Plasmodium falciparum protein kinases: from database mining to drug discovery Inserm team (U 609) Wellcome Centre for Molecular Parasitology University of Glasgow U 609
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 Gx. Gxx. G K E I II III HRDxxxx. N IV V VIA VIB DFG APE D VIII IX R X XI Kinase assay N-ter lobe Protein kinase ATP P C-ter lobe ATPPP Human CDK 2 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 receptor Heterotrimeric G-proteins CDK 7 Cyclin H Ste 20 Ste 5 KSS 1 Ste 11 Ste 7 Fus 3 CDK 4 Cyclin D CDK 1 Cyclin B (MAPKKK) (MAPK) CDK 6 Cyclin D CDK 3 Cyclin ? Cyclin A CDK 1 CDK 2 Cyclin E CDK 2 Cyclin A CDKs
CDKs of P. falciparum Cyclin A CDK 2 PSTAIRE T 161 Pf. PK 5 CDK 1 Cdk 1 PSTTIRE Cyclin A (Cdk 1) AKTYIRE T 161 Pf. PK 6 S (Cdk 1/MAPK) M Pfcrk-4 (Cdk 1/MAPK) CDK 1 CDK 3 CDK 6 Cyclin D T 161 Cyclin B G 1 Cyclin ? EEFAVNE G 2 CDK 2 Pfcrk-3 (Cdk 1) KCILRE Cyclin E CDK 4 Cyclin D CDK 7 Cyclin H Cell cycle control elements AMTSLRE T 161 Pfcrk-1 (p 58) NFVLLRE T 161 Pfmrk (CDK 7) . . . and four potential cyclins CDKs
Activation of Pf. PK 5 (CDK 1) by human cyclin H Pfmrk + Pf. PK 5 + hcyc. H Pfmrk histone H 1 Relaxed cyclin specifity for Pf. PK 5? CAK-independent activation Pf. PK 5 + Pfmrk + hcyc. H Pf. PK 5 Histone H 1 Human cyclin H Pfcyc-1, -3 p 25 RINGO (A. Nebreda) Human cyclin A (J. Endicott) Autophosphorylation of Pf. PK 5 Le Roch et al. (2000), JBC 275, 8952; Merckx et al. , JBC (2003) CDKs
Pf. PK 5: CDK Pfmap-1: MAPK Pf. PK 6: CDK-MAPK? sw|P 43063|CC 28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2. 7. 1. . . 194 3 e-49 sw|Q 07785|CC 2 H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E. . . 592 e-169 sw|Q 11179|YPC 2_CAEEL PUTATIVE SERINE/THREONINE-PROTEIN KINASE C. . . 328 1 e-89 sw|P 23437|CDK 2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -. . . 192 8 e-49 sw|P 34117|CC 2 H_DICDI CDC 2 -LIKE SERINE/THREONINE-PROTEIN KINASE. . . 411 e-115 sw|P 28869|ERK 1_CANAL EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E. . . 254 2 e-67 sw|Q 39026|MPK 6_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6. . . 190 2 e-48 sw|P 29618|CC 21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 382 e-106 sw|P 27638|SPK 1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE SPK 1 (EC. . . 254 2 e-67 sw|Q 63699|CDK 2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). 190 2 e-48 sw|Q 38772|CC 2 A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A. . . 379 e-105 sw|P 23111|CC 2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|P 42525|ERK 1_DICDI EXTRACELLULAR SIGNAL-REGULATED KINASE 1 (E. . . 253 5 e-67 sw|P 23573|CC 2 C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE (E. . . 190 2 e-48 (EC. . . 379 e-105 sw|Q 39024|MPK 4_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4. . . 251 2 e-66 sw|P 24941|CDK 2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -. . . 190 3 e-48 sw|P 24100|CC 2 A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A. . . 374 e-103 sw|O 55076|CDK 2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). 190 4 e-48 sw|P 52389|CC 2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|Q 39025|MPK 5_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5. . . 248 1 e-65 (EC. . . 372 e-103 sw|Q 07176|MMK 1_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M. . . 247 sw|P 48963|CDK 2_MESAU CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). 188 1 e-47 2 e-65 sw|P 29619|CC 22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 370 e-102 3 e-65 sw|P 43450|CDK 2_CARAU CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). sw|Q 40353|MMK 2_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M. . . 247 sw|P 43450|CDK 2_CARAU CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). 188 1 e-47 369 e-102 sw|Q 63699|CDK 2_RAT CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). sw|P 27361|MK 03_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2. 7. . . 247 3 e-65 sw|Q 39023|MPK 3_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3. . . 187 3 e-47 367 e-101 sw|Q 07176|MMK 1_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M. . . 187 3 e-47 sw|O 55076|CDK 2_CRIGR CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). sw|Q 40531|NTF 6_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 246 5 e-65 367 e-101 sw|P 47812|MK 14_XENLA MITOGEN-ACTIVATED PROTEIN KINASE 2 (EC 2. 7. . . 246 5 e-65 sw|Q 40532|NTF 4_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 186 6 e-47 sw|P 24941|CDK 2_HUMAN CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -. . . 366 e-101 sw|P 48963|CDK 2_MESAU CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). sw|P 21708|MK 03_RAT MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2. 7. 1. . . 246 6 e-65 sw|P 54666|CC 23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 3. . . 185 1 e-46 366 e-101 sw|Q 06060|MAPK_PEA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D 5. . . 245 8 e-65 sw|Q 06060|MAPK_PEA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG D 5. . . 185 1 e-46 sw|Q 05006|CC 22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 365 e-101 sw|P 23437|CDK 2_XENLA CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -. . . sw|P 26696|MK 01_XENLA MITOGEN-ACTIVATED PROTEIN KINASE (EC 2. 7. 1. . . 245 1 e-64 sw|P 24100|CC 2 A_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A. . . 184 2 e-46 360 2 e-99 sw|Q 39026|MPK 6_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 6. . . 245 1 e-64 sw|P 23111|CC 2_MAIZE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 184 2 e-46 sw|Q 00526|CDK 3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2. 7. 1. -). 356 2 e-98 sw|Q 39021|MPK 1_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1. . . 243 3 e-64 sw|P 29618|CC 21_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 183 4 e-46 sw|P 24923|CC 21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 356 4 e-98 sw|Q 40532|NTF 4_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 243 4 e-64 sw|P 39951|CC 2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2. . . 182 7 e-46 sw|Q 00535|CDK 5_HUMAN CELL DIVISION PROTEIN KINASE 5 (EC 2. 7. 1. -. . . 355 5 e-98 sw|Q 38772|CC 2 A_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG A. . . 182 7 e-46 sw|Q 02399|CDK 5_BOVIN CELL DIVISION PROTEIN KINASE 5 (EC 2. 7. 1. -. . . sw|Q 39027|MPK 7_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7. . . 243 4 e-64 355 7 e-98 sw|P 51166|CDK 5_XENLA CELL DIVISION PROTEIN KINASE 5 (EC 2. 7. 1. -. . . sw|P 39745|SUR 1_CAEEL MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC. . . 243 5 e-64 sw|P 06493|CC 2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 182 9 e-46 354 1 e-97 sw|Q 40517|NTF 3_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 242 7 e-64 sw|P 48734|CC 2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 181 1 e-45 sw|P 49615|CDK 5_MOUSE CELL DIVISION PROTEIN KINASE 5 (EC 2. 7. 1. -. . . 354 1 e-97 sw|P 27703|MK 01_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2. 7. . . 242 7 e-64 sw|P 34117|CC 2 H_DICDI CDC 2 -LIKE SERINE/THREONINE-PROTEIN KINASE. . . 181 2 e-45 sw|P 43063|CC 28_CANAL CELL DIVISION CONTROL PROTEIN 28 (EC 2. 7. 1. . . 353 3 e-97 sw|P 28482|MK 01_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2. 7. . . 242 7 e-64 sw|P 52389|CC 2_VIGUN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 180 3 e-45 sw|Q 38773|CC 2 B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B. . . 353 3 e-97 sw|Q 03114|CDK 5_RAT CELL DIVISION PROTEIN KINASE 5 (EC 2. 7. 1. -). . . sw|P 46196|MK 01_BOVIN MITOGEN-ACTIVATED PROTEIN KINASE 1 (EC 2. 7. . . 242 7 e-64 sw|P 11440|CC 2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 180 3 e-45 352 6 e-97 sw|Q 39022|MPK 2_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2. . . 242 9 e-64 sw|P 34556|CC 2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 180 3 e-45 sw|P 06493|CC 2_HUMAN CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 351 1 e-96 sw|Q 40884|MAPK_PETHY MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1. . . 242 1 e-63 sw|Q 04770|CDK 2_ENTHI CELL DIVISION PROTEIN KINASE 2 HOMOLOG (EC. . . 179 6 e-45 sw|P 39951|CC 2_RAT CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC 2. . . 351 1 e-96 sw|Q 63844|MK 03_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 3 (EC 2. 7. . . 240 3 e-63 sw|Q 05006|CC 22_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 179 7 e-45 sw|P 11440|CC 2_MOUSE CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 350 2 e-96 sw|P 29619|CC 22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 178 1 e-44 sw|P 48734|CC 2_BOVIN CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|Q 00772|SLT 2_YEAST MITOGEN-ACTIVATED PROTEIN KINASE SLT 2/MPK 1. . . 240 5 e-63 (EC. . . 350 2 e-96 sw|P 70618|MK 14_RAT MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2. 7. . 239 8 e-63 sw|Q 40517|NTF 3_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 178 2 e-44 sw|P 51958|CC 2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 348 7 e-96 sw|P 97377|CDK 2_MOUSE CELL DIVISION PROTEIN KINASE 2 (EC 2. 7. 1. -). sw|P 47811|MK 14_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2. . 239 8 e-63 sw|Q 39025|MPK 5_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 5. . . 178 2 e-44 344 2 e-94 sw|P 13863|CC 2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|Q 16539|MK 14_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2. . 239 8 e-63 sw|Q 40884|MAPK_PETHY MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1. . . 178 2 e-44 (EC. . . 343 3 e-94 sw|Q 90336|MK 14_CYPCA MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2. . 239 8 e-63 sw|P 13863|CC 2_CHICK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 178 2 e-44 sw|P 35567|CC 21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 341 1 e-93 sw|O 02812|MK 14_CANFA MITOGEN-ACTIVATED PROTEIN KINASE 14 (EC 2. . 238 1 e-62 sw|Q 07785|CC 2 H_PLAFK CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (E. . . 177 2 e-44 sw|P 24033|CC 22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 340 1 e-93 sw|Q 39023|MPK 3_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 3. . . 238 2 e-62 sw|P 24923|CC 21_MEDSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 177 3 e-44 sw|P 48609|CDK 5_DROME CELL DIVISION PROTEIN KINASE 5 HOMOLOG (EC. . . 340 3 e-93 sw|Q 13164|MK 07_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 7 (EC 2. 7. . . 238 2 e-62 sw|P 24033|CC 22_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 176 5 e-44 sw|P 00546|CC 28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2. 7. 1. . . 340 3 e-93 sw|Q 06309|CRK 1_LEIME CELL DIVISION PROTEIN KINASE 2 HOMOLOG sw|O 08666|MK 11_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2. . 236 5 e-62 sw|Q 39027|MPK 7_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 7. . . 176 6 e-44 CRK. . . 339 3 e-93 sw|P 40417|ERKA_DROME MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC. . . 232 8 e-61 sw|P 23572|CC 2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 176 6 e-44 sw|P 23572|CC 2_DROME CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 339 4 e-93 sw|Q 92398|SPM 1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE SPM 1 (EC. . . 231 2 e-60 sw|P 35567|CC 21_XENLA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 176 6 e-44 sw|P 54664|CC 21_TRYCO CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 338 1 e-92 sw|P 43068|MKC 1_CANAL MITOGEN-ACTIVATED PROTEIN KINASE MKC 1 (EC. . . 229 9 e-60 sw|Q 39021|MPK 1_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 1. . . 175 8 e-44 sw|P 34112|CC 2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 335 5 e-92 sw|P 14681|KSS 1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE KSS 1 (EC. . . 229 9 e-60 sw|P 51958|CC 2_CARAU CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 175 8 e-44 sw|P 04551|CC 2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2. 7. 1. -. . . 335 9 e-92 sw|P 54119|CC 2_AJECA CELL DIVISION CONTROL PROTEIN 2 (EC 2. 7. 1. -. . . sw|Q 63538|MK 12_RAT MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2. 7. . 228 1 e-59 sw|P 00546|CC 28_YEAST CELL DIVISION CONTROL PROTEIN 28 (EC 2. 7. 1. . . 175 1 e-43 333 3 e-91 sw|P 16892|FUS 3_YEAST MITOGEN-ACTIVATED PROTEIN KINASE FUS 3 (EC. . . 228 1 e-59 sw|P 04551|CC 2_SCHPO CELL DIVISION CONTROL PROTEIN 2 (EC 2. 7. 1. -. . . 174 2 e-43 sw|Q 00646|CC 2_EMENI CELL DIVISION CONTROL PROTEIN 2 (EC 2. 7. 1. -. . . 332 6 e-91 sw|Q 09892|STY 1_SCHPO MITOGEN-ACTIVATED PROTEIN KINASE STY 1 (EC. . . 228 2 e-59 sw|Q 39022|MPK 2_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2. . . 174 2 e-43 sw|P 38973|CC 21_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 1. . . 332 6 e-91 sw|Q 15759|MK 11_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 11 (EC 2. . 228 2 e-59 sw|P 34112|CC 2_DICDI CELL DIVISION CONTROL PROTEIN 2 HOMOLOG (EC. . . 174 2 e-43 sw|P 17157|PH 85_YEAST NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2. . . 328 6 e-90 sw|O 35831|KPT 2_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2 sw|O 08911|MK 12_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2. . 227 3 e-59 sw|Q 00526|CDK 3_HUMAN CELL DIVISION PROTEIN KINASE 3 (EC 2. 7. 1. -). 173 3 e-43 (E. . . 324 2 e-88 sw|P 53778|MK 12_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 12 (EC 2. . 225 1 e-58 sw|Q 40353|MMK 2_MEDSA MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG M. . . 173 4 e-43 sw|P 23573|CC 2 C_DROME CELL DIVISION CONTROL PROTEIN 2 COGNATE (E. . . 324 2 e-88 sw|O 15264|MK 13_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 13 (EC 2. . 222 1 e-57 sw|Q 00532|KKIA_HUMAN SERINE/THREONINE-PROTEIN KINASE KKIALRE (E. . . 173 6 e-43 sw|Q 00537|KPT 2_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-2. . . 323 4 e-88 sw|Q 92207|HOG 1_CANAL MITOGEN-ACTIVATED PROTEIN KINASE HOG 1 (EC. . . 220 5 e-57 sw|P 32485|HOG 1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE HOG 1 (EC. . . 172 7 e-43 sw|Q 92241|PH 85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2. . . 320 2 e-87 sw|P 32485|HOG 1_YEAST MITOGEN-ACTIVATED PROTEIN KINASE HOG 1 (EC. . . 217 4 e-56 sw|Q 15131|CDKA_HUMAN CELL DIVISION PROTEIN KINASE 10 (EC 2. 7. 1. . 172 7 e-43 sw|Q 04899|KPT 3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3. . . 315 6 e-86 sw|Q 39024|MPK 4_ARATH MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 4. . . 171 1 e-42 sw|O 35832|KPT 3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 sw|P 36005|KKQ 1_YEAST PROBABLE SERINE/THREONINE-PROTEIN KINASE Y. . . 213 4 e-55 (E. . . 314 2 e-85 sw|P 45983|MK 08_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2. 7. . . 205 2 e-52 sw|Q 04899|KPT 3_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3. . . 171 1 e-42 sw|Q 00536|KPT 1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1. . . 312 7 e-85 sw|P 49187|MK 10_RAT MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2. 7. . 204 3 e-52 sw|P 24788|KP 58_MOUSE GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K. . . 171 2 e-42 sw|Q 04735|KPT 1_MOUSE SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1. . . 310 2 e-84 sw|P 49185|MK 08_RAT MITOGEN-ACTIVATED PROTEIN KINASE 8 (EC 2. 7. 1. . . 204 3 e-52 sw|P 39745|SUR 1_CAEEL MITOGEN-ACTIVATED PROTEIN KINASE SUR-1 (EC. . . 171 2 e-42 sw|Q 07002|KPT 3_HUMAN SERINE/THREONINE PROTEIN KINASE PCTAIRE-3. . . 307 1 e-83 sw|P 34556|CC 2_CAEEL CELL DIVISION CONTROL PROTEIN 2 HOMOLOGsw|P 53779|MK 10_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2. . 201 1 e-51 sw|Q 92241|PH 85_KLULA NEGATIVE REGULATOR OF THE PHO SYSTEM (EC 2. . . 171 2 e-42 (EC. . . 305 5 e-83 sw|Q 61831|MK 10_MOUSE MITOGEN-ACTIVATED PROTEIN KINASE 10 (EC 2. . 200 3 e-51 sw|Q 40531|NTF 6_TOBAC MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG N. . . 171 2 e-42 sw|P 54666|CC 23_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 3. . . 300 2 e-81 sw|P 45984|MK 09_HUMAN MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2. 7. . . 199 6 e-51 sw|Q 38773|CC 2 B_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B. . . 171 2 e-42 sw|P 25859|CC 2 B_ARATH CELL DIVISION CONTROL PROTEIN 2 HOMOLOG B. . . 293 2 e-79 sw|P 49186|MK 09_RAT MITOGEN-ACTIVATED PROTEIN KINASE 9 (EC 2. 7. 1. . . 199 7 e-51 sw|O 35832|KPT 3_RAT SERINE/THREONINE-PROTEIN KINASE PCTAIRE-3 (E. . . 170 3 e-42 sw|Q 38774|CC 2 C_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG C. . . 286 4 e-77 sw|P 41808|SMK 1_YEAST SPORULATION-SPECIFIC MITOGEN-ACTIVATED PRO. . . 199 1 e-50 sw|P 21127|KP 58_HUMAN GALACTOSYLTRANSFERASE ASSOCIATED PROTEIN K. . . 170 3 e-42 sw|Q 38775|CC 2 D_ANTMA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG D. . . 284 1 e-76 sw|P 29619|CC 22_ORYSA CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 194 3 e-49 sw|Q 00536|KPT 1_HUMAN SERINE/THREONINE-PROTEIN KINASE PCTAIRE-1. . . 170 4 e-42 sw|P 54665|CC 22_TRYBB CELL DIVISION CONTROL PROTEIN 2 HOMOLOG 2. . . 284 2 e-76 sw|P 40417|ERKA_DROME MITOGEN-ACTIVATED PROTEIN KINASE ERK-A (EC. . . 170 4 e-42 CDKs
CDKs Pf. CRK 4 Pf. PK 6 MAPKs CDKs
MAP kinase pathway R TK ras MAP kinase MAP kinase Cyclin A ? Cyclin E Cyclin A CDK 2 MAP kinase CDK 1 S G 2 CDK 2 M Cyclin B CDK 1 G 1 CDK 3 Pf. PK 6 Pfcrk-4 Cyclin ? ? CDK 6 Cyclin D CDK 4 Cyclin D CDK 7 Cyclin H Cell cycle control elements CDKs
Aim : To identify components of MAPK modules. pheromone G-proteins receptor Ste 20 Two MAP kinase homologues Ste 11 Ste 7 KSS 1 Fus 3 Ste 5 (MAPKKK) (MAPK) 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 MBP Dorin et al. (1999), JBC 274, 29912. MAPK pathway
Atypical MAPK pathway in Plasmodium Typical MAPK module P. falciparum MEKK SMANS TEY MEK 1/2 MAPK (ERK 1/2) ? TSH TDY Pfmap-2 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 MEK 1 as a query No hit in Plasmo. DB 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 MEK 1 IKLCDFGVS--GQLIDSMANSFVGTRSYMSPERLQGTHYSV--QSDIWSM : : : : Pfnek-1 AKIGDFGLSK-NIGIESMAHSCVGTPYYWSPELLLHETKSYDDKSDMWAL : : : : : : NIMA VKLGDFGLSKLMHSH-DF. STYVGTPFYMSPEIC--AAEKSTLRSDIWAV ( SMANS MEK 1/2 ) SMAHS Pfnek-1 MAPK pathway
Typical MAPK module P. falciparum MEKK SMANS MEK 1/2 SMAHS Pfnek-1 ? TEY MAPK (ERK 1/2) TSH TDY Pfmap-2 Pfmap-1 MAPK pathway
Atypical MAPK pathway in Plasmodium Pfnek-1: - closely related to NIMA kinases; MAPKK–like activation site. SMANS MEK 1/2 SMAHS Pfnek-1 (100%) Dxx. T NIMA/Nek (42%) (30%) Phosphorylation of Pfmap-2 by Pfnek-1: ERK 2 Pfmap-2 + - + Autoradiogram Synergistic activities of Pfnek-1 and Pfmap-2 m. ERK 2 Pfmap-2 Coomassie m. ERK 2 nek-1 + map-2 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! Ste 20 Ste 11 Ste 7 KSS 1 Fus 3 Ste 5 - Experimental (PCR) approaches - Targeted database mining (BLASTP) (MAPKKK) (MAPK) - Genome-wide “kinome” characterisation (with P. Ward, WCMP, and J. Packer, Abbott laboratories) Plasmo. DB 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 Tyr. K, no PKC, no STE Kinome
pfb 0150 c map 2 k 1 map 3 k 3 map 4 k 1 pak 1 csnk 1 g 1 Pf 110377 (Pf. CK 1) csnk 1 d pf 140320 Casein kinase 1 Ste 7/11/20 pf 110220 mal 6 p 1191 pf 130258 tgfbr 1 Pfb 0520 w (Pf. Raf) zak limk 1 raf 1 src insr egfr pfi 1280 c pfi 1290 w Mal 6 p 1146 (Pf. PK 4) pfa 0380 w pf 140423 pfl 0080 c mal 6 p 156 Pfl 1370 w (Pfnek-1) nek 7 Pfe 1290 w nek 1 Mal 7 p 1100 (Pfnek-4) Mal 7 p 191 (Pf. EST) pf 110488 mal 13 p 1196 Pfc 0755 c (Pfcrk-4) Mal 6 p 1271 (Pfcrk-5) Pf 130206 (Pf. PK 6) Pfd 0740 w (Pfcrk-3) Mal 13 p 1279 (Pf. PK 5) cdk 2 Pf 100141 (Pfmrk) Pfd 0865 c (Pfcrk-1) Pf 110147 (Pfmap-2) Pf 140294 (Pfmap-1) mapk 1 Pf 110096 (Pf. CK 2) Pf 080044 (Pf. PK 1) mal 13 p 184 Pfc 0525 c (Pf. GSK 3) gsk 3 b pf 110156 Pf 140431(Pflammer) clk 1 pfc 0105 w pf 140408 pf 140392 mal 7 p 173 Pfl 1885 c (Pf. PK 2) pf 110242 Pfb 0815 w (Pf. CDPK 1) pf 070072 Pf 130211 Mal 6 p 1108 (Pf. CDPK 2) Pfc 0420 w (Pf. CDPK 3) mapkapk 2 camk 2 a camk 1 dapk 1 pf 110239 pf 140227 pfc 0485 w pfb 0665 w mal 7 p 118 pf 110060 pf 110464 pfc 0385 c mal 13 p 1278 pf 110227 Pf 140346 (Pf. PKG) rock 1 Pfl 2250 c prkca akt 1 prkaca Pfi 1685 w (Pf. PKA) Pf 140516 (Pf. KIN) Pf 140476 pf 130085 Pfb 0605 w (Pf. PK 7) Pfl 2280 w Tyrosine kinase-like Tyrosine kinase Nim. A CMGC Camk AGC Kinome pfi 1415 w
A novel, Apicomplexa-specific family of PK-like proteins Gx. Gxx. G K E I II III W HRDxxxx. N IV V FIKKxxxxx. W GExxxxxx. Nxx. MExxxxxx. L VIA DFG D VII VIB APE VIII IX LHxx. Gxx. HLDxxxx. Nx. L Px. PE Yxx. Vxxx. Dxx. M R X WP XI Lxxxxx. R PW CD[FL][GA]xxxxx. Px. Y • 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
Pf. EMP 1 (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 Pyr. R Pyr. S Pyr selection targeted gene Check for the presence of the desired genotype (PCR) Pyr. R Clone parasites Analyse phenotype Kinases as drug targets
Pbcrk-1 cannot be knocked-out… Msc. I 1734 Pbcrk-1 202 Pst I 1027 Msc. I h. DHFR Homologous recombination Msc. I episome Rangajaran et al. , submitted WT Kinases as drug targets
…but the Pbcrk-1 locus is amenable to genetic modification. Nde. I Pbcrk-1 802 Xho. I Nde. I 1734 h. DHFR Homologous recombination Nde. I h. DHFR Nde. I 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 Cdk 2 CDC 28 CAMKII FUS 3 332 -EPIYIVIEYMSK--GSLLDFLKGEM 333 -EPIYIVTEYMNK--GSLLDFLKDGE 309 -PPFYIITEFMTY--GNLLDYLRECN 83 -NKLYLVFEFLHQ---DLKKFMDASA 92 -HKLYLVFEFLDL---DLKRYMEGIP 84 -GHHYLIFDLVTG--GELFEDIVARE 87 -NEVYIIQELMQT---DLHRVISTQM- Pfmap-2 Pfmap-1 Pf. PK 5 Pf. PK 6 Pf. GSK-3 Etc. . . 171 -DELYIVLEIADS---DLKKLFKTPI 107 -NDIYLIFDFMET---DLHEVIKADL 73 -KRLVLVFEHLDQ---DLKKLLDVCE 109 -SCLYLAFEYCDI---DLFNLIKKHN 151 -IFLNVVMEYIPQ---TVHKYMKYYS- Bishop et al. , Nature 407: 395 -401 3. Parasite PKs as targets: validation
Validation by chemical genetics Selective inhibition of mutagenised Pf. PK 5 -Pfcyc-1 by N-M-PP 1 0 1 10 0 0 000 10 1 10 0 0 000 n. M 1 N-M-PP 1 10 1 1 Histone H 1 100% 50 50 His-Pf. PK 5 WT His-Pf. PK 5 F 79 G Next: allele replacement in the parasite Kinases as drug targets
Chemical library screening Medium-throughput screening Preliminary inhibition assay (Pf. GSK 3). 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 Nu 6102 binding to Pf. PK 5 and Cdk 2 • Mode of binding highly conserved from Cdk 2 • Subtle differences in ribose binding site may afford greater potency for Pf. PK 5 selective inhibitors • IC 50: Pf. PK 5, 215 n. M CDK 2, 5 n. M 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 U 609: Pauline Ward Dominique Dorin Karine Le Roch Anais Merckx Leila Equinet Marie-Paule Nivez Luc Reiniger Caroline Doerig
Скачать презентацию Plasmodium falciparum protein kinases from database mining to
b2d527a4827d55e147e48566071d25d2.ppt