ste7
Fus3
CDK4
Cyclin D
CDK1
Cyclin B
(MAPKKK)
(MAPKK)
(MAPK)
CDK6
Cyclin D
CDK3
Cyclin ?
Cyclin A
CDK1
CDK2
Cyclin E
CDK2
Cyclin A
CDKs
CDKs of P. falciparum
PSTAIRE
T161
Cdk1
PSTTIRE
T161
PfPK5
(Cdk1)
AKTYIRE
T161
Pfcrk-3 (Cdk1)
KCILRE
T161
PfPK6
(Cdk1/MAPK)
Cyclin E
Cyclin A
Cyclin A
CDK2
CDK1
S
CDK2
M
Pfcrk-4 (Cdk1/MAPK)
CDK1
CDK3
CDK6
Cyclin D
T161
Cyclin B
G1
Cyclin ?
EEFAVNE
G2
CDK4
Cyclin D
CDK7
Cyclin H
Cell cycle control elements
AMTSLRE
T161
Pfcrk-1 (p58)
NFVLLRE
T161
Pfmrk
(CDK7)
...and four potential cyclins
CDKs
Activation of PfPK5 (CDK1) by human cyclin H
Pfmrk + PfPK5 +
hcycH
hcycH
Pfmrk
hcycH
Pfmrk
histone H1
Relaxed cyclin specifity for PfPK5?
CAK-independent activation
PfPK5 +
Pfmrk +
hcycH
PfPK5
Histone H1
Human cyclin H
Pfcyc-1, -3
p25
RINGO (A. Nebreda)
Human cyclin A (J. Endicott)
Autophosphorylation of PfPK5
Le Roch et al. (2000), JBC 275, 8952; Merckx et al., JBC (2003)
CDKs
PfPK5: CDK
Pfmap-1: MAPK
PfPK6: CDK-MAPK?
sw|Q07785|CC2H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E... 592 e-169
sw|P43063|CC28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 194 3e-49
sw|Q11179|YPC2_CAEEL
PUTATIVE SERINE/THREONINE-PROTEIN KINASE C... 328 1e-89sw|P23437|CDK2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 192 8e-49
sw|P34117|CC2H_DICDI CDC2-LIKE SERINE/THREONINE-PROTEIN KINASE
... 411 e-115
sw|P28869|ERK1_CANAL
EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E... 254 2e-67
sw|P29618|CC21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
1 ... 382 e-106
sw|Q39026|MPK6_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6... 190 2e-48
sw|P27638|SPK1_SCHPO
MITOGEN-ACTIVATED PROTEIN KINASE SPK1 (EC ... 254 2e-67 sw|Q63699|CDK2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 190 2e-48
sw|Q38772|CC2A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
A ... 379 e-105
EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E... 253 5e-67sw|P23573|CC2C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE (E... 190 2e-48
sw|P23111|CC2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|P42525|ERK1_DICDI
(EC... 379 e-105
sw|Q39024|MPK4_ARATH
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4... 251 2e-66
sw|P24100|CC2A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
A ... 374 e-103
sw|P24941|CDK2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-... 190 3e-48
sw|Q39025|MPK5_ARATH
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5... 248 1e-65
sw|P52389|CC2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 372 e-103
sw|O55076|CDK2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 190 4e-48
sw|Q07176|MMK1_MEDSA
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 247 sw|P48963|CDK2_MESAU
2e-65
sw|P29619|CC22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
2 ... 370 e-102
CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 188 1e-47
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 247 sw|P43450|CDK2_CARAU
3e-65
sw|P43450|CDK2_CARAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-).sw|Q40353|MMK2_MEDSA
369 e-102
CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). 188 1e-47
MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2.7... 247 3e-65 sw|Q39023|MPK3_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3... 187 3e-47
sw|Q63699|CDK2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). sw|P27361|MK03_HUMAN
367 e-101
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 246 5e-65
sw|O55076|CDK2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-). sw|Q40531|NTF6_TOBAC
367 e-101
sw|Q07176|MMK1_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 187 3e-47
sw|P47812|MK14_XENLA
MITOGEN-ACTIVATED PROTEIN KINASE 2 (EC 2.7... 246 5e-65 sw|Q40532|NTF4_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 186 6e-47
sw|P24941|CDK2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-...
366 e-101
MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2.7.1... 246 6e-65 sw|P54666|CC23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 3 ... 185 1e-46
sw|P48963|CDK2_MESAU CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-).sw|P21708|MK03_RAT
366 e-101
sw|Q06060|MAPK_PEA
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D5 ... 245 8e-65
sw|Q05006|CC22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
2 ... 365 e-101
sw|Q06060|MAPK_PEA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D5 ... 185 1e-46
MITOGEN-ACTIVATED PROTEIN KINASE (EC 2.7.1... 245 1e-64 sw|P24100|CC2A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 184 2e-46
sw|P23437|CDK2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-...sw|P26696|MK01_XENLA
360 2e-99
sw|Q39026|MPK6_ARATH
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6... 245 1e-64
sw|Q00526|CDK3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2.7.1.-).
356 2e-98
sw|P23111|CC2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 184 2e-46
sw|Q39021|MPK1_ARATH
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 243 3e-64
sw|P24923|CC21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
1 ... 356 4e-98
sw|P29618|CC21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 183 4e-46
sw|Q40532|NTF4_TOBAC
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 243 4e-64
sw|Q00535|CDK5_HUMAN CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-...
355 5e-98
sw|P39951|CC2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2... 182 7e-46
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7... 243 4e-64
sw|Q02399|CDK5_BOVIN CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-...sw|Q39027|MPK7_ARATH
355 7e-98
sw|Q38772|CC2A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A ... 182 7e-46
MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC... 243 5e-64 sw|P06493|CC2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 182 9e-46
sw|P51166|CDK5_XENLA CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-...sw|P39745|SUR1_CAEEL
354 1e-97
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 242 7e-64
sw|P49615|CDK5_MOUSE CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-...sw|Q40517|NTF3_TOBAC
354 1e-97
sw|P48734|CC2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 181 1e-45
sw|P27703|MK01_MOUSE
MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64 sw|P34117|CC2H_DICDI CDC2-LIKE SERINE/THREONINE-PROTEIN KINASE ... 181 2e-45
sw|P43063|CC28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1...
353 3e-97
sw|P28482|MK01_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64 sw|P52389|CC2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45
sw|Q38773|CC2B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
B ... 353 3e-97
MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2.7... 242 7e-64 sw|P11440|CC2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45
sw|Q03114|CDK5_RAT CELL DIVISION PROTEIN KINASE 5 (EC 2.7.1.-) ... sw|P46196|MK01_BOVIN
352 6e-97
sw|Q39022|MPK2_ARATH
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2... 242 9e-64
sw|P06493|CC2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
(EC... 351 1e-96
sw|P34556|CC2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 180 3e-45
sw|Q40884|MAPK_PETHY
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 242 1e-63
sw|P39951|CC2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC
2... 351 1e-96
sw|Q04770|CDK2_ENTHI CELL DIVISION PROTEIN KINASE 2 HOMOLOG (EC... 179 6e-45
sw|Q63844|MK03_MOUSE
MITOGEN-ACTIVATED
PROTEIN
KINASE
3
(EC
2.7...
240
3e-63
sw|P11440|CC2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 350 2e-96
sw|Q05006|CC22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 179 7e-45
MITOGEN-ACTIVATED PROTEIN KINASE SLT2/MPK1... 240 5e-63sw|P29619|CC22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 178 1e-44
sw|P48734|CC2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|Q00772|SLT2_YEAST
(EC... 350 2e-96
MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.7.... 239 8e-63 sw|Q40517|NTF3_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 178 2e-44
sw|P51958|CC2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|P70618|MK14_RAT
(EC... 348 7e-96
MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63 sw|Q39025|MPK5_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5... 178 2e-44
sw|P97377|CDK2_MOUSE CELL DIVISION PROTEIN KINASE 2 (EC 2.7.1.-).sw|P47811|MK14_MOUSE
344 2e-94
MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63 sw|Q40884|MAPK_PETHY MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 178 2e-44
sw|P13863|CC2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|Q16539|MK14_HUMAN
(EC... 343 3e-94
sw|Q90336|MK14_CYPCA
MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 239 8e-63 sw|P13863|CC2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 178 2e-44
sw|P35567|CC21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 341 1e-93
sw|O02812|MK14_CANFA
MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2.... 238 1e-62 sw|Q07785|CC2H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E... 177 2e-44
sw|P24033|CC22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
2 ... 340 1e-93
MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3... 238 2e-62
sw|P48609|CDK5_DROME CELL DIVISION PROTEIN KINASE 5 HOMOLOGsw|Q39023|MPK3_ARATH
(EC... 340 3e-93
sw|P24923|CC21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 177 3e-44
sw|Q13164|MK07_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 7 (EC 2.7... 238 2e-62 sw|P24033|CC22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 176 5e-44
sw|P00546|CC28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1...
340 3e-93
MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2.... 236 5e-62 sw|Q39027|MPK7_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7... 176 6e-44
sw|Q06309|CRK1_LEIME CELL DIVISION PROTEIN KINASE 2 HOMOLOG sw|O08666|MK11_MOUSE
CRK... 339 3e-93
sw|P40417|ERKA_DROME
MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC... 232 8e-61
sw|P23572|CC2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
(EC... 339 4e-93
sw|P23572|CC2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 176 6e-44
sw|Q92398|SPM1_SCHPO
MITOGEN-ACTIVATED PROTEIN KINASE SPM1 (EC ... 231 2e-60sw|P35567|CC21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1 ... 176 6e-44
sw|P54664|CC21_TRYCO CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
1 ... 338 1e-92
MITOGEN-ACTIVATED PROTEIN KINASE MKC1 (EC ... 229 9e-60
sw|P34112|CC2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG sw|P43068|MKC1_CANAL
(EC... 335 5e-92
sw|Q39021|MPK1_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1... 175 8e-44
MITOGEN-ACTIVATED PROTEIN KINASE KSS1 (EC ... 229 9e-60 sw|P51958|CC2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 175 8e-44
sw|P04551|CC2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-...sw|P14681|KSS1_YEAST
335 9e-92
MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.7.... 228 1e-59 sw|P00546|CC28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2.7.1... 175 1e-43
sw|P54119|CC2_AJECA CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-...sw|Q63538|MK12_RAT
333 3e-91
MITOGEN-ACTIVATED PROTEIN KINASE FUS3 (EC ... 228 1e-59 sw|P04551|CC2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-... 174 2e-43
sw|Q00646|CC2_EMENI CELL DIVISION CONTROL PROTEIN 2 (EC 2.7.1.-...sw|P16892|FUS3_YEAST
332 6e-91
sw|Q09892|STY1_SCHPO
MITOGEN-ACTIVATED PROTEIN KINASE STY1 (EC ... 228 2e-59 sw|Q39022|MPK2_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2... 174 2e-43
sw|P38973|CC21_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
1 ... 332 6e-91
sw|Q15759|MK11_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2.... 228 2e-59 sw|P34112|CC2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC... 174 2e-43
sw|P17157|PH85_YEAST NEGATIVE REGULATOR OF THE PHO SYSTEM (EC
2... 328 6e-90
MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.... 227 3e-59 sw|Q00526|CDK3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2.7.1.-). 173 3e-43
sw|O35831|KPT2_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2sw|O08911|MK12_MOUSE
(E... 324 2e-88
sw|P53778|MK12_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2.... 225 1e-58 sw|Q40353|MMK2_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M... 173 4e-43
sw|P23573|CC2C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE
(E... 324 2e-88
sw|O15264|MK13_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 13 (EC 2.... 222 1e-57 sw|Q00532|KKIA_HUMAN SERINE/THREONINE-PROTEIN KINASE KKIALRE (E... 173 6e-43
sw|Q00537|KPT2_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2
... 323 4e-88
MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 220 5e-57
sw|Q92241|PH85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM sw|Q92207|HOG1_CANAL
(EC 2... 320 2e-87
sw|P32485|HOG1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 172 7e-43
sw|P32485|HOG1_YEAST
MITOGEN-ACTIVATED PROTEIN KINASE HOG1 (EC ... 217 4e-56sw|Q15131|CDKA_HUMAN CELL DIVISION PROTEIN KINASE 10 (EC 2.7.1.... 172 7e-43
sw|Q04899|KPT3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3
... 315 6e-86
PROBABLE SERINE/THREONINE-PROTEIN KINASE Y... 213 4e-55sw|Q39024|MPK4_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4... 171 1e-42
sw|O35832|KPT3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3sw|P36005|KKQ1_YEAST
(E... 314 2e-85
sw|P45983|MK08_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2.7... 205 2e-52 sw|Q04899|KPT3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 ... 171 1e-42
sw|Q00536|KPT1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1
... 312 7e-85
sw|P49187|MK10_RAT
MITOGEN-ACTIVATED
PROTEIN KINASE 10 (EC 2.7.... 204 3e-52 sw|P24788|KP58_MOUSE GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K... 171 2e-42
sw|Q04735|KPT1_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1 ... 310 2e-84
sw|P49185|MK08_RAT
MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2.7.1... 204 3e-52 sw|P39745|SUR1_CAEEL MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC... 171 2e-42
sw|Q07002|KPT3_HUMAN SERINE/THREONINE PROTEIN KINASE PCTAIRE-3
... 307 1e-83
MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2.... 201 1e-51 sw|Q92241|PH85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2... 171 2e-42
sw|P34556|CC2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|P53779|MK10_HUMAN
(EC... 305 5e-83
sw|Q61831|MK10_MOUSE
MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2.... 200 3e-51 sw|Q40531|NTF6_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N... 171 2e-42
sw|P54666|CC23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
3 ... 300 2e-81
sw|P45984|MK09_HUMAN
MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2.7... 199 6e-51 sw|Q38773|CC2B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B ... 171 2e-42
sw|P25859|CC2B_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
B ... 293 2e-79
sw|P49186|MK09_RAT
MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2.7.1... 199 7e-51 sw|O35832|KPT3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 (E... 170 3e-42
sw|Q38774|CC2C_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
C ... 286 4e-77
sw|P41808|SMK1_YEAST
SPORULATION-SPECIFIC MITOGEN-ACTIVATED PRO... 199 1e-50
sw|Q38775|CC2D_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
D ... 284 1e-76
sw|P21127|KP58_HUMAN GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K... 170 3e-42
sw|P29619|CC22_ORYSA
CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2 ... 194 3e-49 sw|Q00536|KPT1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1 ... 170 4e-42
sw|P54665|CC22_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG
2 ... 284 2e-76
sw|P40417|ERKA_DROME MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC... 170 4e-42
CDKs
HsCDK1
SpCdc2
PfPK5
PfCRK4
PfCRK4
PfPK6
PfPK6
MmErk2
ScFus3
PfMAP1
PfcPKA
702.2
500
200 100
50
20
10
5
2 1
CDKs
MAPKs
0
CDKs
MAP kinase pathway
R
TK
ras
MAP kinase kinase kinase
MAP kinase kinase
MAP kinase
?
Cyclin E
Cyclin A
Cyclin A
CDK2
CDK1
S
G2
CDK2
M
Cyclin B
CDK1
G1
CDK3
PfPK6
Cyclin ?
CDK6
Cyclin D
Pfcrk-4
?
CDK4
Cyclin D
CDK7
Cyclin H
Cell cycle control elements
CDKs
Aim : To identify components of MAPK modules.
pheromone
G-proteins
receptor
Ste20
Two MAP kinase homologues
Ste11
Ste7
KSS1 Fus3
Ste5
(MAPKKK)
(MAPKK)
(MAPK)
F
Y
L
K N C
TXY
F
Y
L
K N C
TXY
F
Y
L
K N C
TSH
Mammalian/yeast MAPK
Pfmap-1
(IKEQ)5 (KKYVDGSN)12
Pfmap-2
MAPK pathway
Activation of GST-Pfmap-2 by gametocyte extracts
Activity of
recombinant Pfmap-2
Pfmap-2
MBP
% activity
600
400
200
100
Lane 1
2
3
4-5
6-7
Dorin et al. (1999), JBC 274, 29912.
MAPK pathway
Atypical MAPK pathway in Plasmodium
Typical MAPK module
P. falciparum
MEKK
SMANS
TEY
MEK1/2
MAPK
(ERK1/2)
?
TSH
Pfmap-2
TDY
Pfmap-1
MAPK pathway
?
Can we use genomic databases to solve black boxes ?
?
MAPK pathway
…it works well for reconstruction of
metabolic pathways...
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 ».
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!
Query:
1 GVSGQL-IDSMANSFVGTRSYMSP 23
G+S + I+SMA+S VGT Y SP
Subject: 591 GLSKNIGIESMAHSCVGTPYYWSP 662
30%
100%
42%
Mm MEK1 IKLCDFGVS--GQLIDSMANSFVGTRSYMSPERLQGTHYSV--QSDIWSM
: ::: :
:.:::.: ::: : ::: :
:
:: :
Pfnek-1 AKIGDFGLSK-NIGIESMAHSCVGTPYYWSPELLLHETKSYDDKSDMWAL
: :::::::
:
:::: : :::
:
:: ::
NIMA
VKLGDFGLSKLMHSH-DF.STYVGTPFYMSPEIC--AAEKSTLRSDIWAV
(
SMANS
MEK1/2 )
SMAHS
Pfnek-1
MAPK pathway
Typical MAPK module
P. falciparum
MEKK
SMANS
MEK1/2
SMAHS
Pfnek-1
?
TEY
MAPK
(ERK1/2)
TSH
Pfmap-2
TDY
Pfmap-1
MAPK pathway
Atypical MAPK pathway in Plasmodium
Pfnek-1: - closely related to NIMA kinases; MAPKK–like activation site.
SMANS
MEK1/2
SMAHS
Pfnek-1 (100%)
DxxT
NIMA/Nek (42%)
(30%)
Phosphorylation of Pfmap-2 by Pfnek-1
Pfnek-1:
ERK2 Pfmap-2
+ - + - +
Autoradiogram
Synergistic activities of
Pfnek-1 and Pfmap-2
mERK2
Pfmap-2
Coomassie
mERK2
map-2 nek-1
nek-1 +
map-2
MBP
Pfmap-2
Dorin et al. (2001), EJB 268:2600-2608
MAPK pathway
Aim : To identify components of MAPK modules.
pheromone
G-proteins
receptor
No typical MAPKK homologues!
Ste20
Ste11
Ste7
KSS1 Fus3
Ste5
- Experimental (PCR) approaches
- Targeted database mining (BLASTP)
(MAPKKK)
(MAPKK)
(MAPK)
- Genome-wide “kinome” characterisation
(with P. Ward, WCMP, and J. Packer, Abbott laboratories)
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
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
pfb0150c
map2k1
map3k3
map4k1
pak1
csnk1g1
Pf110377 (PfCK1)
csnk1d
Casein kinase 1
Ste7/11/20
pf140320
pf110220
mal6p1191
pf130258
tgfbr1
Pfb0520w (PfRaf)
zak
limk1
raf1
src
insr
egfr
pfi1280c
pfi1290w
Mal6p1146 (PfPK4)
pfa0380w
pf140423
pfl0080c
mal6p156
Pfl1370w (Pfnek-1)
nek7
Pfe1290w
nek1
Mal7p1100 (Pfnek-4)
Mal7p191 (PfEST)
pf110488
mal13p1196
Pfc0755c (Pfcrk-4)
Mal6p1271 (Pfcrk-5)
Pf130206 (PfPK6)
Pfd0740w (Pfcrk-3)
Mal13p1279 (PfPK5)
cdk2
Pf100141 (Pfmrk)
Pfd0865c (Pfcrk-1)
Pf110147 (Pfmap-2)
Pf140294 (Pfmap-1)
mapk1
Pf110096 (PfCK2)
Pf080044 (PfPK1)
mal13p184
Pfc0525c (PfGSK3)
gsk3b
pf110156
Pf140431(Pflammer)
clk1
pfc0105w
pf140408
pf140392
mal7p173
Pfl1885c (PfPK2)
pf110242
Pfb0815w (PfCDPK1)
pf070072
Pf130211
Mal6p1108 (PfCDPK2)
Pfc0420w (PfCDPK3)
mapkapk2
camk2a
camk1
dapk1
pf110239
pf140227
pfc0485w
pfb0665w
mal7p118
pf110060
pf110464
pfc0385c
mal13p1278
pf110227
Pf140346 (PfPKG)
rock1
Pfl2250c
prkca
akt1
prkaca
Pfi1685w (PfPKA)
Pf140516 (PfKIN)
Pf140476
pf130085
Pfb0605w (PfPK7)
Pfl2280w
Tyrosine kinase-like
Tyrosine kinase
NimA
CMGC
Camk
AGC
Kinome
pfi1415w
A novel, Apicomplexa-specific family of PK-like proteins
GxGxxG
K
E
I
II
III
W
HRDxxxxN
IV
V
FIKKxxxxxW
GExxxxxxNxxMExxxxxxL
VIA
VIB
LHxxGxxHLDxxxxNxL
DFG
APE
D
VII
VIII
IX
PxPE
YxxVxxxDxxM
R
X
WP
XI
LxxxxxR PW
CD[FL][GA]xxxxxPxY
• 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
Reconstructing metabolic pathways
Model eukaryote
metabolites
Plasmodium
Enzyme A
Enzyme B
Enzyme C
Kinome
Problems in reconstructing regulatory pathways:
recognisable
not recognisable
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
Validation by gene knock-out
Malaria parasites:
- Extremely low transfection rate
- No inducible expression system yet
PyrR
PyrS
Pyr selection
targeted gene
Check for the presence of
the desired genotype (PCR)
PyrR
Clone parasites
Analyse phenotype
Kinases as drug targets
Pbcrk-1 cannot be knocked-out…
MscI
MscI
1734
Pbcrk-1
202
Pst I
1027
MscI
hDHFR
Homologous recombination
MscI
MscI
MscI
episome
Rangajaran et al., submitted
WT
Kinases as drug targets
…but the Pbcrk-1 locus is amenable to genetic modification.
NdeI
NdeI
Pbcrk-1
802
XhoI
NdeI
1734
hDHFR
Homologous recombination
NdeI
hDHFR
NdeI
1734
episome
recombinant
Rangajaran et al., submitted
WT
Kinases as drug targets
Problem: detailed studies of the phenotype are impossible using KO approach.
a possible solution: chemical genetics ?
Validation by chemical genetics
ATP
Inhibitor
ACTIVE
Wild-type kinase
Large gatekeeper: F, L...
ATP
Inhibitor
INACTIVE
Mutated kinase
Small gatekeeper: G, A...
Kinases as drug targets
338
V-Src
C-Fyn
C-Abl
Cdk2
CDC28
CAMKII
FUS3
332-EPIYIVIEYMSK--GSLLDFLKGEM333-EPIYIVTEYMNK--GSLLDFLKDGE309-PPFYIITEFMTY--GNLLDYLRECN83-NKLYLVFEFLHQ---DLKKFMDASA92-HKLYLVFEFLDL---DLKRYMEGIP84-GHHYLIFDLVTG--GELFEDIVARE87-NEVYIIQELMQT---DLHRVISTQMBishop et al., Nature 407:395-401
Pfmap-2
Pfmap-1
PfPK5
PfPK6
PfGSK-3
Etc...
171-DELYIVLEIADS---DLKKLFKTPI107-NDIYLIFDFMET---DLHEVIKADL73-KRLVLVFEHLDQ---DLKKLLDVCE109-SCLYLAFEYCDI---DLFNLIKKHN151-IFLNVVMEYIPQ---TVHKYMKYYS3. Parasite PKs as targets: validation
Validation by chemical genetics
Selective inhibition of mutagenised PfPK5-Pfcyc-1 by N-M-PP1
nM 1 N-M-PP1
Histone H1
100%
100%
50
50
His-PfPK5 WT
His-PfPK5 F79G
Next: allele replacement in the parasite
Kinases as drug targets
Chemical library screening
Medium-throughput
screening
Preliminary inhibition
assay (PfGSK3).
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0,01
A
0,1
1
10
Concentration (µM)
Paullone
Gwennpaullone
100
Kenpaullone
Alsterpaullone
next: - selectivity studies
- cellular effects of the hits
Kinases as drug targets
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
Acknowledgements
Jane Endicott, Univ. of Oxford
Kavita Shah, Novartis, San Diego
Laurent Meijer, CNRS, Roscoff/Rockefeller
Debopam Chakrabarti, UCF, Orlando
Robert Menard, Institut Pasteur
Ali Sultan, Harvard School of Public Health
Jeremy Packer, Abbott Laboratories
WCMP-INSERM U609:
Pauline Ward
Dominique Dorin
Karine Le Roch
Anais Merckx
Leila Equinet
Marie-Paule Nivez
Luc Reiniger
Caroline Doerig