                             SEQUENCE LISTING

<110>  Takeda Pharmaceutical Company Limited
 
<120>  MAPK/ERK KINASE INHIBITORS

<130>  MEK-5009-WO

<150>  60/987,279
<151>  2007-11-12

<160>  6     

<170>  PatentIn version 3.3

<210>  1
<211>  400
<212>  PRT
<213>  Artificial

<220>
<223>  Residues 2-393 of human MEK1 with deletion from residues 32-51, 
       mutations S218E and S222D, and an N-terminal 6-histidine tag.


<220>
<221>  MISC_FEATURE
<222>  (1)..(28)
<223>  N-terminal 6-histidine tag

<220>
<221>  MISC_FEATURE
<222>  (29)..(400)
<223>  Residues 2-393 of human MEK1 with deletion from residues 32-51, 
       and mutations S218E and S222D

<400>  1

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr 
1               5                   10                  15      


Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Gly Ser Pro Lys Lys Lys 
            20                  25                  30          


Pro Thr Pro Ile Gln Leu Asn Pro Ala Pro Asp Gly Ser Ala Val Asn 
        35                  40                  45              


Gly Thr Ser Ser Ala Glu Thr Asn Leu Glu Ala Phe Leu Thr Gln Lys 
    50                  55                  60                  


Gln Lys Val Gly Glu Leu Lys Asp Asp Asp Phe Glu Lys Ile Ser Glu 
65                  70                  75                  80  


Leu Gly Ala Gly Asn Gly Gly Val Val Phe Lys Val Ser His Lys Pro 
                85                  90                  95      


Ser Gly Leu Val Met Ala Arg Lys Leu Ile His Leu Glu Ile Lys Pro 
            100                 105                 110         


Ala Ile Arg Asn Gln Ile Ile Arg Glu Leu Gln Val Leu His Glu Cys 
        115                 120                 125             


Asn Ser Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly 
    130                 135                 140                 


Glu Ile Ser Ile Cys Met Glu His Met Asp Gly Gly Ser Leu Asp Gln 
145                 150                 155                 160 


Val Leu Lys Lys Ala Gly Arg Ile Pro Glu Gln Ile Leu Gly Lys Val 
                165                 170                 175     


Ser Ile Ala Val Ile Lys Gly Leu Thr Tyr Leu Arg Glu Lys His Lys 
            180                 185                 190         


Ile Met His Arg Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser Arg 
        195                 200                 205             


Gly Glu Ile Lys Leu Cys Asp Phe Gly Val Ser Gly Gln Leu Ile Asp 
    210                 215                 220                 


Glu Met Ala Asn Asp Phe Val Gly Thr Arg Ser Tyr Met Ser Pro Glu 
225                 230                 235                 240 


Arg Leu Gln Gly Thr His Tyr Ser Val Gln Ser Asp Ile Trp Ser Met 
                245                 250                 255     


Gly Leu Ser Leu Val Glu Met Ala Val Gly Arg Tyr Pro Ile Pro Pro 
            260                 265                 270         


Pro Asp Ala Lys Glu Leu Glu Leu Met Phe Gly Cys Gln Val Glu Gly 
        275                 280                 285             


Asp Ala Ala Glu Thr Pro Pro Arg Pro Arg Thr Pro Gly Arg Pro Leu 
    290                 295                 300                 


Ser Ser Tyr Gly Met Asp Ser Arg Pro Pro Met Ala Ile Phe Glu Leu 
305                 310                 315                 320 


Leu Asp Tyr Ile Val Asn Glu Pro Pro Pro Lys Leu Pro Ser Gly Val 
                325                 330                 335     


Phe Ser Leu Glu Phe Gln Asp Phe Val Asn Lys Cys Leu Ile Lys Asn 
            340                 345                 350         


Pro Ala Glu Arg Ala Asp Leu Lys Gln Leu Met Val His Ala Phe Ile 
        355                 360                 365             


Lys Arg Ser Asp Ala Glu Glu Val Asp Phe Ala Gly Trp Leu Cys Ser 
    370                 375                 380                 


Thr Ile Gly Leu Asn Gln Pro Ser Thr Pro Thr His Ala Ala Gly Val 
385                 390                 395                 400 


<210>  2
<211>  1203
<212>  DNA
<213>  Artificial

<220>
<223>  DNA sequence encoding SEQ ID NO: 1.

<400>  2
atgtcgtact accatcacca tcaccatcac gattacgata tcccaacgac cgaaaacctg     60

tattttcagg gcgccatggg atcccccaag aagaagccga cgcccatcca gctgaacccg    120

gcccccgacg gctctgcagt taacgggacc agctctgcgg agaccaactt ggaggccttt    180

cttacccaga agcagaaggt gggagaactg aaggatgacg actttgagaa gatcagtgag    240

ctgggggctg gcaatggcgg tgtggtgttc aaggtctccc acaagccttc tggcctggtc    300

atggccagaa agctaattca tctggagatc aaacccgcaa tccggaacca gatcataagg    360

gagctgcagg ttctgcatga gtgcaactct ccgtacatcg tgggcttcta tggtgcgttc    420

tacagcgatg gcgagatcag tatctgcatg gagcacatgg atggaggttc tctggatcaa    480

gtcctgaaga aagctggaag aattcctgaa caaattttag gaaaagttag cattgctgta    540

ataaaaggcc tgacatatct gagggagaag cacaagatca tgcacagaga tgtcaagccc    600

tccaacatcc tagtcaactc ccgtggggag atcaagctct gtgactttgg ggtcagcggg    660

cagctcatcg acgaaatggc caacgacttc gtgggcacaa ggtcctacat gtcgccagaa    720

agactccagg ggactcatta ctctgtgcag tcagacatct ggagcatggg actgtctctg    780

gtagagatgg cggttgggag gtatcccatc cctcctccag atgccaagga gctggagctg    840

atgtttgggt gccaggtgga aggagatgcg gctgagaccc cacccaggcc aaggaccccc    900

gggaggcccc ttagctcata cggaatggac agccgacctc ccatggcaat ttttgagttg    960

ttggattaca tagtcaacga gcctcctcca aaactgccca gtggagtgtt cagtctggaa   1020

tttcaagatt ttgtgaataa atgcttaata aaaaaccccg cagagagagc agatttgaag   1080

caactcatgg ttcatgcttt tatcaagaga tctgatgctg aggaagtgga ttttgcaggt   1140

tggctctgct ccaccatcgg ccttaaccag cccagcacac caacccatgc tgctggcgtc   1200

taa                                                                 1203


<210>  3
<211>  428
<212>  PRT
<213>  Artificial

<220>
<223>  Residues 1-400 of human MEK2 with mutations S222E and S226D, and 
       an N-terminal 6-histidine tag.


<220>
<221>  MISC_FEATURE
<222>  (1)..(28)
<223>  N-terminal 6-histidine tag

<220>
<221>  MISC_FEATURE
<222>  (29)..(428)
<223>  Residues 1-400 of human MEK2 with mutations S222E and S226D

<400>  3

Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr 
1               5                   10                  15      


Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Glu Pro Met Leu Ala Arg 
            20                  25                  30          


Arg Lys Pro Val Leu Pro Ala Leu Thr Ile Asn Pro Thr Ile Ala Glu 
        35                  40                  45              


Gly Pro Ser Pro Thr Ser Glu Gly Ala Ser Glu Ala Asn Leu Val Asp 
    50                  55                  60                  


Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln Gln Lys Lys 
65                  70                  75                  80  


Arg Leu Glu Ala Phe Leu Thr Gln Lys Ala Lys Val Gly Glu Leu Lys 
                85                  90                  95      


Asp Asp Asp Phe Glu Arg Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly 
            100                 105                 110         


Val Val Thr Lys Val Gln His Arg Pro Ser Gly Leu Ile Met Ala Arg 
        115                 120                 125             


Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile Ile 
    130                 135                 140                 


Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr Ile Val Gly 
145                 150                 155                 160 


Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu 
                165                 170                 175     


His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Glu Ala Lys Arg 
            180                 185                 190         


Ile Pro Glu Glu Ile Leu Gly Lys Val Ser Ile Ala Val Leu Arg Gly 
        195                 200                 205             


Leu Ala Tyr Leu Arg Glu Lys His Gln Ile Met His Arg Asp Val Lys 
    210                 215                 220                 


Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp 
225                 230                 235                 240 


Phe Gly Val Ser Gly Gln Leu Ile Asp Glu Met Ala Asn Asp Phe Val 
                245                 250                 255     


Gly Thr Arg Ser Tyr Met Ala Pro Glu Arg Leu Gln Gly Thr His Tyr 
            260                 265                 270         


Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Leu 
        275                 280                 285             


Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys Glu Leu Glu 
    290                 295                 300                 


Ala Ile Phe Gly Arg Pro Val Val Asp Gly Glu Glu Gly Glu Pro His 
305                 310                 315                 320 


Ser Ile Ser Pro Arg Pro Arg Pro Pro Gly Arg Pro Val Ser Gly His 
                325                 330                 335     


Gly Met Asp Ser Arg Pro Ala Met Ala Ile Phe Glu Leu Leu Asp Tyr 
            340                 345                 350         


Ile Val Asn Glu Pro Pro Pro Lys Leu Pro Asn Gly Val Phe Thr Pro 
        355                 360                 365             


Asp Phe Gln Glu Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu 
    370                 375                 380                 


Arg Ala Asp Leu Lys Met Leu Thr Asn His Thr Phe Ile Lys Arg Ser 
385                 390                 395                 400 


Glu Val Glu Glu Val Asp Phe Ala Gly Trp Leu Cys Lys Thr Leu Arg 
                405                 410                 415     


Leu Asn Gln Pro Gly Thr Pro Thr Arg Thr Ala Val 
            420                 425             


<210>  4
<211>  1287
<212>  DNA
<213>  Artificial

<220>
<223>  DNA sequence encoding SEQ. I.D. No. 3.

<400>  4
atgtcgtact accatcacca tcaccatcac gattacgata tcccaacgac cgaaaacctg     60

tattttcagg gcgccatgga acccatgctg gcccggagga agccggtgct gccggcgctc    120

accatcaacc ctaccatcgc cgagggccca tcccctacca gcgagggcgc ctccgaggca    180

aacctggtgg acctgcagaa gaagctggag gagctggaac ttgacgagca gcagaagaag    240

cggctggaag cctttctcac ccagaaagcc aaggtcggcg aactcaaaga cgatgacttc    300

gaaaggatct cagagctggg cgcgggcaac ggcggggtgg tcaccaaagt ccagcacaga    360

ccctcgggcc tcatcatggc caggaagctg atccaccttg agatcaagcc ggccatccgg    420

aaccagatca tccgcgagct gcaggtcctg cacgaatgca actcgccgta catcgtgggc    480

ttctacgggg ccttctacag tgacggggag atcagcattt gcatggaaca catggacggc    540

ggctccctgg accaggtgct gaaagaggcc aagaggattc ccgaggagat cctggggaaa    600

gtcagcatcg cggttctccg gggcttggcg tacctccgag agaagcacca gatcatgcac    660

cgagatgtga agccctccaa catcctcgtg aactctagag gggagatcaa gctgtgtgac    720

ttcggggtga gcggccagct catagacgaa atggccaacg acttcgtggg cacgcgctcc    780

tacatggctc cggagcggtt gcagggcaca cattactcgg tgcagtcgga catctggagc    840

atgggcctgt ccctggtgga gctggccgtc ggaaggtacc ccatcccccc gcccgacgcc    900

aaagagctgg aggccatctt tggccggccc gtggtcgacg gggaagaagg agagcctcac    960

agcatctcgc ctcggccgag gccccccggg cgccccgtca gcggtcacgg gatggatagc   1020

cggcctgcca tggccatctt tgaactcctg gactatattg tgaacgagcc acctcctaag   1080

ctgcccaacg gtgtgttcac ccccgacttc caggagtttg tcaataaatg cctcatcaag   1140

aacccagcgg agcgggcgga cctgaagatg ctcacaaacc acaccttcat caagcggtcc   1200

gaggtggaag aagtggattt tgccggctgg ttgtgtaaaa ccctgcggct gaaccagccc   1260

ggcacaccca cgcgcaccgc cgtgtaa                                       1287


<210>  5
<211>  614
<212>  PRT
<213>  Artificial

<220>
<223>  Residues 1-379 of human ERK1 with an N-terminal GST tag.


<220>
<221>  MISC_FEATURE
<222>  (1)..(235)
<223>  N-terminal GST tag

<220>
<221>  MISC_FEATURE
<222>  (236)..(614)
<223>  Residues 1-379 of human ERK1

<400>  5

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 
1               5                   10                  15      


Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 
            20                  25                  30          


Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 
        35                  40                  45              


Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 
    50                  55                  60                  


Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 
65                  70                  75                  80  


Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 
                85                  90                  95      


Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 
            100                 105                 110         


Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 
        115                 120                 125             


Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 
    130                 135                 140                 


Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 
145                 150                 155                 160 


Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 
                165                 170                 175     


Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 
            180                 185                 190         


Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 
        195                 200                 205             


Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu 
    210                 215                 220                 


Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Gly Met Ala Ala Ala Ala 
225                 230                 235                 240 


Ala Gln Gly Gly Gly Gly Gly Glu Pro Arg Arg Thr Glu Gly Val Gly 
                245                 250                 255     


Pro Gly Val Pro Gly Glu Val Glu Met Val Lys Gly Gln Pro Phe Asp 
            260                 265                 270         


Val Gly Pro Arg Tyr Thr Gln Leu Gln Tyr Ile Gly Glu Gly Ala Tyr 
        275                 280                 285             


Gly Met Val Ser Ser Ala Tyr Asp His Val Arg Lys Thr Arg Val Ala 
    290                 295                 300                 


Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr Tyr Cys Gln Arg Thr 
305                 310                 315                 320 


Leu Arg Glu Ile Gln Ile Leu Leu Arg Phe Arg His Glu Asn Val Ile 
                325                 330                 335     


Gly Ile Arg Asp Ile Leu Arg Ala Ser Thr Leu Glu Ala Met Arg Asp 
            340                 345                 350         


Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp Leu Tyr Lys Leu Leu 
        355                 360                 365             


Lys Ser Gln Gln Leu Ser Asn Asp His Ile Cys Tyr Phe Leu Tyr Gln 
    370                 375                 380                 


Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn Val Leu His Arg 
385                 390                 395                 400 


Asp Leu Lys Pro Ser Asn Leu Leu Ile Asn Thr Thr Cys Asp Leu Lys 
                405                 410                 415     


Ile Cys Asp Phe Gly Leu Ala Arg Ile Ala Asp Pro Glu His Asp His 
            420                 425                 430         


Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro 
        435                 440                 445             


Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys Ser Ile Asp Ile Trp 
    450                 455                 460                 


Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser Asn Arg Pro Ile Phe 
465                 470                 475                 480 


Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His Ile Leu Gly Ile Leu 
                485                 490                 495     


Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile Ile Asn Met Lys Ala 
            500                 505                 510         


Arg Asn Tyr Leu Gln Ser Leu Pro Ser Lys Thr Lys Val Ala Trp Ala 
        515                 520                 525             


Lys Leu Phe Pro Lys Ser Asp Ser Lys Ala Leu Asp Leu Leu Asp Arg 
    530                 535                 540                 


Met Leu Thr Phe Asn Pro Asn Lys Arg Ile Thr Val Glu Glu Ala Leu 
545                 550                 555                 560 


Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro Thr Asp Glu Pro Val 
                565                 570                 575     


Ala Glu Glu Pro Phe Thr Phe Ala Met Glu Leu Asp Asp Leu Pro Lys 
            580                 585                 590         


Glu Arg Leu Lys Glu Leu Ile Phe Gln Glu Thr Ala Arg Phe Gln Pro 
        595                 600                 605             


Gly Val Leu Glu Ala Pro 
    610                 


<210>  6
<211>  1845
<212>  DNA
<213>  Artificial

<220>
<223>  DNA sequence encoding SEQ ID NO: 5.

<400>  6
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt     60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa    120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat    180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac    240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg    300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt    360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa    420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat    480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa    540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca    600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat    660

ctggaagttc tgttccaggg gcccctggga tccccgaatt ccgggatggc ggcggcggcg    720

gctcaggggg gcgggggcgg ggagccccgt agaaccgagg gggtcggccc gggggtcccg    780

ggggaggtgg agatggtgaa ggggcagccg ttcgacgtgg gcccgcgcta cacgcagttg    840

cagtacatcg gcgagggcgc gtacggcatg gtcagctcgg cctatgacca cgtgcgcaag    900

actcgcgtgg ccatcaagaa gatcagcccc ttcgaacatc agacctactg ccagcgcacg    960

ctccgggaga tccagatcct gctgcgcttc cgccatgaga atgtcatcgg catccgagac   1020

attctgcggg cgtccaccct ggaagccatg agagatgtct acattgtgca ggacctgatg   1080

gagactgacc tgtacaagtt gctgaaaagc cagcagctga gcaatgacca tatctgctac   1140

ttcctctacc agatcctgcg gggcctcaag tacatccact ccgccaacgt gctccaccga   1200

gatctaaagc cctccaacct gctcatcaac accacctgcg accttaagat ttgtgatttc   1260

ggcctggccc ggattgccga tcctgagcat gaccacaccg gcttcctgac ggagtatgtg   1320

gctacgcgct ggtaccgggc cccagagatc atgctgaact ccaagggcta taccaagtcc   1380

atcgacatct ggtctgtggg ctgcattctg gctgagatgc tctctaaccg gcccatcttc   1440

cctggcaagc actacctgga tcagctcaac cacattctgg gcatcctggg ctccccatcc   1500

caggaggacc tgaattgtat catcaacatg aaggcccgaa actacctaca gtctctgccc   1560

tccaagacca aggtggcttg ggccaagctt ttccccaagt cagactccaa agcccttgac   1620

ctgctggacc ggatgttaac ctttaacccc aataaacgga tcacagtgga ggaagcgctg   1680

gctcacccct acctggagca gtactatgac ccgacggatg agccagtggc cgaggagccc   1740

ttcaccttcg ccatggagct ggatgaccta cctaaggagc ggctgaagga gctcatcttc   1800

caggagacag cacgcttcca gcccggagtg ctggaggccc cctag                   1845


