1111111111111111 IIIIII IIIII 11111 1111111111 11111 lllll 111111111111111 111111111111111 11111111
`US 20040067234Al
`
`(19) United States
`(12) Patent Application Publication
`Einat et al.
`
`(10) Pub. No.: US 2004/0067234 Al
`Apr. 8, 2004
`(43) Pub. Date:
`
`(54)
`
`ISOCITRATE DEHYDROGENASE AND USES
`THEREOF
`
`(76)
`
`Inventors: Paz Einat, Nes Zionna (IL); Louis
`Deiss, Chicago, IL (US); Ruth Maya,
`Moshav Rinnatia (IL)
`
`Correspondence Address:
`John P. White
`Cooper & Dunham LLP
`1185 Avenue of the Americas
`New York, NY 10036 (US)
`
`(21) Appl. No.:
`
`10/618,143
`
`(22) Filed:
`
`Jul. 11, 2003
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/395,364, filed on Jul.
`11, 2002. Provisional application No. 60/428,805,
`filed on Nov. 25, 2002.
`
`Publication Classification
`
`Int. Cl.7 .................................................. A61K 39/395
`(51)
`(52) U.S. Cl. ............................ 424/155.1; 310/36; 514/12
`
`(57)
`
`ABSTRACT
`
`The present invention discloses uses for the IDH gene and/or
`polypeptide and/or modulators thereof in the diagnosis and
`treatment of apoptosis-related diseases.
`
`Rigel Exhibit 1032
`Page 1 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 1 of 12
`
`US 2004/0067234 Al
`
`Figure 1:
`ORF of lsocitrate dehydrogenase 1 (XM_055088)
`(nucleic acid sequence: SEQ ID N0:1: amino acid sequence: SEQ ID N0:2)
`
`1
`46
`91
`136
`181
`226
`
`271
`9
`316
`24
`361
`39
`406
`54
`451
`69
`496
`84
`541
`99
`586
`114
`631
`129
`676
`144
`721
`159
`766
`174
`811
`189
`856
`204
`901
`219
`946
`234
`991
`249
`1036
`264
`1081
`279
`1126
`294
`1171
`309
`1216
`324
`1261
`339
`1306
`354
`1351
`369
`
`GGC GGC GAA GCG GGG GCA CGC CCT CGC ACA CGC AGA GAT AAA TTG
`TGC TCC CAT GAC CTT TAT TTG GAA AGT GCC TGC GGG CCT AAA ATT
`GGC CTT TGT CCC ACC GAG TAC ACT CAG CAC TGT ACT TTA AAC CGG
`ATA AAC TGG GCT GTC TGG CAG GCG ATA AAC TAC ATT CAG TTG AGT
`CTG CAA GAC TGG GAG GAA CTG GGG TGA TAA GAA ATC TAT TCA CTG
`TCA AGG TTT ATT GAA GTC AAA ATG TCC AAA AAA ATC AGT GGC GGT
`G
`G
`K K I S
`MS
`TCT GTG GTA GAG ATG CAA GGA GAT GAA ATG ACA CGA ATC ATT TGG
`W
`I
`I
`R
`T
`M
`Q
`E
`D
`G
`M
`E
`V
`V
`S
`GAA TTG ATT AAA GAG AAA CTC ATT TTT CCC TAC GTG GAA T~G GAT
`D
`L
`V
`Y
`P
`E
`F
`I
`L
`K
`E
`K
`I
`L
`E
`CTA CAT AGC TAT GAT TTA GGC ATA GAG AAT CGT GAT GCC ACC AAC
`N
`T
`A
`D
`R
`N
`E
`I
`G
`L
`D
`Y
`S
`H
`L
`GAC CAA GTC ACC AAG GAT GCT GCA GAA GCT ATA AAG AAG CAT AAT
`N
`H
`K
`K
`I
`A
`E
`A
`A
`D
`K
`T
`V
`D
`Q
`GTT GGC GTC AAA TGT GCC ACT ATC ACT CCT GAT GAG AAG AGG GTT
`V
`R
`K
`E
`D
`P
`T
`I
`T
`A
`C
`K
`V
`G
`V
`GAG GAG TTC AAG TTG AAA CAA ATG TGG AAA TCA CCA AAT GGC ACC
`T
`G
`N
`P
`S
`Q M W K
`K
`L
`K
`F
`E
`E
`ATA CGA AAT ATT CTG GGT GGC ACG GTC TTC AGA GAA GCC ATT ATC
`I
`I
`A
`E
`R
`F
`V
`T
`G
`G
`L
`I
`N
`R
`I
`TGC AAA AAT ATC CCC CGG CTT GTG AGT GGA TGG GTA AAA CCT ATC
`I
`P
`K
`V
`W
`G
`S
`V
`R . L
`P
`I
`N
`K
`C
`ATC ATA GGT CGT CAT GCT TAT GGG GAT CAA TAC AGA GCA ACT GAT
`D
`T
`A
`R
`Y
`D
`Q
`G
`Y
`A
`H
`R
`G
`I
`I
`TTT GTT GTT CCT GGG CCT GGA AAA GTA GAG ATA ACC TAC ACA CCA
`P
`T
`Y
`T
`I
`E
`K V
`G
`P
`G
`P
`V V
`F
`AGT GAC GGA ACC CAA AAG GTG ACA TAC CTG GTA CAT AAC TTT GAA
`E
`F
`N
`H
`V
`L
`Y
`T
`Q
`V
`K
`T
`G
`D
`S
`GAA GGT GGT GGT GTT GCC ATG GGG ATG TAT AAT CAA GAT AAG TCA
`S
`K
`D
`N
`A M G M Y
`Q
`V
`G
`G
`G
`E
`ATT GAA GAT TTT GCA CAC AGT TCC TTC CAA ATG GCT CTG TCT AAG
`K
`S
`L
`A
`M
`F
`S
`Q
`S
`H
`A
`F
`D
`E
`I
`GGT TGG CCT TTG TAT CTG AGC ACC AAA AAC ACT ATT CTG AAG AAA
`s
`K
`L
`K
`y
`T
`N
`K
`T
`p
`G W
`L
`L
`TAT GAT GGG CGT TTT AAA GAC ATC TTT CAG GAG ATA TAT GAC AAG
`K
`D
`Y
`I
`E
`F
`I
`D
`K
`F
`R
`Q
`G
`D
`Y
`CAG TAC AAG TCC CAG TTT GAA GCT CAA AAG ATC TGG TAT GAG CAT
`H
`E
`I W Y
`K
`Q
`A
`E
`F
`Q
`S
`K
`Y
`Q
`AGG CTC ATC GAC GAC ATG GTG GCC CAA GCT ATG AAA TCA GAG GGA
`G
`E
`S
`K
`A
`M
`D M V
`A
`Q
`D
`I
`L
`R
`GGC TTC ATC TGG GCC TGT AAA AAC TAT GAT GGT GAC GTG CAG TCG
`S
`V
`Q
`D
`G
`D
`Y
`N
`K
`C
`I W A
`F
`G
`GAC TCT GTG GCC CAA GGG. TAT GGC TCT CTC GGC ATG ATG ACC AGC
`S
`T
`G M M
`L
`S
`G
`Y
`G
`A
`V
`S
`D
`Q
`GTG CTG GTT TGT CCA GAT GGC AAG ACA GTA GAA GCA GAG GCT GCC
`A
`A
`E
`A
`E
`V
`T
`K
`D G
`P
`C
`V
`L
`V
`CAC GGG ACT GTA ACC CGT CAC TAC CGC ATG TAC CAG AAA GGA CAG
`Q
`G
`K
`Q
`R M Y
`Y
`H
`R
`T
`V
`T
`G
`H
`GAG ACG TCC ACC AAT CCC ATT GCT TCC ATT TTT GCC TGG ACC AGA
`R
`T
`A W
`F
`I
`S
`A
`I
`P
`N
`T
`S
`T
`E
`GGG TTA GCC CAC AGA GCA AAG CTT GAT AAC AAT AAA GAG CTT GCC
`A
`L
`E
`K
`N
`N
`D
`L
`K
`A
`R
`H
`A
`L
`G
`TTC TTT GCA AAT GCT TTG GAA GAA GTC TCT ATT GAG ACA ATT GAG
`E
`I
`T
`E
`I
`S
`V
`E
`E
`L
`A
`N
`A
`F
`F
`GCT GGC TTC ATG ACC AAG GAC TTG GCT GCT TGC ATT AAA GGT TTA
`L
`G
`K
`A
`I
`A
`C
`L
`D
`K
`T
`F M
`G
`A
`
`45
`90
`135
`180
`225
`270
`8
`315
`23
`360
`38
`405
`53
`450
`68
`495
`83
`540
`98
`585
`113
`630
`128
`675
`143
`720
`158
`765
`173
`810
`188
`855
`203
`900
`218
`945
`233
`990
`248
`1035
`263
`1080
`278
`1125
`293
`1170
`308
`1215
`323
`1260
`338
`1305
`353
`1350
`368
`1395
`383
`
`Rigel Exhibit 1032
`Page 2 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 2 of 12
`
`US 2004/0067234 Al
`
`1396 CCC MT GTG CAA CGT TCT GAC TAC TTG AAT ACA TTT GAG TTC ATG
`y
`s
`T
`F
`E
`F
`M
`p
`D
`L
`N
`R
`Q
`N
`V
`384
`1441 GAT AM CTT GGA GM AAC_ TTG MG ATC AM CTA GCT CAG GCC AM
`A
`A
`K
`G
`L
`K
`Q
`I
`K
`L
`E
`L
`N
`D
`K
`399
`1486 CTT TAA GTT CAT ACC TGA GCT AAG AAG GAT AAT TGT CTT TTG GTA
`414
`*
`L
`1531 ACT AGG TCT ACA GGT TTA CAT TTT TCT GTG TTA CAC TCA AGG ATA
`1576 AAG GCA AM TCA ATT TTG TAA TTT GTT TAG AAG CCA GAG TTT ATC
`1621 TTT TCT ATA AGT TTA CAG CCT TTT TCT TAT ATA TAC AGT TAT TGC
`1666 CAC CTT TGT GAA CAT GGC AAG GGA CTT TTT TAC AAT TTT TAT TTT
`1711 ATT TTC TAG TAC CAG CCT AGG AAT TCG GTT AGT ACT CAT TTG TAT
`1756 TCA CTG TCA CTT TTT CTC ATG TTC TAA TTA TAA ATG ACC AM ATC
`1801 AAG ATT GCT CAA AAG GGT AM TGA TAG CCA CAG TAT TGC TCC CTA
`1846 AM TAT GCA TAA AGT AGA AAT TCA CTG CCT TCC CCT CCT GTC CAT
`1891 GAC CTT GGG CAC AGG GAA GTT CTG GTG TCA TAG ATA TCC CGT TTT
`1936 GTG AGG TAG AGC TGT GCA TTA AAC TTG CAC ATG ACT GGA ACG AAG
`1981 TAG GAG TGC AAC TCA AAT GTG TTG AAG ATA CTG CAG TCA TTT TTG
`2026 TAA AGA CCT TGC TGA ATG TTT CCA ATA GAC TM ATA CTG TTT AGG
`2071 CCG CAG GAG AGT TTG GAA TCC GGA ATA AAT ACT ACC TGG AGG TTT
`2116 GTC CTC TCC ATT TTT CTC TTT CTC CTC CTG GCC TGG CCT GAA TAT
`2161 TAT ACT ACT CTA AAT AGC ATA TTT CAT CCA AGT GCA ATA ATG TAA
`2206 GCT GAA TCT TTT TTG GAC TTC TGC TGG CCT GTT TTA TTT CTT TTA
`2251 TAT AAA TGT GAT TTC TCA GAA ATT GAT ATT AAA CAC TAT CT.T ATC
`2296 TTC TCC
`2301
`
`1440
`398
`1485
`413
`1530
`
`1575
`1620
`1665
`1710
`1755
`1800
`1845
`1890
`1935
`1980
`2025
`2070
`2115
`2160
`2205
`2250
`2295
`
`Rigel Exhibit 1032
`Page 3 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 3 of 12
`
`US 2004/0067234 Al
`
`Figure 2:
`ORF of lsocitrate dehydrogenase 2 (NM_002168)
`(nucleic acid sequence: SEQ ID N0:3: amino acid sequence: SEQ ID N0:4)
`
`1 ccagcgttagcccgcggccaggcagccgggaggagcggcgcgcgctcggacctctcccgc
`61 cctgctcgttcgctctccagcttggg~tgqccggctacctgcgggtcgtgcgctcgctct
`M A G Y L R V V R S L
`1
`121 gcagagcctcaggctcgcggccggcctgggcgccggcggccctgacagcccccacctcgc
`S
`P T
`T A
`L
`S G S R P . A W A P A A
`12 C R A
`181 aagagcagccgcggcgccactatgccgacaaaaggatcaaggtggcgaagcccgtggtgg
`I K V A K P V V
`32 Q E Q P R R H Y A D K R
`241 agatggatggtgatgagatgacccgtattatctggcagttcatcaaggagaagctcatcc
`I
`I K E K L
`F
`I W Q
`I
`52 E M D G D E M T R
`301 tgccccacgtggacatccagctaaagtattttgacctcgggctcccaaaccgtgaccaga
`I Q L K Y F D L G L P N R D Q
`72 L P H V D
`361 ctgatgaccaggtcaccattgactctgcactggccacccagaagtacagtgtggctgtca
`I· D S A L A T Q K Y S V A V
`92 T D D Q V T
`421 agtgtgccaccatcacccctgatgaggcccgtgtggaagagttcaagctgaagaagatgt
`T P D E A R V E E F K L K K M
`I
`112 K C A T
`481 ggaaaagtcccaatggaactatccggaacatcctgggggggactgtcttccgggagccca
`I L G G T V F R E P
`I R N
`132 W K S P N G T
`541 tcatctgcaaaaacatcccacgcctagtccctggctggaccaagcccatcaccattggca
`I G
`T
`I
`P R L V P G W T K P
`I
`I C K N
`152 I
`601 ggcacgcccatggcgaccagtacaaggccacagactttgtggcagaccgggccggcactt
`172 R H A H G D Q Y K A T D F V A D R A G T
`661 tcaaaatggtcttcaccccaaaagatggcagtggtgtcaaggagtgggaagtgtacaact
`192 F K M V F T P K D G S G V K E W E V Y N
`721 tccccgcaggcggcgtgggcatgggcatgtacaacaccgacgagtccatctcaggttttg
`S G F
`I
`212 F P A G G V G M G M Y N T D E S
`781 cgcacagctgcttccagtatgccatccagaagaaatggccgctgtacatgagcaccaaga
`I Q K K W P L Y M S T K
`232 A H S C F Q Y A
`841 acaccatactgaaagcctacgatgggcgtttcaaggacatcttccaggagatctttgaca
`F D
`I
`F Q E
`I
`L K A Y D G R F K D
`I
`252 N T
`901 agcactataagaccgacttcgacaagaataagatctggtatgagcaccggctcattgatg
`I D
`I W Y E H R L
`272 K H Y K T D F D K N K
`961 acatggtggctcaggtcctcaagtcttcgggtggctttgtgtgggcctgcaagaactatg
`292 D M V A Q V L K S S G G F V W A C K N Y
`1021 acggagatgtgcagtcagacatcctggcccagggctttggctcccttggcctgatgacgt
`I L A Q G F G S L G L M T
`312 D G D V Q S D
`1081 ccgtcctggtctgccctgatgggaagacgattgaggctgaggccgctcatgggaccgtca
`I E A E A A H G T V
`332 S V L V C P D G K T
`1141 cccgccactatcgggagcaccagaagggccggcccaccagcaccaaccccatcgccagca
`I A S
`352 T R H Y R E H Q K G R P T S T N P
`1201 tctttgcctggacacgtggcctggagcaccgggggaagctggatgggaaccaagacctca
`F A W T R G L E H R G K L D G N Q D L
`372 I
`1261 tcaggtttgcccagatgctggagaaggtgtgcgtggagacggtggagagtggagccatga
`392 I R F A Q M L E K V C V E T V E S G A M
`1321 ccaaggacctggcgggctgcattcacggcctcagcaatgtgaagctgaacgagcacttcc
`I H G L S N V K L N E H F
`412 T K D L A G C
`1381 tgaacaccacggacttcctcgacaccatcaagagcaacctggacagagccctgggcaggc
`I K S N L D R A L G R
`432 L N T T D F L D T
`1441 ag~9ggggaggcgccacccatggctgcagtggaggggccagggctgagccggcgggtcc
`*
`452 Q
`1501 tcctgagcgcggcagagggtgagcctcacagcccctctctggaggcctttctaggggatg
`1561 tttttttataagccagatgtttttaaaagcatatgtgtgtttcccctcatggtgacgtga
`1621 ggcaggagcagtgcgttttacctcagccagtcagtatgttttgcatactgtaatttatat
`1681 tgcccttggaacacatggtgccatatttagctactaaaaagctcttcacaaaaaaaaaaa
`
`Rigel Exhibit 1032
`Page 4 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 4 of 12
`
`US 2004/0067234 Al
`
`Figure 3:
`lsocitrate dehydrogenas anti sense fragment
`(SEQ ID NO: 5)
`
`S'TGCTCTGTGGGCTAACCCTCTGGTCCAGGCAAAAATGGAAGCAATGGGATTGGTGGACGTCTCCTGT
`CCTTTCTGGTACATGCGGTAGTGACGGGTTACAGTCCCGTGGGCAGCCTCTGCTTCTACCGTCTTGCCA
`TCTGGACAAACCAGCACGCTGGTCATCATGCCGAGAGAGCCATACCCTTGGGCCACAGAGTCCGACTGC
`ACGTCACCATCATAGTTTTTACAGGCCCAGATGAAGCCTCCCTCTGATCTCATAGCTGGGGCCACCATG
`TCGTCGATGAGCCTATGCTCATACCAGATCTTTTGAGCTTCAAACTGGGACTTGTACTGCTTGTCATAT
`ATCTCCTGAAAGATGTCTTTAAAACGCCCATCATATTTCTTCAGAATGGTGTTTTTGGTGCTCAGArAC
`AAAGGCCAACCCTTAGACAGAGCCATTTGGAAGGAACTGTGTGCAAAATCTTCAATTGACTTATCTTGA
`TTATACATCCCCATGACAACACCACCACCTTCTTCAAGTTATGTACCAGG'3
`
`Rigel Exhibit 1032
`Page 5 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 5 of 12
`
`US 2004/0067234 Al
`
`Figure 4:
`IDH siRNA sequence
`(SEQ ID N0:6)
`
`5' AATCGTGATGCCACCAACGAC
`
`'3
`
`Rigel Exhibit 1032
`Page 6 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 6 of 12
`
`US 2004/0067234 Al
`
`Figure 5:
`Alignment between lsocitrate dehydrogenase 1 (XM_055088) and ·As
`
`fragment
`
`ICD
`IRT-4Cl
`
`1
`1
`
`ICD
`IRT-4Cl
`
`61
`1
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`ICD
`IRT-4Cl
`
`121
`1
`
`181
`1
`
`241
`1
`
`301
`1
`
`361
`1
`
`421
`1
`
`481
`1
`
`541
`1
`
`601
`1
`
`661
`1
`
`721
`1
`
`781
`34
`
`841
`94
`
`901
`154
`
`961
`214
`
`ICD
`IRT-4Cl
`
`1021
`274
`
`ICD
`IRT-4Cl
`
`1081
`334
`
`GGCGGCGAAGCGGGGGCACGCCCTCGCACACGCAGAGATAAATTGTGCTCCCATGACCTT
`
`TATTTGGAAAGTGCCTGCGGGCCTAAAATTGGCCTTTGTCCCACCGAGTACACTCAGCAC
`
`TGTACTTTAAACCGGATAAACTGGGCTGTCTGGCAGGCGATAAACTACATTCAGTTGAGT
`
`CTGCAAGACTGGGAGGAACTGGGGTGATAAGAAATCTATTCACTGTCAAGGTTTATTGAA
`
`GTCAAAATGTCCAAAAAAATCAGTGGCGGTTCTGTGGTAGAGATGCAAGGAGATGAAATG
`
`ACACGAATCATTTGGGAATTGATTAAAGAGAAACTCATTTTTCCCTACGTGGAATTGGAT
`
`CTACATAGCTATGATTTAGGCATAGAGAATCGTGATGCCACCAACGACCAAGTCACCAAG
`
`GATGCTGCAGAAGCTATAAAGAAGCATAATGTTGGCGTCAAATGTGCCACTATCACTCCT
`
`GATGAGAAGAGGGTTGAGGAGTTCAAGTTGAAACAAATGTGGAAATCACCAAATGGCACC
`
`ATACGAAATATTCTGGGTGGCACGGTCTTCAGAGAAGCCATTATCTGCAAAAATATCCCC
`
`CGGCTTGTGAGTGGATGGGTAAAACCTATCATCATAGGTCGTCATGCTTATGGGGATCAA
`
`TACAGAGCAACTGATTTTGTTGTTCCTGGGCCTGGAAAAGTAGAGATAACCTACACACCA
`
`AGTGACGGAACCCAAAAGGTGACATAl!ll!l'll'IHll'
`jjjf-TTGAAGAAGGTGGTGGTGTT
`--------------------------
`
`TTGAAGAAGGTGGTGGTGTT
`
`CATGGGGATGTATAATCAAGATAAGTCAATTGAAGATTTTGCACACAGTTCCTTCC
`CATGGGGATGTATAATCAAGATAAGTCAATTGAAGATTTTGCACACAGTTCCTTCC
`
`TGGCTCTGTCTAAGGGTTGGCCTTTGTATCTGAGCACCAAAAACAC
`
`TGGCTCTGTCTAAGGGTTGGCCTTTGTATCTGAGCACCAAAAACAC 11111111■
`
`TATGATGGGCGTTTTAAAGACATCTTTCAGGAGATATATGACAAGCAGTACAAGTCCCAG
`TATGATGGGCGTTTTAAAGACATCTTTCAGGAGATATATGACAAGCAGTACAAGTCCCAG
`
`TTTGAAGCTCAAAAGATCTGGTATGAGCATAGGCTCATCGACGACATGGTGGCCC• GCT
`TTTGAAGCTCAAAAGATCTGGTATGAGCATAGGCTCATCGACGACATGGTGGCCC GCT
`
`• TCAGAGGGAGGCTTCATCTGGGCCTGTAAAAACTATGATGGTGACGTGCAGTCG
`
`TCAGAGGGAGGCTTCATCTGGGCCTGTAAAAACTATGATGGTGACGTGCAGTCG
`
`GACTCTGTGGCCCAAGGGTATGGCTCTCTCGGCATGATGACCAGCGTGCTGGTTTGTCC
`GACTCTGTGGCCCAAGGGTATGGCTCTCTCGGCATGATGACCAGCGTGCTGGTTTGTCC
`
`ICD
`IRT-4Cl
`
`1141
`394
`
`GATGGCAAGAC•GTAGAAGCAGAGGCTGCCCACGGGACTGTAACCCGTCACTACCGCATG
`GATGGCAAGAC GTAGAAGCAGAGGCTGCCCACGGGACTGTAACCCGTCACTACCGCATG
`
`ICD
`
`1201
`
`TACCAGAAAGGACAGGAGACGTCCACCAATCCCATTGCTTCCATTTTTGCCTGGACCAG
`
`Rigel Exhibit 1032
`Page 7 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 7 of 12
`
`US 2004/0067234 Al
`
`IRT-4Cl
`
`454 TACCAGAAAGGACAGGAGACGTCCACCAATCCCATTGCTTCCATTTTTGCCTGGACCAG
`
`ICD
`IRT-4Cl
`
`1261
`514
`
`ICD
`IRT-4Cl
`
`1321 TTGGAAGAAGTCTCTATTGAGACAATTGAGGCTGGCTTCATGACCAAGGACTTGGCTGCT
`531
`
`ICD
`IRT-4Cl
`
`1381 TGCATTAAAGGTTTACCCAATGTGCAACGTTCTGACTACTTGAATACATTTGAGTTCATG
`531
`
`ICD
`IRT-4Cl
`
`1441 GATAAACTTGGAGAAAACTTGAAGATCAAACTAGCTCAGGCCAAACTTTAAGTTCATACC
`531
`
`ICD
`IRT-4Cl
`
`1501 TGAGCTAAGAAGGATAATTGTCTTTTGGTAACTAGGTCTACAGGTTTACATTTTTCTGTG
`531
`
`ICD
`IRT-4Cl
`
`1561 TTACACTCAAGGATAAAGGCAAAATCAATTTTGTAATTTGTTTAGAAGCCAGAGTTTATC
`531
`
`ICD
`IRT-4Cl
`
`1621 TTTTCTATAAGTTTACAGCCTTTTTCTTATATATACAGTTATTGCCACCTTTGTGAACAT
`531
`
`ICD
`IRT-4Cl
`
`1681 GGCAAGGGACTTTTTTACAATTTTTATTTTATTTTCTAGTACCAGCCTAGGAATTCGGTT
`531
`
`ICD
`IRT-4Cl
`
`1741 AGTACTCATTTGTATTCACTGTCACTTTTTCTCATGTTCTAATTATAAATGACCAAAATC
`531
`
`ICD
`IRT-4Cl
`
`1801 AAGATTGCTCAAAAGGGTAAATGATAGCCACAGTATTGCTCCCTAAAATATGCATAAAGT
`531
`
`ICD
`IRT-4Cl
`
`1861 AGAAATTCACTGCCTTCCCCTCCTGTCCATGACCTTGGGCACAGGGAAGTTCTGGTGTCA
`531
`
`ICD
`IRT-4Cl
`
`1921 TAGATATCCCGTTTTGTGAGGTAGAGCTGTGCATTAAACTTGCACATGACTGGAACGAAG
`531
`
`ICD
`IRT-4Cl
`
`1981 TAGGAGTGCAACTCAAATGTGTTGAAGATACTGCAGTCATTTTTGTAAAGACCTTGCTGA
`531
`
`ICD
`IRT-4Cl
`
`2041 ATGTTTCCAATAGACTAAATACTGTTTAGGCCGCAGGAGAGTTTGGAATCCGGAATAAAT
`531
`
`ICD
`IRT-4Cl
`
`2101 ACTACCTGGAGGTTTGTCCTCTCCATTTTTCTCTTTCTCCTCCTGGCCTGGCCTGAATAT
`531
`
`ICD
`IRT-4Cl
`
`2161 TATACTACTCTAAATAGCATATTTCATCCAAGTGCAATAATGTAAGCTGAATCTTTTTTG
`531
`
`ICD
`IRT-4Cl
`
`2221 GACTTCTGCTGGCCTGTTTTATTTCTTTTATATAAATGTGATTTCTCAGAAATTGATATT
`531
`
`ICD
`IRT-4Cl
`
`2281 AAACACTATCTTATCTTCTCCTG
`531 -----------------------
`
`Rigel Exhibit 1032
`Page 8 of 39
`
`

`

`Patent Application Publication
`
`Apr. 8, 2004 Sheet 8 of 12
`
`US 2004/0067234 Al
`
`Figure 6
`Alignment between IDH2 and IDH1 amino acid sequences
`
`Score= 584 bits (1505), Expect= e-165
`281/397 (70%), Positives = 328/397 (81%) ,· Gaps
`Identities
`(0%)
`
`2/397
`
`.----u
`-'
`□
`IDH2: 50 WEMDGDEMTRIIWQFIKEKLILPHVDIQLKYFDLGLPNRDQTDDQVTIDSALATQKYSV 109
`WEM GDEMTRIIW+ IKEKLI P+V++ L +DLG+ NRD T+DQVT D+A A +K++V
`IDHl: 10 WEMQGDEMTRIIWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKHNV 69
`
`'----~' 0...
`DD...
`
`IDH2: 110 AVKCATITPDEARVEEFKLKKMWKSPNGTIRNILGGTVFREPIICKNIPRLVPGWTKPIT 169
`VKCATITPDE RVEEFKLK+MWKSPNGTIRNILGGTVFRE IICKNIPRLV GW KPI
`IDHl: 70 GVKCATITPDEKRVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPII 129
`
`IDH2: 170 IGRHAHGDQYKATDFVADRAGTFKMVFTPKDGSGVKEWEVYNFP-AGGVGMGMYNTDESI 228
`GGV MGMYN D+SI
`+ V+NF
`G ++ +TP DG+
`IGRHA+GDQY+ATDFV
`IDHl: 130 IGRHAYGDQYRATDFWPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGMYNQDKSI 189
`
`IDH2: 229 SGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKIWYEHR 288
`FAHS FQ A+ K WPLY+STKNTILK YDGRFKDIFQEI+DK YK+ F+ KIWYEHR
`IDHl: 190 EDFAHSSFQMALSKGWPLYLSTKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKIWYEHR 249
`
`IDH2: 289 LIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAAH 348
`LIDDMVAQ +KS GGF+WACKNYDGDVQSD +AQG+GSLG+MTSVLVCPDGKT+EAEAAH
`IDHl: 250 LIDDMVAQAMKSEGGFIWACKNYDGDVQSDSVAQGYGSLGMMTSVLVCPDGKTVEAEAAH 309
`
`IDH2: 349 GTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVES 408
`FA LE+V +ET+E+
`GTVTRHYR +QKG+ TSTNPIASIFAWTRGL HR KLD N++L
`IDHl: 310 GTVTRHYRMYQKGQETSTNPIASIFAWTRGLAHRAKLDNNKELAFFANALEEVSIETIEA 369
`
`IDH2: 409 GAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNL 445
`G MTKDLA CI GL NV+++ +LNT +F+D + NL
`IDH1: 370 GFMTKDLAACIKGLPNVQRSD-YLNTFEFMDKLGENL 405
`
`Rigel Exhibit 1032
`Page 9 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 9 of 12
`
`US 2004/0067234 Al
`
`Figure 7
`
`3
`
`2.5
`
`2
`
`e
`c
`0
`u
`m
`>
`0
`en 1.5
`·u5
`_g
`0..
`0
`0..
`(1)
`"O
`0
`LL 0.5
`
`-
`
`T
`.L
`
`T
`J_
`.
`
`1
`
`0 -
`
`FAS
`
`FAS+ INF
`
`Treatmen1
`
`Rigel Exhibit 1032
`Page 10 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 10 of 12 US 2004/0067234 Al
`Figure 8
`
`4.00
`
`3.50
`
`~·-
`0
`t..) ,_
`Q) > 0
`en
`"in
`.9 a.
`
`0 a. co -0
`
`"'O
`0
`LL
`
`e 3.00
`2.50 -
`
`2.00
`
`1.50
`
`1.00
`
`0.50 -
`
`0.00 -,
`
`_ ..
`
`IRT_4C1
`Treatment
`
`Rigel Exhibit 1032
`Page 11 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 11 of 12 US 2004/0067234 Al
`
`Figure 9
`
`A) apoptosis protection
`
`40.0
`
`30.0
`
`%
`ap
`0
`pt
`OS 20.0
`is
`
`~
`
`~
`
`"T"
`
`-
`
`10.0 ·
`
`0.0 -,
`
`Control
`
`IDH
`
`Full Length ORF
`
`Bl viability assay
`
`ro
`> -~
`
`::J
`(/)
`<1>
`Ol
`
`cu c
`cii a.
`
`<1>
`(.)
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`~
`
`-
`
`,-
`..L
`
`.
`
`Control
`
`IDH
`
`Rigel Exhibit 1032
`Page 12 of 39
`
`

`

`Patent Application Publication Apr. 8, 2004 Sheet 12 of 12 US 2004/0067234 Al
`
`Figure 10
`
`6 -
`
`5
`
`4 -
`
`3
`
`2 -
`
`0 -
`
`-
`
`T
`. I
`
`1
`
`IDH1 siRNA
`
`0
`
`0
`UJ
`·;;;
`
`0 ... -C
`(.) ... Cl) >
`0 -C.
`ctl -0
`
`0
`C.
`
`"C
`0
`LL
`
`Rigel Exhibit 1032
`Page 13 of 39
`
`

`

`US 2004/0067234 Al
`
`Apr. 8, 2004
`
`1
`
`ISOCITRATE DEHYDROGENASE AND USES
`THEREOF
`
`PRIORITY
`
`[0001] This application claims the benefit of U.S. provi(cid:173)
`sional patent application No. 60/395364, filed 11 Jul. 2002,
`and of U.S. provisional patent application No. 60/428805,
`filed 25 Nov. 2002, which are both hereby incorporated by
`reference in their entirety.
`
`FIELD OF THE INVENTION
`
`[0002] This invention relates to the field of treatment of
`apoptosis-related diseases, and screening for novel modula(cid:173)
`tors of such diseases.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Apoptosis, also known as 'programmed cell death',
`is an intrinsic program of cell self-destruction or "suicide",
`which is inherent in every eukaryotic cell. In response to a
`triggering stimulus, cells undergo a highly characteristic
`cascade of events manifested by cell shrinkage, blebbing of
`cell membranes, chromatin condensation and fragmentation,
`culminating in cell conversion to clusters of membrane(cid:173)
`bound particles ( a pop to tic bodies), which are thereafter
`engulfed by macrophages (Wyllie AH., et al., Int Rev. Cytol
`68:251-306, 1980).
`
`[0004] Apoptosis is now recognized as one of the more
`important biological processes, having a major role in nor(cid:173)
`mal
`tissue development and homeostasis. Moreover,
`derangement of apoptosis control has a role in the patho(cid:173)
`genesis of numerous medical disorders, ranging from dis(cid:173)
`orders of excessive apoptosis such as neurodegenerative
`disorders ( e.g., Alzheimer's disease or Parkinson's disease),
`to disorders wherein death of defective cells is inappropri(cid:173)
`ately inhibited, such as cancer (Bursch, W., et al., Trends
`Pharmacol. Sci., 13:245-251, 1992).
`
`[0005] Tumor drug resistance is a major problem in the
`treatment of cancer by chemotherapy. In the common epi(cid:173)
`thelial malignancies of adult life----carcinomas of the breast,
`colon and lung-the impact of chemotherapy has been
`disappointing. In the last few years, increasing efforts have
`been invested in obtaining a greater understanding of the
`response and resistance of cancer cells to chemotherapy by
`focussing on the role of apoptosis. The rationale behind this
`approach is that a mechanistic understanding of apoptosis
`will improve the chances of overcoming tumor drug resis(cid:173)
`tance.
`
`[0006] Apoptosis can be thought of as a "default" process,
`intrinsic to all cells, which is abrogated by the provision of
`survival signals. A framework for drug-induced apoptosis
`can be described in which a balance exists between intrinsic
`and extrinsic survival signals and drug-induced death sig(cid:173)
`nals. Pro- and anti-apoptotic signals impact upon apoptotic
`proteins which ultimately control the apoptotic process. This
`framework suggests multiple points at which therapeutic
`interventions could be made to overcome drug resistance
`and, in addition, generates novel molecular targets for the
`induction of apoptosis in cancer and other cells. Two areas
`of fundamental importance are the identification of novel
`agents, informed by a mechanistic understanding of the
`process of drug-induced apoptosis, and the modulation of
`
`cellular resistance to conventional agents, which would
`derive from a knowledge of the mechanisms that allow
`cancer cells to evade apoptosis after drug-induced damage
`(Makin, G. and Dive, C. Trends in Cell Biology 11:S22-S26,
`2001).
`
`SUMMARY OF THE INVENTION
`
`[0007] Applicants have unexpectedly discovered that the
`IDHl and IDH2 gene and/or polypeptide products play a
`role in preventing apoptosis, are anti-apoptopic, and provide
`a positive viability signal to the FAS induced apoptotic
`pathway. Furthermore, applicants have discovered that inhi(cid:173)
`bition of expression of the IDHl or IDH2 gene or neutral(cid:173)
`ization of the expression products promotes cell death.
`
`[0008]
`In accordance with these discoveries, the present
`invention provides methods for treating apoptosis related
`diseases, pharmaceutical compositions for treating apoptosis
`related diseases, diagnostic and prognostic processes in
`connection with apoptosis relates diseases, and screening
`processes aimed at obtaining IDH modulators.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0009]
`In the following description and claims use will be
`made, at times, of a variety of terms, and the meaning of
`such terms as they should be construed in accordance with
`the invention is as follows:
`
`[0010] "apoptosis"-a physiological type of cell death
`which results from activation of some cellular mechanisms,
`i.e. death which is controlled by the machinery of the cell.
`Apoptosis may, for example, be the result of activation of the
`cell machinery by an external trigger, e.g. a cytokine or
`anti-FAS antibody, which leads to cell death or by an internal
`signal. The term "programmed cell death" may also be used
`interchangeably with "apoptosis".
`
`[0011]
`"apoptosis-related disease" -a disease whose eti(cid:173)
`ology is related either wholly or partially to the process of
`apoptosis. The disease may be caused either by a malfunc(cid:173)
`tion of the apoptotic process (such as in cancer or an
`autoimmune disease) or by overactivity of the a pop to tic
`process (such as in certain neurodegenerative diseases).
`[0012]
`"Cancer" or "Tumor"-an uncontrolled growing
`mass of abnormal cells. These terms include both primary
`tumors, which may be benign or malignant, as well as
`secondary tumors, or metastases which have spread to other
`sites in the body. Examples of cancer-type diseases include,
`inter alia: carcinoma (e.g.: breast, colon and lung), leukemia
`such as B cell leukemia, lymphoma such as B-cell lym(cid:173)
`phoma, blastoma such as neuroblastoma and melanoma.
`
`[0013] The term "polynucleotide" refers to any molecule
`composed of DNA nucleotides, RNA nucleotides or a com(cid:173)
`bination of both types, i.e. that comprises two or more of the
`bases guanidine, citosine, timidine, adenine, uracil or
`inosine, inter alia. A polynucleotide may include natural
`nucleotides, chemically modified nucleotides and synthetic
`nucleotides, or chemical analogs thereof. The term encom(cid:173)
`passes "oligonucleotides" and "nucleic acids". A polynucle(cid:173)
`otide generally has from about 75 to 10,000 nucleotides,
`optionally from about 100 to 3,500 nucleotides. An oligo(cid:173)
`nucleotide refers generally to a chain of nucleotides extend(cid:173)
`ing from 2-500 nucleotides.
`
`Rigel Exhibit 1032
`Page 14 of 39
`
`

`

`US 2004/0067234 Al
`
`Apr. 8, 2004
`
`2
`
`[0014]
`"Amino acid"-a molecule which consists of any
`one of the 20 naturally occurring amino acids, amino acids
`which have been chemically modified (see below), or syn(cid:173)
`thetic amino acids.
`
`[0015] "Polypeptide"-a molecule composed of amino
`acids. The term includes peptides, polypeptides, proteins
`and peptidomimetics,
`
`[0016] A "peptidomimetic" is a compound containing
`non-peptidic structural elements that is capable of mimick(cid:173)
`ing the biological action(s) of a natural parent peptide. Some
`of the classical peptide characteristics such as enzymatically
`scissille peptidic bonds are normally not present in a pep(cid:173)
`tidomimetic.
`
`[0017] By "silencing RNA" (siRNA) is meant an RNA
`molecule which decreases or silences the expression of a
`gene/mRNA of its endogenous or cellular counterpart. The
`term is understood to encompass "RNA interference"
`(RNAi), and "double-stranded RNA" (dsRNA). For recent
`information on these terms and proposed mechanisms, see
`Bernstein E., Denli AM., Hannon G J: The rest is silence.
`RNA. 2001 November; 7(11):1509-21; and Nishikura K.: A
`short primer on RNAi: RNA-directed RNA polymerase acts
`as a key catalyst. Cell. 2001 Nov. 16; 107(4):415-8. One
`example of an siRNA, is the siRNA depicted in FIG. 4,
`which is a novel siRNAfor the IDH gene, and is considered
`part of the present invention. In one embodiment, the present
`invention therefore comprises an siRNA molecule for the
`IDH gene, having the sequence set forth in FIG. 4 (SEQ ID
`NO:6), and a vector comprising said siRNA.
`
`[0018] By the term "antisense" (AS) or "antisense frag(cid:173)
`ment" is meant a nucleic acid fragment having inhibitory
`antisense activity, said activity causing a decrease in the
`expression of the endogenous genomic copy of the corre(cid:173)
`sponding gene (in this case IDH). The sequence of the AS is
`designed to complement a target mRNA of interest and form
`an RNA:AS duplex. This duplex formation can prevent
`processing, splicing, transport or translation of the relevant
`mRNA. Moreover, certain AS nucleotide sequences can
`elicit cellular RNase H activity when hybridized with their
`target mRNA, resulting in mRNAdegradation (Calabretta et
`al, 1996: Antisense strategies in the treatment of leukemias.
`Semin Oneal. 23(1):78-87). In that case, RNase H will
`cleave the RNA component of the duplex and can potentially
`release the AS to further hybridize with additional molecules
`of the target RNA An additional mode of action results from
`the interaction of AS with genomic DNA to form a triple
`helix which can be transcriptionally inactive. The AS frag(cid:173)
`ment of the present invention optionally has the sequence
`depicted in FIG. 3 or a homologous sequence thereof.
`Particular AS fragments are the AS of the DNA encoding the
`particular fragments of IDH described herein.
`
`[0019]
`"Conservative substitution"-refers to the substi(cid:173)
`tution of an amino acid in one class by an amino acid of the
`same class, where a class is defined by common physico(cid:173)
`chemical amino acid side chain properties and high substi(cid:173)
`tution frequencies in homologous polypeptides found in
`nature, as determined, for example, by a standard Dayhoff
`frequency exchange matrix or BLOSUM matrix. Six general
`classes of amino acid side chains have been categorized and
`include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly);
`Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys);
`Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp).
`
`For example, substitution of an Asp for another class III
`residue such as Asn, Gin, or Glu, is a conservative substi(cid:173)
`tution.
`
`[0020]
`the
`"Non-conservative substitution" -refers to
`substitution of an amino acid in one class with an amino acid
`from another class; for example, substitution of an Ala, a
`class II residue, with a class III residue such as Asp, Asn,
`Glu, or Gin.
`
`[0021]
`"Chemically modified"-when referring to the
`product of the invention, means a product (polypeptide)
`where at least one of its amino acid residues is modified
`either by natural processes, such as processing or other
`post-translational modifications, or by chemical modifica(cid:173)
`tion techniques which are well known in the art. Among the
`numerous known modifications typical, but not exclusive
`examples include: acetylation, acylation, amidation, ADP(cid:173)
`ribosylation, glycosylation, GPI anchor formation, covalent
`attachment of a lipid or lipid derivative, methylation, myris(cid:173)
`tlyation, pegylation, prenylation, phosphorylation, ubiquti(cid:173)
`nation, or any similar process.
`
`[0022] "IDH gene"-the isocitrate dehydrogenase 1 cod(cid:173)
`ing sequence open reading frame, as shown in FIG. 1 (SEQ
`ID NO:1), or the
`isocitrate dehydrogenase 2 coding
`sequence open reading frame, as shown in FIG. 2 (SEQ ID
`NO:3), or any homologous sequence thereof preferably
`having at least 70% identity, more preferable 80% identity,
`even more preferably 90% or 95% identity. This encom(cid:173)
`passes any sequences derived from SEQ ID NO: 1 or SEQ ID
`NO:3 which have undergone mutations as described herein.
`
`[0023]
`"IDH polypeptide" refers to the polypeptide of the
`IDHl or IDH2 gene, and is understood to include, for the
`purposes of the instant invention, the terms "oxalosuccinate
`decarboxylase", "ICD", "CID", "IDP", "IDPS", "PICD",
`"ICDH", "HCID", "IDHM", "ICD-M", "mNADP-IDH" and
`"NADP+-specific IDHH", derived from any organism,
`optionally man, splice variants and fragments thereof retain(cid:173)
`ing viability activity, and homologs thereof, preferably hav(cid:173)
`ing at least 70%, more preferably at least 80%, even more
`preferably at least 90% or 95% homology thereto. In addi(cid:173)
`tion, this term is understood to encompass polypeptides
`resulting from minor alterations in the IDHl or IDH2 coding
`sequence, such as, inter alia, point mutations, substitutions
`deletions and insertions which may cause a difference in a
`few amino acids between the resultant polypeptide and the
`naturally occurring IDHl or IDH2. Polypeptides encoded by
`nucleic acid sequences which bind to the IDHl or IDH2
`coding sequence or genomic sequence under conditions of
`highly stringent hybridization, which are well-known in the
`art (for example Ausubel et al., Current Protocols in Molecu(cid:173)
`lar Biology, John Wiley and Sons, Baltimore, Md. (1988),
`updated in 1995 and 1998), are also encompassed by this
`term. Chemically modified IDHl or IDH2 or chemically
`modified fragments of IDHl or IDH2 are also included in
`the term, so long as the viability activity is retained. The
`polypeptide sequence of IDHl is depicted in FIG. 1 (SEQ
`ID NO: 2). The polypeptide sequence ofIDH2 is depicted in
`FIG. 2 (SEQ ID NO:4).
`
`[0024] While mostly IDHl is exemplified herein, it is to be
`understood that for all embodiments, IDH2 can effectively
`replace IDHl, or act in conjunction with or in addition to
`IDHl.
`
`Rigel Exhibit 1032
`Page 15 of 39
`
`

`

`US 2004/0067234 Al
`
`Apr. 8, 2004
`
`3
`
`[0025]
`"Viability activity" defines the capability of the
`IDH polypeptide to interfere with the apoptotic process in a
`cell thereby promoting the survival and viability of the cell.
`
`[0026]
`"Biologically active"-the capability of a molecule
`to modulate the apoptotic process.
`
`[0027] "Modulator"-any molecule that is capable of
`modulation, i.e. that either increases (promotes) or decreases
`(prevents). The term is understood to include partial or full
`inhibition, stimulation and enhancement. In the case of a
`modulator of a polypeptide, such as a the IDH polypeptide,
`the modulator may be a direct modulator of the biological
`activity of IDH, or it may be a modulator of the IDH gene;
`in the latter case, the viability activity of IDH is indirectly
`modulated by a modulator that affects the transcription or
`translation of the gene ( and does not directly act on the
`polypeptide). Modulators can include AS fragments, siR(cid:173)
`NAs, ribozymes, polypeptides, small chemical molecules
`and pigments, inte