targeting the untargeted kinome: the gsk published kinase inhibitor set … · 2016-01-08 ·...

Targeting the untargeted kinome: The GSK Published Kinase Inhibitor Set

Supplementary Material

Additional Files 1. PKIS Information. PKIS identifying codes, SMILES, and literature references. Available as an Excel

spreadsheet. 2. PKIS Compound structures. Available as an SDF file. 3. Heat map of Nanosyn screening data. Available as an Excel spreadsheet. 4. Heat map of DSF screening data. Available as an Excel spreadsheet. 5. GPCR screening data. Available as an Excel spreadsheet. 6. NCI-60 screening data. Available as an Excel spreadsheet. 7. Angiogenesis screening data. Available as an Excel spreadsheet.

Contents Supplementary Table 1. PKIS series information .............................................................................................3

Supplementary Table 2. Nanosyn kinase assay panel ......................................................................................4

Supplementary Table 3. Average hit rate for kinase sub-families ...................................................................5

Supplementary Table 4. Enhanced selectivity profiles of previously described inhibitors ..............................6

Supplementary Table 5. Example chemical probe starting points obtained by simple filtering. Kinases inhibited >40% inhibition at 0.1 µM, where the compound inhibits no more than 22 kinases (10%) at 1.0 µM, and where the kinase is in the top 5 hits for the compound. ...................................................................7

Supplementary Table 6. Example inhibitors of previously untargeted kinases from inhibition seen at 0.1 μM compound ............................................................................................................................................... 12

Supplementary Table 7. Example inhibitors of previously untargeted kinases from inhibition seen at 1.0 μM compound ............................................................................................................................................... 13

Supplementary Table 8. Example PLK1 inhibitors that also inhibit NEK9 ..................................................... 14

Supplementary Table 9. List of GPCRs tested for the PKIS ............................................................................ 15

Supplementary Table 10. Selectivity analysis of optimised LOK (STK10) inhibitors against a panel of 255 kinases (Nanosyn) .......................................................................................................................................... 16

Supplementary Table 11. Data collection and refinement statistics for co-crystal structures ..................... 23

Supplementary Figure 1. PKIS compound classes ......................................................................................... 24

Supplementary Figure 2. Correlation plots of kinase inhibition replicates ................................................... 25

Supplementary Figure 3. Frequency of inhibition of kinases by the PKIS ..................................................... 26

Supplementary Figure 4. Kinase selectivity profiles for selected prolific compounds .................................. 27

Supplementary Figure 5. Examples of specific inhibition of original targets ................................................ 28

Nature Biotechnology: doi:10.1038/nbt.3374

Supplementary Figure 6. Kinase target coverage by the PKIS compounds ................................................... 29

Supplementary Figure 7. Analysis of oxindole chemotype ........................................................................... 30

Supplementary Figure 8. Survey analysis for effects of substituents across multiple kinase targets ........... 31

Supplementary Figure 9. Correlation plots of BRSK1 and BRSK2 inhibition ................................................. 33

Supplementary Figure 10. GW768505A inhibits TrkA auto-phosphorylation in KM12 cells ........................ 34

Supplementary Figure 11. Evaluation of the selective LOK/SLK chemical probe GSK3037619A .................. 35

Supplementary Figure 12. Automated image analysis for angiogenesis screen ........................................... 36

Supplementary Material References ............................................................................................................. 38


Supplementary Table 1. PKIS series information

Series Series name Count Original kinase target(s) & subfamily References

1 4-Pyrimidinyl_ortho-aryl_azoles 31 p38 CMGC 1-4 2 Oxindoles 30 cRaf, CDK2, TrkA TK, CMGC 5-7 3 Furazan_benzimidazoles 25 MSK1, ROCK, Akt AGC 8-13 4 4-Anilino_quinazolines_and_related 25 EGFR/ErbB2 TK 14-18

5 Benzimidazole_N-thiophenes 21 IKKε, PLK1 Other 19-21

6 4-Pyridyl_ortho-aryl_azoles 18 p38, TGFβR1, BRAF CMGC, TK 1, 22-28

7 2H-3-Pyrimidinyl_pyrazolopyridazines 16 GSK3, CDK4 CMGC 29, 30

8 2-Amino_oxazoles 15 VEGFR2 TK 31 9 4-Hydrazinyl_pyrazolopyrimidines 15 GSK3 CMGC 32, 33

10 2,4-Dianilino_pyrrolopyrimidines 15 IGF-1R TK 34-36

11 Biaryl_amides 14 p38α CMGC 37-41

12 3-Vinyl_pyridines 13 cRAF TKL 42 13 Anilino_thienopyrimidines 12 EGFR/ErbB2 TK 43, 44

14 Benzimidazolyl_diaryl_ureas 12 TIE2/VEGFR2 TK 45 15 2-Aryl-3-pyridimidinyl_pyrazolopyridazines 12 GSK3 CMGC 30 16 2,4-Diamino_pyrimidines 12 VEGFR2, LCK TK 16, 46, 47

17 Maleimides 11 GSK3 CMGC 48, 49

18 Furopyrimidines_and_related 9 VEGFR2, TIE2 TK 50-53

19 Indazole-3-carboxamides 7 GSK3 CMGC 54, 55

20 3-Amino_pyrazolopyridines 7 GSK3 CMGC 54, 55

21 2-Pyridinyl_imidazoles_and_related 7 TGFBR TK 22, 27

22 4-Anilino-5-alkynyl_pyrimidines 7 EGFR/ErbB2 TK 56 23 3-Cyano_thiophenes 6 JNK2, JNK3 CMGC 57 24 Phenyl_carboxamides 6 IKKα, IKKβ Other 58 25 Indazole-5-carboxamides 6 ROCK AGC 59, 60

26 3-Amino_pyrazolopyridazines 4 GSK3 CMGC 55 27 3-Amino_pyrazoles 3 CDK2 CMGC 61 28 Imidazotriazines 3 PLK1 Other 23 29 4-Anilino_quinolines 2 RET TK 62 30 6-Phenyl_isoquinolines 2 IKKβ Other 63 31 3-Benzyl_pyrimidines 1 CSF1R TK 64


Supplementary Table 2. Nanosyn kinase assay panel

Protein kinase subfamily

Size of human kinome

subfamily 65

Number of unique kinases in assay panel

% of subfamily covered

Additional assays/notes

AGC 63 34 54 CAMK 74 34 46

CK1 12 4 33

CMGC 64 24 38 2 CDK2 assays with different cyclins

STE 48 13 27

TK 90 69 78 19 disease relevant mutants and 2 LYN

splice variants

TKL 43 4 9 2 disease relevant mutants

Atypical 38 0 0 Other 86 14 16

Lipid kinases Phosphoinositide66 19 3 16

Sphingosine67 2 2 10


Supplementary Table 3. Average hit rate for kinase sub-families

Kinome group Hit rate* at 0.1 M Hit rate at 1.0 M AGC 0.9% 3.4%

CAMK 0.4% 2.7% CK1 0.3% 2.5%

CMGC 2.2% 6.9% STE 1.9% 13.5% TK 1.9% 7.0% TKL 0.5% 2.1%

Atypical N/A N/A Other 0.9% 4.4% Lipid 0% 1.5%

Sphingosine 0% 0% *Hit rate is defined as #(hits >50%)/(#inhibitors * #(kinases in sub-family))


Supplementary Table 4. Enhanced selectivity profiles of previously described inhibitors

Compound Original target %I at 100 nM Selectivity notes

SB-686709-A GSK3β GSK3α: 85% GSK3β: 81%

No other kinases above 15% I at 100 nM; 2 kinases above 40% at 1 M

GW632580X cFMS (CSFR) 78% 3 kinases above 20% I at 100 nM and 1 M (TRK A/B/C)

SB-590885-AAD BRAF BRAF: 63%

BRAF V599E: 72%

No other kinases above 25% I at 100 nM; 11 additional kinases above 20% at 1 M

SB-245392 p38 (MAPK14)

80% 1 additional kinase above 20% at 100 nM

(MAPKAPK3); 6 additional kinases above 20 % at 1 M

GSK317314A PLK1 95% 2 additional kinases above 20% at 100 nM (NEK9, LOK); 8 additional kinases above 20

% at 1 M


Supplementary Table 5. Example chemical probe starting points obtained by simple filtering. Kinases inhibited >40% inhibition at 0.1 µM, where the compound inhibits no more than 22 kinases (10%) at 1.0 µM, and where the kinase is in the top 5 hits for the compound.

Kinase Compound Rank %I

ABL-H396P GW711782X 1 47

ABL-H396P GW813360X 1 47

ABL-H396P GW643971X 4 52

ABL-H396P GW817394X 5 51

ABL-M351T GW711782X 2 44

ABL-M351T GW813360X 2 45

ABL-M351T GW643971X 3 54

ABL-M351T GW817394X 4 52

ABL-M351T GW817396X 5 52

ABL-Q252H GW817394X 1 61

ABL-Q252H GW643971X 2 58

ABL-Q252H GW817396X 2 64

ABL-Q252H GW813360X 4 40

ABL-Y253F GW817394X 2 58

ABL-Y253F GW813360X 3 45

ABL-Y253F GW817396X 3 59

ABL-Y253F GW643971X 5 52

AKT1 GSK554170A 4 61

AKT2 GSK554170A 3 74

AKT2 GSK614526A 3 80

AKT3 GSK554170A 5 58

ALK GSK1511931A 2 52

ALK GSK1751853A 2 63

ALK GSK1392956A 3 83

ALK GSK1713088A 3 56

ALK GSK1819799A 3 68

ALK GSK2186269A 3 90

ARG GW829877X 1 64

BRAF SB-614067-R 1 50

BRAF SB-590885-AAD 2 63

BRAF SB-682330-A 2 49

BRAF-V599E SB-590885-AAD 1 72

BRAF-V599E SB-682330-A 1 56

BRAF-V599E SB-614067-R 2 42

BRSK1 GSK204925A 2 42

BRSK2 GSK204925A 1 58

CDK2/cyclinA GW280670X 2 47



CDK2/cyclinA GW352430A 3 51

CDK2/cyclinA SB-675259-M 5 66

CDK2/cyclinE GW280670X 1 58



CDK2/cyclinE GW352430A 2 53



CDK4/cyclinD SB-732881 4 48

CDK4/cyclinD SB-732881-H 4 52

CK1a SB-220025-A 2 67

CK1a SB-220025-R 2 61

CK1a SB-223133 2 62

CK1a SB-236687 2 53

CLK2 GW807982X 1 81

CLK2 GW810372X 1 95

CLK2 GW819077X 1 57

DDR2 GW589961A 1 84

DDR2 GW694234A 1 98

DDR2 GW694590A 1 77

DDR2 GW700494A 2 74

DYRK1A SB-675259-M 3 74

DYRK1B SB-675259-M 4 69

EGFR GI261520A 1 63

EGFR GSK300014A 1 61


EGFR GW282449A 1 87

EGFR GW284372X 1 64

EGFR GW566221A 1 80

EGFR GW568377A 1 67

EGFR GW574783B 1 82

EGFR GW576924A 1 84

EGFR GW580496A 1 69

EGFR GW615311X 1 72

EGFR GW616030X 1 69

EGFR GW621823A 1 78

EGFR GW633459A 1 65

EGFR GW659893X 1 45

EGFR GW684626B 1 45

EGFR GW693881A 1 66

EGFR GW703087X 1 57

EGFR GW772405X 1 62

EGFR GW794726X 1 70

EGFR GW799251X 1 48


EGFR GW807930X 1 61






EGFR GW282974X 2 88

EGFR GW301888X 2 67

EGFR GW458787A 2 84

EGFR GW583373A 2 74

EGFR GW784684X 2 87

EGFR GW869810X 2 77

EGFR GW461104A 3 77

EGFR GW576609A 3 81

EGFR GW680191X 3 76

EGFR GW784684X 3 84

EGFR-L858R GW461104A 1 100

EGFR-L858R GW583373A 1 75

EGFR-L858R GW643971X 1 64

EGFR-L858R GW680191X 1 97

EGFR-L858R GW574783B 2 62

EGFR-L858R GW576609A 2 88

EGFR-L858R GW580496A 2 49

EGFR-L858R GW282449A 3 72

EGFR-L858R GW282974X 3 65

EGFR-L858R GW576924A 3 43

EGFR-L858R GW621823A 3 53

EGFR-L858R GW829055X 3 44

EGFR-L858R GW869810X 4 41

EGFR-L858R GW784684X 5 78

EGFR-L861Q GW461104A 2 94

EGFR-L861Q GW616030X 2 42

EGFR-L861Q GW680191X 2 92

EGFR-L861Q GW580496A 3 48

EGFR-L861Q GW869810X 3 43

EGFR-L861Q GW282449A 4 56

EGFR-L861Q GW574783B 4 53

EGFR-L861Q GW583373A 4 67

EGFR-L861Q GW621823A 4 53

EGFR-L861Q GW282974X 5 50

EGFR-L861Q GW576609A 5 77

ERBB2 GW549390X 1 44

ERBB2 GW633459A 2 44

ERBB2 GW282974X 4 53

ERBB2 GW576609A 4 78

ERBB4 GR269666A 1 98

ERBB4 GSK182497A 1 93

ERBB4 GSK192082A 1 94

ERBB4 GSK200398A 1 107

ERBB4 GSK238063A 1 107

ERBB4 GSK238583A 1 112

ERBB4 GSK259178A 1 87

ERBB4 GW282974X 1 91

ERBB4 GW301888X 1 101

ERBB4 GW458787A 1 85

ERBB4 GW576609A 1 89

ERBB4 GW784684X 1 91

ERBB4 GW827102X 1 48

ERBB4 GW869810X 1 82

ERBB4 GI261520A 2 56

ERBB4 GW282449A 2 84

ERBB4 GW566221A 2 55

ERBB4 GW576924A 2 69

ERBB4 GW621823A 2 76

ERBB4 GW574783B 3 60

ERBB4 GW583373A 3 70

ERBB4 GW461104A 4 47

ERBB4 GW580496A 4 43

ERBB4 GW784684X 4 82

FLT3 GW352430A 1 64

FLT3 GW575533A 2 77

FLT3 GW577921A 2 61

FLT3 GW631581B 2 72

FLT3 GW627512B 3 62

FLT-3-D835Y GW352430A 4 51

FMS GW572401X 1 42

FMS GW632580X 1 78

FMS GW830365A 1 80

GSK3A GW817396X 1 64

GSK3A SB-358518 1 48

GSK3A SB-675259-M 1 81

GSK3A SB-678557-A 1 80

GSK3A SB-686709-A 1 85

GSK3A SB-698596-AC 1 89

GSK3A SB-732881 1 78

GSK3A SB-732881-H 1 80

GSK3A SB-739245-AC 1 47

GSK3A GW817394X 3 54

GSK3A GW352430A 5 50

GSK3B SB-675259-M 2 80

GSK3B SB-678557-A 2 71

GSK3B SB-686709-A 2 81


GSK3B SB-698596-AC 2 85

GSK3B SB-732881 2 75

GSK3B SB-732881-H 2 77

GSK3B GW817396X 4 55

HIPK1 GW810372X 2 85

HIPK4 GW810372X 3 51

IGF1R GSK2186269A 1 103

IGF1R GSK1713088A 2 57

IGF1R GSK1751853A 3 58

IGF1R GSK1392956A 4 81

IGF1R GSK1819799A 5 66

IKKE GSK554170A 1 90

IKKE GSK614526A 1 90

INSR GSK1392956A 1 110

INSR GSK1713088A 1 64

INSR GSK1751853A 1 69

INSR GSK2219385A 1 53

INSR GSK1819799A 2 83

INSR GSK2186269A 2 93

IRR GSK2186269A 5 72

KDR GW786460X 1 61

KDR SB-476429-A 1 69

KIT GW575533A 1 79

KIT GW577921A 1 78

KIT GW627512B 1 85

KIT GW631581B 1 78

KIT GW759710A 1 42

KIT GW694234A 2 85

KIT GW280670X 3 41

KIT GW589961A 4 73

KIT GW700494A 4 49

KIT GW830365A 4 60

KIT-T670I GW627512B 2 69

KIT-T670I GW575533A 4 46

KIT-V560G GW575533A 5 46

KIT-V560G GW589961A 5 70

KIT-V560G GW830365A 5 57

LOK GSK237700A 2 54

LOK GSK312948A 2 76

LOK GW843682X 2 76

LOK GW852849X 2 67

LOK SB-630812 2 52

LOK SB-633825 2 44

LOK GSK579289A 3 50

LOK GSK978744A 3 48

LOK GSK571989A 4 63

LRRK2-G2019S GW405841X 1 58

LRRK2-G2019S GSK2219385A 2 46

LRRK2-G2019S GW429374A 3 47

LRRK2-G2019S GSK1751853A 5 42

LTK GSK1819799A 4 66

LTK GSK2186269A 4 76

MAP4K4 GW335962X 1 45

MAP4K4 GW429374A 1 69

MAP4K4 GW405841X 2 45

MAP4K4 GW711782X 3 43

MAPK3 GW301789X 1 93

MAPKAPK3 SB-284847-BT 2 41

MER GW576924A 4 42

MINK GW429374A 4 45

MKNK1 SB-739245-AC 2 42

MKNK1 SB-732881 3 55

MKNK1 SB-732881-H 3 58

MSK1 GSK466317A 3 56

MSK1 SB-772077-B 4 68

MSK1 GSK248233A 5 60

MSK2 GSK248233A 4 61

MST1 GW416469X 1 44

MUSK GSK466317A 4 50

MUSK GSK571989A 5 42

NEK9 GW837331X 1 55

NEK9 GSK579289A 2 62

NEK9 GSK978744A 2 62

NEK9 GSK571989A 3 77

P38α GW434756X 1 41

P38α GW561436X 1 75

P38α GW569293E 1 64

P38α GW581744X 1 75

P38α GW743024X 1 50

P38α GW829055X 1 46

P38α SB-220025-A 1 73

P38α SB-220025-R 1 67

P38α SB-220455 1 50

P38α SB-221466 1 64

P38α SB-223133 1 71

P38α SB-226879 1 66

P38α SB-236687 1 84

P38α SB-239272 1 81

P38α SB-242717 1 59

P38α SB-242718 1 53

P38α SB-242719 1 87

P38α SB-245392 1 80


P38α SB-250715 1 77

P38α SB-251505 1 66

P38α SB-251527 1 81

P38α SB-253228 1 48

P38α SB-264866 1 63

P38α SB-278538 1 89

P38α SB-278539 1 68

P38α SB-284847-BT 1 87

P38α GW775608X 2 65

P38α SB-476429-A 3 41

P38β GW769076X 1 41

P38β GW775608X 1 69

P38β GW561436X 2 45

P38β GW581744X 2 50

P38β SB-242719 2 41

P38β SB-250715 2 52

P38β SB-278538 2 48

P38β SB-236687 3 51

P70S6K1 GSK248233A 3 72

PAK5 GSK938890A 2 87

PAK6 GSK938890A 1 91

PDGFRα GW659386A 1 49

PDGFRα GW843682X 3 47

PDGFRα GW852849X 3 47

PDGFRα GW694234A 5 75

PDGFRα-T674I GW589961A 2 77




PDGFRα-V561D GW659386A 2 41






PDGFRβ GW843682X 4 45

PDGFRβ GW852849X 4 41

PI4-K-β GW405841X 3 44

PIM1 SB-772077-B 2 74

PKA GSK614526A 5 69

PKA GSK938890A 5 77

PKC-η GSK949675A 1 79

PKC-η GSK554170A 2 84

PKC-η GSK614526A 2 87

PKC-η GSK938890A 3 81

PLK1 GSK1023156A 1 78

PLK1 GSK237700A 1 91

PLK1 GSK312948A 1 90

PLK1 GSK317314A 1 95

PLK1 GSK317315A 1 97

PLK1 GSK571989A 1 98

PLK1 GSK579289A 1 96

PLK1 GSK978744A 1 92

PLK1 GW804482X 1 67

PLK1 GW843682X 1 93

PLK1 GW852849X 1 94

PLK1 GW853606X 1 81

PLK1 GW837331X 2 47

PRKG2 SB-772077-B 3 73

PRKX GSK614526A 4 77



PYK2 GSK1819799A 1 91

PYK2 GSK1392956A 2 87

PYK2 GSK1751853A 4 58

RET GW700494A 1 76

RET SB-476429-A 2 48

RET-V804L GW441756X 1 46

RET-Y791F GW700494A 3 66

ROCK1 GSK180736A 2 62

ROCK1 GSK248233A 2 85

ROCK1 GSK270822A 2 74

ROCK1 GSK299115A 2 69

ROCK1 GSK466314A 2 68

ROCK1 GSK466317A 2 71

ROCK1 SB-772077-B 5 65

ROCK2 GSK180736A 1 64

ROCK2 GSK248233A 1 86

ROCK2 GSK270822A 1 77

ROCK2 GSK299115A 1 71

ROCK2 GSK466314A 1 70

ROCK2 GSK466317A 1 72

ROCK2 SB-772077-B 1 75

SNF1LK (SIK) GW432441X 1 47

SNF1LK (SIK) GW435821X 1 49

TIE2 SB-437013 1 53

TIE2 SB-630812 1 77

TIE2 SB-633825 1 75

TNK1 GW429374A 2 53

TNK1 GW442130X 2 40

TRKC GW442130X 1 42

TTK GSK1511931A 1 52


TTK GSK571989A 2 94

TTK GSK1392956A 5 74

TXK GSK625137A 1 45

YES GW829055X 2 45


Supplementary Table 6. Example inhibitors of previously untargeted kinases from inhibition seen at 0.1 μM compound

Compound Structure Kinase %I @100 nM

Additional kinases with significant

inhibition @ 100 nM

Additional kinases

with >50%I @ 1.0 M

SB-739245-AC

MKNK1: 42% (IC50 = 50 nM)

3CDK6: 31%

(IC50 = 57 nM) GSK3α: 47%

(IC50 = 57 nM) GSK3β: 26%

(IC50 = 78 nM)

3 (CDK6, GSK3α, GSK3β)

GSK204925A

BRSK1: 42% (IC50 = 53 nM) BRSK2: 58%

(IC50 = 27 nM)

2 PLK1: 32%

(IC50 = 110 nM) LOK: 25%

(IC50 = 190 nM)

4 (PLK1, LOK,

PDGFRα, PDGFRα)

SB-633825 LOK: 44% (IC50 = 66 nM)

2 TIE2: 75%

(IC50 = 3.5 nM) BRK: 29%

(IC50 = 150 nM)

2 (TIE2, BRK)


Supplementary Table 7. Example inhibitors of previously untargeted kinases from inhibition seen at 1.0 μM compound

Compound Structure Top Kinases inhibited at 1.0 μM (% inhibition)

SB-711237

CDK6 38MAP4K4 33ABL1 28

GW861893X

NEK9 81LRRK2 47AURKA 44

SB-242721

F

NN

HN

N

O

N

N

MAPK14 74MAPKAPK3 31PIK3CA 20PIK3CD 18

GW445015X

SIK1 40ABL1 32MKNK1 29EPHA4 26


Supplementary Table 8. Example PLK1 inhibitors that also inhibit NEK9

Compound Structure Top Kinases inhibited at 1.0 μM (% inhibition)

GSK317314A PLK1 NEK9 STK10

95 35 20

GSK571989A PLK1 TTK

NEK9

98 94 77


97 36 35

GW837331X

O

NH

NN

FF

F

NN

OO

NEK9 PLK1

NUAK1

55 47 35


92 88 65


Supplementary Table 9. List of GPCRs tested for the PKIS

Receptor Reference agonist used M1, muscarinic acetylcholine receptor Acetylcholine M3, muscarinic acetylcholine receptor Acetylcholine M3D, M3 DREADD68 Clozapine N-Oxide (CNO) M5, muscarinic acetylcholine receptor Acetylcholine H1, Histamine receptor Histamine 1A, 1A adrenergic receptor Norepinephrine 1D, 1D adrenergic receptor Norepinephrine 5-HT2A, serotonin receptor 5-HT 5-HT2B, serotonin receptor 5-HT 5-HT2C, serotonin receptor 5-HT V1a, vasopressin receptor [Arg8]-Vasopressin V1b, vasopressin receptor [Arg8]-Vasopressin V2, vasopressin receptor [Arg8]-Vasopressin OT, oxytocin receptor Oxytocin P2Y1, purinergic receptor ADPP2Y2, purinergic receptor ATP or UTP P2Y4, purinergic receptor UTP P2Y6, purinergic receptor UDP P2Y11, purinergic receptor ATP NK1, tachykinin receptor Substance P NK2, tachykinin receptor Neurokinin A NK3, tachykinin receptor Neurokinin B PAR1, proteinase-activated receptor TRAP-6


Supplementary Table 10. Selectivity analysis of optimised LOK (STK10) inhibitors against a panel of 255 kinases (Nanosyn)

Kinase SB-440719 Kinase GSK3037619A

LOK 34 LOK 68

SLK 21 SLK 47

SGK1 18 RIPK2 24

SGK2 17 HCK 20

STK25 16 STK16 20

HCK 15 SGK1 19

CHEK1 13 TNIK 19

MST4 12 SGK2 18

STK16 11 MINK 17

JAK1 11 GRK6 12

SIK 10 BRK 12

EPH-B2 9 P38-ALPHA 11

PKC-ZETA 9 STK25 11

PI4-K-BETA 9 IRAK1 11

ZAP70 9 MST4 11

PKN2 9 EPH-B2 10

SRC 9 PKN2 10

CDK2 9 MUSK 10

MUSK 8 CDK2 10

FGFR4 8 PI3-KINASE-DELTA 9

PLK2 8 TRKA 9

PHK-GAMMA1 8 RET 9

MINK 7 JAK1 9

RET 7 BLK 8

TEC 7 ZAP70 8 CDK6-CYCLIND3 7 SRC 8

TRKA 7 PHK-GAMMA1 7 SNF1LK2 6 LRRK2-G2019S 7

GRK7 6 ERB-B4 7 CLK4 6 YES 7 NEK6 6 FGFR2 7 MELK 6 FGFR4 7

LRRK2-G2019S 6 TSSK2 6 CAMK4 6 MELK 6

MAP4K4 6 SNF1LK2 6 MST1 6 CAMK4 6

PHK-GAMMA2 5 BRSK1 6


PAR-1B-ALPHA 5 SIK 6 FGFR2 5 MSK2 6

LCK 5 TNK2 6 BRK 5 LCK 6 FES 5 EPH-A8 6

PRKD3 5 PKC-ETA 6 IRAK1 5 TEC 6 CLK2 5 PKC-BETA2 5 BLK 5 FAK 5

TSSK2 5 MRCK-ALPHA 5 MARK1 5 TAOK3 5 EPH-A8 5 FGR 5 MST3 5 CDK6-CYCLIND3 5 TNIK 5 KDR 5

PRKD2 5 CHEK1 5 DYRK4 5 DCAMKL2 5 HASPIN 5 PKC-THETA 5 NDRG1 5 PRKD3 5

PKC-BETA1 5 NEK6 5 ITK 5 NEK1 5

MRCK-ALPHA 4 DYRK4 5 AURORA-B 4 FES 5 PKC-BETA2 4 AURORA-B 5

PKC-ETA 4 LTK 5 MAPKAPK-2 4 INSR 4

ERB-B4 4 PKC-BETA1 4 YES 4 CAMK1D 4

EPH-A5 4 MST3 4 CK1-GAMMA3 4 TBK1 4

FLT-4 4 PHK-GAMMA2 4 PKC-THETA 4 CSK 4

LATS2 4 HIPK3 4 TXK 4 HIPK1 4

ARK5 4 CDK4-CYCLIND 4 DYRK3 4 BMX 4 CHEK2 4 EPH-A4 4

CSK 4 EPH-A1 4 PLK1 4 PERK 4

CDK2-CYCLINE 4 FYN 4 CAMK1D 4 CAMK2A 4

FLT-3 4 CLK2 4 EPH-A1 4 PKN1 4 HIPK1 4 DDR2 4


GRK6 4 MST1 4 INSR 4 PRKX 4

CAMK1A 3 RSK3 4 RSK1 3 MER 4 FYN 3 MAP4K4 4

SRPK2 3 PAK6 4 CAMK2D 3 TXK 4

JAK3 3 EPH-A5 4 MSK2 3 MARK1 3 TNK2 3 TRKB 3 PAK3 3 FGFR3 3

TAK1-TAB1 3 PLK2 3 PKC-EPSILON 3 RSK1 3

HIPK3 3 PKC-ZETA 3 AMP-A1B1G1 3 DAPK1 3

EPH-A4 3 FLT-3 3 LTK 3 CDK7 3

PI3-KINASE-GAMMA 3 PIM3 3 ARG 3 TYRO3 3 TRKB 3 RON 3 AKT2 3 EPH-A2 3

EPH-A2 3 HIPK2 3 MAPKAPK-3 3 ARK5 3

FRAP1 3 MST2 3 PRKX 3 PI4-K-BETA 3 HIPK2 3 CK1-GAMMA3 3 TSSK1 3 CAMK2D 3 JNK2 3 PRKD2 3 CRAF 3 LATS2 3 AKT3 3 PI3-KINASE-GAMMA 3

DAPK1 3 TAOK2 3 PKN1 3 MAPKAPK-2 3 BRSK2 3 MKNK1 3

CAMK2A 3 LYNB 3 GSK-3-BETA 2 AKT3 3

CK1-GAMMA1 2 AXL 3 NEK1 2 MNK2 3 MNK2 2 CHEK2 3

CDK5-P25 2 TRKC 2 CDK9-CYCLINT1 2 MRCK-BETA 2

HIPK4 2 TAK1-TAB1 2 FGR 2 CAMK1A 2 ALK 2 CDK1 2


MARK3 2 EPH-B1 2 MRCK-BETA 2 PAK3 2

RSK3 2 TSSK1 2 DYRK1A 2 HASPIN 2

FER 2 MAPKAPK-3 2 CDK1 2 AMP-A1B1G1 2

CDK4-CYCLIND 2 BTK 2 PRKG1 2 GSK-3-BETA 2 MKNK1 2 PAK1 2

FMS 2 ROCK2 2 ROCK2 2 AKT2 2

GSK-3-ALPHA 2 PLK1 2 JNK3 2 DYRK3 2 FAK 2 CDK5-P25 2

TRKC 2 ITK 2 EPH-B1 2 NEK9 2

RSK4 2 P70S6K1 1 CLK3 2 BRSK2 1 AXL 2 CDK9-CYCLINT1 1

PI3-KINASE-DELTA 2 DYRK1A 1 TBK1 1 EPH-A3 1

MAP4K5 1 HIPK4 1 RON 1 RSK4 1

ROCK1 1 MARK4 1 MER 1 AKT1 1 PTK5 1 PTK5 1

P38-ALPHA 1 TTK 1 MARK4 1 ARG 1

ROS 1 IGF1R 1 PAK5 1 SYK 1 MET 1 CLK1 1 NEK9 1 MAP4K5 1 PLK3 1 JAK3 1 DDR2 1 AURORA-C 1 PAK1 1 CK1-GAMMA1 1 SYK 1 PAR-1B-ALPHA 1

FGFR3 1 PYK2-FAK2 1 EGFR 1 P38-BETA 1 LYNB 1 CRAF 1 CDK7 1 ROS 0 IRR 1 PKC-EPSILON 0

MAP4K2 1 ROCK1 0 BTK 1 CLK3 0


CLK1 1 GRK7 0 MEK1 1 NDRG1 0 PAK6 1 IRR 0

DCAMKL2 1 EGFR 0 CK1-GAMMA2 0 SRPK1 0

AKT1 0 FLT-1 0 P38-DELTA 0 PKC-GAMMA 0

FLT-1 0 MEK2 0 IGF1R 0 PDGFR-ALPHA 0

EPH-A3 0 AMP-A2B1G1 0 MST2 0 PDK1 0 NEK2 0 MARK3 0 PASK 0 IKK-ALPHA 0

PKACB 0 NEK2 0 PRAK 0 CK1-GAMMA2 0 NDR2 0 FLT-4 0 BMX 0 MAP4K2 0

FGFR1 0 EPH-B3 -1 PIM3 0 PAK4 -1

CAMK2B 0 MAPK1 -1 BRAF 0 CLK4 -1

AURORA-A -1 PASK -1 CDK5 -1 PRAK -1 ABL1 -1 SPHK2 -1

AURORA-C -1 ALK -1 PKC-ALPHA -1 EPH-B4 -1 IKK-ALPHA -1 LYNA -1

PIM-1-KINASE -1 SPHK1 -1 P38-BETA -1 PRKD1 -1

EPH-B3 -1 BRAF -1 KDR -1 MET -1

SPHK2 -1 PRKG1 -1 MAPK1 -1 CAMK2B -1

JNK1 -1 MSK1 -1 SRPK1 -1 PI3KB -1

PDGFR-BETA -1 MEK1 -1 TIE2 -1 PKACB -1 TTK -1 FMS -2

TYRO3 -1 ABL1 -2 PIM2 -1 JNK1 -2

PDGFR-ALPHA -1 GSK-3-ALPHA -2 SRMS -1 PAK5 -2 PDK1 -2 PIM2 -2


EPH-B4 -2 FRAP1 -2 MSK1 -2 MSSK1 -2 TAOK2 -2 P38-DELTA -2

PKC-GAMMA -2 RSK2 -2 TYK2 -2 CK1 -2

P38-GAMMA -2 CAMK2G -2 P70S6K1 -2 ERB-B2 -2

RSK2 -2 PDGFR-BETA -2 PRKD1 -2 TNK1 -2

PYK2-FAK2 -2 AURORA-A -3 TAOK3 -2 PKC-IOTA -3 SPHK1 -2 CDK5 -3

CAMK2G -2 TYK2 -3 PKA -2 JNK2 -3 CK1 -2 P38-GAMMA -4

MEK2 -2 PKA -4 RIPK2 -2 DYRK1B -4 PERK -2 PIM-1-KINASE -4

ERB-B2 -2 SRMS -4 PAK2 -3 PAK2 -4 CK2 -3 JAK2 -4

PKC-IOTA -3 FGFR1 -4 MSSK1 -3 MAPK3 -4 LYNA -3 NEK7 -5

MAPK3 -3 IKK-BETA -5 TNK1 -3 FER -5 BRSK1 -3 IRAK4 -5 DAPK3 -4 P70S6K2 -5

IKK-BETA -4 DAPK3 -5 CDK3-CYCLINE -4 PLK4 -5

PRKG2 -4 PRKG2 -5 JAK2 -5 JNK3 -5

P70S6K2 -5 CK2 -5 NEK7 -5 PKC-ALPHA -5

PI3-KINASE-ALPHA -5 TIE2 -5 PI3KB -5 PI3-KINASE-ALPHA -5 IRAK4 -5 NDR2 -5 DYRK2 -5 CDK3-CYCLINE -6

DYRK1B -6 PLK3 -6 PAK4 -6 CDK2-CYCLINE -6 PLK4 -7 SRPK2 -7 KIT -8 DYRK2 -9

AMP-A2B1G1 -8 SGK3 -9


IKK-EPSILON -10 KIT -11 SGK3 -10 IKK-EPSILON -14

The percentage inhibition values were measured in duplicate at 0.1 μM compound. Kinases are listed in descending order of percentage inhibition, and the 22 most inhibited kinases for each compound are shown.


Supplementary Table 11. Data collection and refinement statistics for co-crystal structures

STK10 : SB-633825 (form 1)

STK10 : SB-633825 (form 2)

SLK : SB-440719

PDB ID 4USD 4USE 4USF

Crystallisation conditions 0.2M NH4Cl, 0.1M Tris pH 8.0, 20% PEG 6000,

10% Ethylene glycol

50% (v/v) PEG 300, 0.1M cacodylate pH

6.5, 0.2M MgCl2

20% PEG6000, 10% ethylene glycol, 0.1 M

MES pH 6.0, 0.1 M MgCl2

Space group P212121 P21 P22121

No. of molecules in the asymmetric unit

2 2 1

Unit cell dimensions a, b, c (Å) , , (°)

46.7, 99.6, 151.8 90.0, 90.0, 90.0

62.4, 50.3, 133.7 90.0, 101.2, 90.0

49.9, 75.3, 189.6 90.0, 90.0, 90.0

Data collection

Beamline Diamond I04-1 Diamond I03 Diamond I03

Resolution range (Å)a 60.36-3.05(3.34-3.05)

65.59-2.65(2.79-2.65)

48.40-1.75 (1.78-1.75)

Unique observationsa 12981 (3009) 23790 (3436) 72706 (3952)

Average multiplicitya 3.7 (3.4) 3.0 (2.9) 4.1 (4.2)

Completeness (%)a 92.8 (91.5) 99.5 (99.5) 99.5 (99.4)

Rmergea 0.11 (0.65) 0.12 (0.91) 0.07 (0.77)

Mean ((I)/σ(I))a 8.3 (2.0) 5.3 (1.8) 10.6 (1.8)

Refinement

Resolution range (Å) 83.41-3.05 131.18-2.65 94.78-1.75

R-value, Rfree 0.23, 0.29 0.22, 0.25 0.20, 0.23

r.m.s. deviation from ideal bond length (Å) 0.011 0.008 0.010

r.m.s. deviation from ideal bond angle ()

1.49 1.27 1.35

a. Values within parentheses refer to the highest resolution shell.


Supplementary Figure 1. PKIS compound classes

Supplementary Figure 1. (a) The size and diversity of the PKIS compound classes, with representative structures. The height of each bar represents the number of compounds within each class. The segments within each indicate different compound clusters, defined by a sphere exclusion method. Compound clustering was based upon Daylight Tanimoto fingerprint similarity (www.daylight.com) with a cluster radius of 0.85, as outlined by Taylor 69 and applied by Martin, Kofron and Traphagen 70. (b) Histograms of molecular weight and cLog P values for PKIS compounds.


Supplementary Figure 2. Correlation plots of kinase inhibition replicates

Supplementary Figure 2. Correlation plots of kinase inhibition responses replicates for (a) and (b).


Supplementary Figure 3. Frequency of inhibition of kinases by the PKIS

Supplementary Figure 3. Frequency of inhibition of kinases by the PKIS. Increasing size and red color of circles is related to the number of PKIS compounds inhibiting each kinase by 50% at 0.1 µM compound.


Supplementary Figure 4. Kinase selectivity profiles for selected prolific compounds

Supplementary Figure 4. Kinase selectivity profiles for selected prolific compounds. Red circles represent kinases inhibited > 50% at 0.1 μM compound.


Supplementary Figure 5. Examples of specific inhibition of original targets

Supplementary Figure 5. Examples of specific inhibition of original targets. (a) Inhibition profiles for selected compounds at 0.1 M compound. (b) Correlation of GSK3 and GSK3 at 0.1 M and 1.0 M compounds.


Supplementary Figure 6. Kinase target coverage by the PKIS compounds

Supplementary Figure 6. Kinase target coverage by the PKIS compounds. (a) Kinases screened by inhibition assay are marked with a blue circle and kinases for which at least one compound provided >50% inhibition at 0.1 µM are marked with red circles; kinases where > 50% was only observed at 1.0 µM are marked with yellow circles. (b) Kinases screened by DSF are marked with a blue circle and kinases for which at least one compound provided a >4 °C stabilisation are marked with yellow circles.


Supplementary Figure 7. Analysis of oxindole chemotype

Supplementary Figure 7 - Analysis of oxindole chemotype. (a) Heatmap of inhibition for oxindoles at 1.0 µM compound. The two compounds with a 7-methyl group (b) show lack of inhibition of most kinases in contrast to the majority of compounds in the chemotype. (c) Example oxindole with 3,5-dihalobenzylidene substituent. (d) Oxindoles with 5- or 4,5- substituents show stronger inhibition of MAP4K4 and MINK1 but lower selectivity. (e) Two oxindoles with an ethyl- or propyl- imidazole that are activators of FER tyrosine kinase.


Supplementary Figure 8. Survey analysis for effects of substituents across multiple kinase targets

Supplementary Figure 8 - Survey analysis for effects of substituents across multiple kinase targets. (a) Inhibition heat maps for selected kinases against chemotype 7 (2H-3- pyrimidinyl pyrazolopyridazines) with the alternating horizontal yellow and blue stripes illustrating promiscuous and selective inhibitors respectively,


according to the absence or presence of a 6-substituent. (b) Addition of a m-CF3 group increases inhibition but decreases selectivity. (c) Also for chemotype 14 (Benzimidazolyl diaryl ureas) a m-substituent on the hydrophobic back pocket binding motif increases inhibition against the primary targets but decreases selectivity.


Supplementary Figure 9. Correlation plots of BRSK1 and BRSK2 inhibition

Supplementary Figure 9. Correlation of BRSK1 and BRSK2 inhibition at 0.1 M and 1.0 M compounds.


Supplementary Figure 10. GW768505A inhibits TrkA auto-phosphorylation in KM12 cells

Supplementary Figure 10. GW768505A inhibits TrkA auto-phosphorylation in KM12 cells and has a slower inhibition kinetic compared to control inhibitor GW441756X. Western blots were probed with anti-TrkA-pY496 and anti-TrkA. A GAPDH control is shown at the bottom panel. A representative experiment out of three repeats is shown.


Supplementary Figure 11. Evaluation of the selective LOK/SLK chemical probe GSK3037619A

Supplementary Figure 11. Evaluation of the selective LOK/SLK chemical probe. (a) and (b) Biolayer Interferometry (BLI) analysis of binding of GSK3037619A to LOK. GSK3037619A bound LOK with a dissociation constant (Kd) of 63 nM. (c) and (d) BLI analysis of binding of GSK3037619A to SLK, which showed a Kd of 199 nM. (e) Western blot to assess the effect of GSK3037619A on ERM (ezrin, radixin or moesin) phosphorylation in Jurkat cells. Blots were probed with anti-phospho-ERM antibody and then stripped and probed with anti-moesin antibody as well as GAPDH. All compounds were used at 1 μM concentration. Erlotinib (previously shown to inhibit LOK but at 10 μM) and Staurosporine were included as controls. Calyculin was used at 80 μM. Note that repeated experiments show some variability. In particular, application of the inhibitors was found to cause variation in the levels of total moesin (a little variation seen in this figure) which can confound the analysis of the phospho- modified proteins. The biological mechanisms of phospho-ERM regulation remain to be investigated.


Supplementary Figure 12. Automated image analysis for angiogenesis screen

Supplementary Figure 12. (a) Region-of-interest is created on 16-bit images and this is followed by an automated image segmentation to determine the tubules (white mask) and branching points (red dots) by Metamorph software (Molecular Devices). (b) Scatter plot distribution showing the results of the high content screening of the PKIS using the endothelial tube formation assay. Dots represent the raw data of total tubule length (pixel), with controls in blue (vehicle, DMSO), positive controls in red (Suramin, 10M) and PKIS compounds in green. Representative fluorescent images showing HUVECs treated with vehicle (DMSO) or positive control are shown to the right of the plot. Vehicle results in a well-developed endothelial capillary-like


network whereas the positive control (Suramin) inhibits the formation of endothelial network. (c) Correlation plot of the two replicates of PKIS compounds used in the screen, R2=0.7.


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