3-benzyl-1-(trans-4-((5-cyanopyridin-2-yl)amino)cy. clohexyl)-1-arylurea derivatives as novel and. selective cyclin-dependent kinase 12 (CDK12)

Átírás

1 SUPPORTING INFORMATION Discovery of 3-benzyl-1-(trans-4-((5-cyanopyridin-2-yl)amino)cy clohexyl)-1-arylurea derivatives as novel and selective cyclin-dependent kinase 12 (CDK12) inhibitors Masahiro Ito,*, Toshio Tanaka,, # Akinori Toita, Noriko Uchiyama, Hironori Kokubo,, # Nao Morishita, Michael G. Klein, Hua Zou, Morio Murakami, Mitsuyo Kondo,,# Tomoya Sameshima, Shinsuke Araki, Satoshi Endo,, Tomohiro Kawamoto,,# Gregg B. Morin,, Samuel A. Aparicio, Atsushi Nakanishi, Hironobu Maezaki,,# and Yasuhiro Imaeda*, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa , Japan Department of Structural Biology, Takeda California Inc., Science Center Dr., San Diego, CA 92121, United States Genome Sciences Centre, British Columbia Cancer Agency, 675 W. 10th Avenue, Vancouver, BC, V5Z 1L3, Canada S1

2 Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada Department of Molecular Oncology, British Columbia Cancer Agency, 675 W. 10th Avenue, Vancouver, BC, V5Z 1L3, Canada Corresponding Author * (M.I.) phone: , masahiro.ito@takeda.com; (Y.I.) phone: , yasuhiro.imaeda@takeda.com Present Addresses # For T. Tanaka, H. Kokubo, M. Kondo, T. Kawamoto, and H. Maezaki: Axcelead Drug Discovery Partners Inc., Fujisawa-shi, Kanagawa, , Japan. For S. Endo: Academic Research and Industrial Collaboration Management Office, Kyushu University, Momochihama, Sawara-ku, Fukuoka, , Japan. TABLE OF CONTENTS Supporting information 1 (Overlay of co-crystal structures (PDB:4NST and PDB: 6CKX)).. S3 Supporting information 2 (The global kinase selectivity of 2)... S4 9 Supporting information 3 (Time dependency for CDK12 inhibition)... S10 11 Supporting information 4 (Co-crystal structure of 29 and CDK12)... S12 16 Supporting information 5 (Single crystal X-ray structure analyses)... S17 22 References... S23 S2

3 Supporting Information 1 (Overlay of co-crystal structures (PDB:4NST and PDB: 6CKX)) Supplementary Figure 1. Overlay of co-crystal structures (PDB:4NST, ligand: ADP, orange; PDB: 6CKX, ligand: compound 29, cyan). The C-terminal extension of CDK12 (PDB:4NST) associates with the bound nucleotide in the ATP cleft. In contrast, the C-terminal extension in the complex of CDK12 and compound 29 was disordered and not defined (PDB:6CKX) and the phenylpyrazole moiety in compound 29 extends out toward the solvent region in which the C-terminal extension could reside. S3

4 Supporting Information 2 (The global kinase selectivity of 2) Z-lyte Life Technologies' SelectScreen Profiling Service: Single Point Results SelectScreen Scientist: Derek Johnston Date: SSBK-Z'-LYTE (Paisley UK) Quality Assurance Review: Andrew Heuze Date: Legend < 40% Inhibition % Phosphorylation Pass 40% - 80% Inhibition Z' Determination Pass 80% Inhibition Project # Compound No. Development 1X Test Compound Difference Between Reaction Concentration [ATP] Tested Kinase Tested % Inhibition % Inhibition Data Points Interference Test Compound Interference (nm) (μm) Point 1 Point 2 mean Point 1 - Point 2 Coumarin Fluorescein SSBK9686_ Km app ABL1 E255K Pass Pass Pass 0.89 PV3864/34528 SSBK9686_ Km app ABL1 G250E Pass Pass Pass 0.90 PV3865/34529 SSBK9686_ Km app ABL1 T315I Pass Pass Pass 0.84 PV3866/39639 SSBK9686_ Km app ABL1 Y253F Pass Pass Pass 0.88 PV3863/34531 SSBK9686_ Km app ABL Pass Pass Pass 0.77 P3049/ SSBK9686_ Km app ABL2 (Arg) Pass Pass Pass 0.86 PV3266/ SSBK9686_ Km app ACVR1B (ALK4) Pass Pass Pass 0.88 PV4312/ SSBK9686_ Km app ADRBK1 (GRK2) Pass Pass Pass 0.85 PV3361/ SSBK9686_ Km app ADRBK2 (GRK3) Pass Pass Pass 0.91 PV3827/38897 SSBK9686_ Km app AKT1 (PKB alpha) Pass Pass Pass 0.85 P2999/ SSBK9686_ Km app AKT2 (PKB beta) Pass Pass Pass 0.87 PV3184/28770 SSBK9686_ Km app AKT3 (PKB gamma) Pass Pass Pass 0.88 PV3185/28771 SSBK9686_ Km app ALK Pass Pass Pass 0.86 PV3867/ SSBK9686_ Km app AMPK A1/B1/G Pass Pass Pass 0.84 PV4672/ SSBK9686_ Km app AMPK A2/B1/G Pass Pass Pass 0.70 PV4674/ SSBK9686_ Km app AURKA (Aurora A) Pass Pass Pass 0.68 PV3612/32155 SSBK9686_ Km app AURKB (Aurora B) Pass Pass Pass 0.90 PV6130/ SSBK9686_ Km app AURKC (Aurora C) Pass Pass Pass 0.74 PV3856/ SSBK9686_ Km app AXL Pass Pass Pass 0.77 PV3971/ SSBK9686_ Km app BLK Pass Pass Pass 0.74 PV3683/33635 SSBK9686_ Km app BMX Pass Pass Pass 0.78 PV3371/ SSBK9686_ BRAF V599E Pass Pass Pass 0.63 PV3849/ SSBK9686_ BRAF Pass Pass Pass 0.81 PV3848/ SSBK9686_ Km app BRSK1 (SAD1) Pass Pass Pass 0.89 PV4333/36097 SSBK9686_ Km app BTK Pass Pass Pass 0.81 PV3363/ SSBK9686_ Km app CAMK1D (CaMKI delta) Pass Pass Pass 0.82 PV3663/ SSBK9686_ Km app CAMK2A (CaMKII alpha) Pass Pass Pass 0.80 PV3142/28192 SSBK9686_ Km app CAMK2B (CaMKII beta) Pass Pass Pass 0.81 PV4205/35330 SSBK9686_ Km app CAMK2D (CaMKII delta) Pass Pass Pass 0.78 PV3373/31647 SSBK9686_ Km app CAMK4 (CaMKIV) Pass Pass Pass 0.68 PV3310/ SSBK9686_ Km app CDC42 BPA (MRCKA) Pass Pass Pass 0.67 PV4398/ SSBK9686_ Km app CDC42 BPB (MRCKB) Pass Pass Pass 0.78 PV4399/36845 SSBK9686_ Km app CDK1/cyclin B Pass Pass Pass 0.91 PV3292/ SSBK9686_ Km app CDK2/cyclin A Pass Pass Pass 0.82 PV3267/ SSBK9686_ Km app CDK5/p Pass Pass Pass 0.91 PV4676/ SSBK9686_ Km app CDK5/p Pass Pass Pass 0.83 PV3000/25348 SSBK9686_ Km app CHEK1 (CHK1) Pass Pass Pass 0.70 P3040/28702 SSBK9686_ Km app CHEK2 (CHK2) Pass Pass Pass 0.74 PV3367/ SSBK9686_ Km app CLK Pass Pass Pass 0.83 PV3315/ SSBK9686_ Km app CLK Pass Pass Pass 0.86 PV4201/ SSBK9686_ Km app CLK Pass Pass Pass 0.92 PV3826/ SSBK9686_ Km app CSF1R (FMS) Pass Pass Pass 0.80 PV3249/ SSBK9686_ Km app CSK Pass Pass Pass 0.90 P2927/ SSBK9686_ Km app CSNK1A1 (CK1 alpha 1) Pass Pass Pass 0.91 PV3850/ SSBK9686_ Km app CSNK1D (CK1 delta) Pass Pass Pass 0.85 PV3665/ SSBK9686_ Km app CSNK1E (CK1 epsilon) Pass Pass Pass 0.85 PV3500/ SSBK9686_ Km app CSNK1G1 (CK1 gamma 1) Pass Pass Pass 0.72 PV3825/34360 SSBK9686_ Km app CSNK1G2 (CK1 gamma 2) Pass Pass Pass 0.83 PV3499/ SSBK9686_ Km app CSNK1G3 (CK1 gamma 3) Pass Pass Pass 0.80 PV3838/ SSBK9686_ Km app CSNK2A1 (CK2 alpha 1) Pass Pass Pass 0.81 PV3248/ SSBK9686_ Km app CSNK2A2 (CK2 alpha 2) Pass Pass Pass 0.76 PV3624/32653 SSBK9686_ Km app DAPK3 (ZIPK) Pass Pass Pass 0.79 PV3686/ SSBK9686_ Km app DCAMKL2 (DCK2) Pass Pass Pass 0.82 PV4297/ SSBK9686_ Km app DNA-PK Pass Pass Pass 0.79 PV5864/ SSBK9686_ Km app DYRK1A Pass Pass Pass 0.89 PV3785/ SSBK9686_ Km app DYRK1B Pass Pass Pass 0.82 PV4649/ SSBK9686_ Km app DYRK Pass Pass Pass 0.88 PV3837/ SSBK9686_ Km app DYRK Pass Pass Pass 0.88 PV3871/37361 SSBK9686_ Km app EEF2K Pass Pass Pass 0.84 PV4559/ SSBK9686_ Km app EGFR (ErbB1) L858R Pass Pass Pass 0.87 PV4128/ SSBK9686_ Km app EGFR (ErbB1) L861Q Pass Pass Pass 0.68 PV3873/34562 SSBK9686_ Km app EGFR (ErbB1) T790M L858R Pass Pass Pass 0.90 PV4879/ SSBK9686_ Km app EGFR (ErbB1) T790M Pass Pass Pass 0.52 PV4803/ SSBK9686_ Km app EGFR (ErbB1) Pass Pass Pass 0.79 PV3872/ SSBK9686_ Km app EPHA Pass Pass Pass 0.76 PV3841/ SSBK9686_ Km app EPHA Pass Pass Pass 0.90 PV3688/ SSBK9686_ Km app EPHA Pass Pass Pass 0.86 PV3651/32933 SSBK9686_ Km app EPHA Pass Pass Pass 0.84 PV3840/34383 SSBK9686_ Km app EPHA Pass Pass Pass 0.92 PV3844/36870 SSBK9686_ Km app EPHB Pass Pass Pass 0.88 PV3786/34225 SSBK9686_ Km app EPHB Pass Pass Pass 0.86 PV3625/ SSBK9686_ Km app EPHB Pass Pass Pass 0.91 PV3658/33066 SSBK9686_ Km app EPHB Pass Pass Pass 0.90 PV3251/29241 SSBK9686_ Km app ERBB2 (HER2) Pass Pass Pass 0.91 PV3366/ SSBK9686_ Km app ERBB4 (HER4) Pass Pass Pass 0.88 PV3626/32657 SSBK9686_ Km app FER Pass Pass Pass 0.91 PV3806/38946 SSBK9686_ Km app FES (FPS) Pass Pass Pass 0.82 PV3354/35734 SSBK9686_ Km app FGFR Pass Pass Pass 0.79 PV3146/ SSBK9686_ Km app FGFR Pass Pass Pass 0.90 PV3368/31517 SSBK9686_ Km app FGFR3 K650E Pass Pass Pass 0.86 PV4392/36445 SSBK9686_ Km app FGFR Pass Pass Pass 0.87 PV3145/28459 SSBK9686_ Km app FGFR Pass Pass Pass 0.79 P3054/26967 SSBK9686_ Km app FGR Pass Pass Pass 0.79 P3041/ SSBK9686_ Km app FLT1 (VEGFR1) Pass Pass Pass 0.89 PV3666/ SSBK9686_ Km app FLT3 D835Y Pass Pass Pass 0.76 PV3967/ SSBK9686_ Km app FLT Pass Pass Pass 0.78 PV3182/ SSBK9686_ Km app FLT4 (VEGFR3) Pass Pass Pass 0.87 PV4129/38454 SSBK9686_ Km app FRAP1 (mtor) Pass Pass Pass 0.70 PV4753/ SSBK9686_ Km app FRK (PTK5) Pass Pass Pass 0.71 PV3874/ SSBK9686_ Km app FYN Pass Pass Pass 0.86 P3042/ SSBK9686_ Km app GRK Pass Pass Pass 0.86 PV3807/ Z' Kinase Part# / Lot# S4

5 SSBK9686_ Km app GRK Pass Pass Pass 0.88 PV3824/ SSBK9686_ Km app GRK Pass Pass Pass 0.85 PV3661/37437 SSBK9686_ Km app GRK Pass Pass Pass 0.93 PV3823/34013 SSBK9686_ Km app GSK3A (GSK3 alpha) Pass Pass Pass 0.86 PV6126/ SSBK9686_ Km app GSK3B (GSK3 beta) Pass Pass Pass 0.79 PV3365/ SSBK9686_ Km app HCK Pass Pass Pass 0.85 PV6128/ SSBK9686_ Km app HIPK1 (Myak) Pass Pass Pass 0.89 PV4561/ SSBK9686_ Km app HIPK Pass Pass Pass 0.92 PV5275/ SSBK9686_ Km app HIPK3 (YAK1) Pass Pass Pass 0.89 PV4209/ SSBK9686_ Km app HIPK Pass Pass Pass 0.90 PV3852/ SSBK9686_ Km app IGF1R Pass Pass Pass 0.83 PV3250/35957 SSBK9686_ Km app IKBKB (IKK beta) Pass Pass Pass 0.60 PV3836/ SSBK9686_ Km app IKBKE (IKK epsilon) Pass Pass Pass 0.63 PV4875/ SSBK9686_ Km app INSR Pass Pass Pass 0.90 PV3781/ SSBK9686_ Km app INSRR (IRR) Pass Pass Pass 0.64 PV3808/34272 SSBK9686_ Km app IRAK Pass Pass Pass 0.57 PV3362/37360 SSBK9686_ Km app ITK Pass Pass Pass 0.70 PV3875/ SSBK9686_ Km app JAK Pass Pass Pass 0.80 PV4774/ SSBK9686_ Km app JAK2 JH1 JH2 V617F Pass Pass Pass 0.82 PV4336/ SSBK9686_ Km app JAK2 JH1 JH Pass Pass Pass 0.72 PV4393/ SSBK9686_ Km app JAK Pass Pass Pass 0.83 PV4210/36301 SSBK9686_ Km app JAK Pass Pass Pass 0.78 PV3855/ SSBK9686_ Km app KDR (VEGFR2) Pass Pass Pass 0.81 PV3660/ SSBK9686_ Km app KIT T670I Pass Pass Pass 0.58 PV3869/34504 SSBK9686_ Km app KIT Pass Pass Pass 0.71 P3081/ SSBK9686_ Km app LCK Pass Pass Pass 0.81 P3043/ SSBK9686_ Km app LTK (TYK1) Pass Pass Pass 0.74 PV4651/ SSBK9686_ Km app LYN A Pass Pass Pass 0.84 P2906/ SSBK9686_ Km app LYN B Pass Pass Pass 0.79 P2907/21076 SSBK9686_ MAP2K1 (MEK1) Pass Pass Pass 0.75 PV3303/ SSBK9686_ MAP2K2 (MEK2) Pass Pass Pass 0.81 PV3615/32519 SSBK9686_ MAP2K6 (MKK6) Pass Pass Pass 0.88 PV3318/ SSBK9686_ MAP3K8 (COT) Pass Pass Pass 0.76 PV4313/ SSBK9686_ Km app MAP3K9 (MLK1) Pass Pass Pass 0.76 PV3787/ SSBK9686_ Km app MAP4K2 (GCK) Pass Pass Pass 0.74 PV4211/ SSBK9686_ Km app MAP4K4 (HGK) Pass Pass Pass 0.66 PV3687/ SSBK9686_ Km app MAP4K5 (KHS1) Pass Pass Pass 0.64 PV3682/ SSBK9686_ Km app MAPK1 (ERK2) Pass Pass Pass 0.89 PV3313/ SSBK9686_ MAPK10 (JNK3) Pass Pass Pass 0.76 PV4563/ SSBK9686_ Km app MAPK11 (p38 beta) Pass Pass Pass 0.82 PV3679/ SSBK9686_ Km app MAPK12 (p38 gamma) Pass Pass Pass 0.82 PV3654/ SSBK9686_ Km app MAPK13 (p38 delta) Pass Pass Pass 0.86 PV3656/ SSBK9686_ Km app MAPK14 (p38 alpha) Direct Pass Pass Pass 0.87 PV3304/ SSBK9686_ MAPK14 (p38 alpha) Pass Pass Pass 0.75 PV3304/ SSBK9686_ Km app MAPK3 (ERK1) Pass Pass Pass 0.77 PV3311/ SSBK9686_ MAPK8 (JNK1) Pass Pass Pass 0.78 PV3319/ SSBK9686_ MAPK9 (JNK2) Pass Pass Pass 0.53 PV3620/32388 SSBK9686_ Km app MAPKAPK Pass Pass Pass 0.86 PV3317/ SSBK9686_ Km app MAPKAPK Pass Pass Pass 0.83 PV3299/38895 SSBK9686_ Km app MAPKAPK5 (PRAK) Pass Pass Pass 0.80 PV3301/ SSBK9686_ Km app MARK1 (MARK) Pass Pass Pass 0.81 PV4395/ SSBK9686_ Km app MARK Pass Pass Pass 0.89 PV3878/ SSBK9686_ Km app MARK Pass Pass Pass 0.78 PV4819/ SSBK9686_ Km app MARK Pass Pass Pass 0.63 PV3851/ SSBK9686_ Km app MATK (HYL) Pass Pass Pass 0.89 PV3370/31553 SSBK9686_ Km app MELK Pass Pass Pass 0.88 PV4823/ SSBK9686_ Km app MERTK (cmer) Pass Pass Pass 0.86 PV3627/32658 SSBK9686_ Km app MET (cmet) Pass Pass Pass 0.67 PV3143/ SSBK9686_ Km app MET M1250T Pass Pass Pass 0.51 PV3968/34718 SSBK9686_ Km app MINK Pass Pass Pass 0.68 PV3810/ SSBK9686_ Km app MKNK1 (MNK1) Pass Pass Pass 0.86 PV6023/ SSBK9686_ Km app MST1R (RON) Pass Pass Pass 0.79 PV4314/ SSBK9686_ Km app MST Pass Pass Pass 0.52 PV3690/ SSBK9686_ Km app MUSK Pass Pass Pass 0.78 PV3834/ SSBK9686_ Km app MYLK2 (skmlck) Pass Pass Pass 0.54 PV3757/ SSBK9686_ Km app NEK Pass Pass Pass 0.78 PV4202/ SSBK9686_ Km app NEK Pass Pass Pass 0.59 PV3360/ SSBK9686_ Km app NEK Pass Pass Pass 0.90 PV4315/ SSBK9686_ Km app NEK Pass Pass Pass 0.83 PV3353/30778 SSBK9686_ Km app NEK Pass Pass Pass 0.92 PV3833/34387 SSBK9686_ Km app NEK Pass Pass Pass 0.79 PV4653/38162 SSBK9686_ Km app NTRK1 (TRKA) Pass Pass Pass 0.84 PV3144/ SSBK9686_ Km app NTRK2 (TRKB) Pass Pass Pass 0.86 PV3616/35706 SSBK9686_ Km app NTRK3 (TRKC) Pass Pass Pass 0.81 PV3617/ SSBK9686_ Km app PAK Pass Pass Pass 0.57 PV3820/35463 SSBK9686_ Km app PAK2 (PAK65) Pass Pass Pass 0.80 PV4565/ SSBK9686_ Km app PAK Pass Pass Pass 0.58 PV3789/34118 SSBK9686_ Km app PAK Pass Pass Pass 0.75 PV4212/ SSBK9686_ Km app PAK Pass Pass Pass 0.54 PV3502/31794 SSBK9686_ Km app PAK7 (KIAA1264) Pass Pass Pass 0.79 PV4405/36846 SSBK9686_ Km app PASK Pass Pass Pass 0.82 PV3972/ SSBK9686_ Km app PDGFRA (PDGFR alpha) Pass Pass Pass 0.82 PV3811/ SSBK9686_ Km app PDGFRA D842V Pass Pass Pass 0.84 PV4203/ SSBK9686_ Km app PDGFRA T674I Pass Pass Pass 0.80 PV3847/35891 SSBK9686_ Km app PDGFRA V561D Pass Pass Pass 0.85 PV4680/ SSBK9686_ Km app PDGFRB (PDGFR beta) Pass Pass Pass 0.94 P3082/27567 SSBK9686_ Km app PDK1 Direct Pass Pass Pass 0.56 P3001/ SSBK9686_ PDK Pass Pass Pass 0.60 P3001/ SSBK9686_ Km app PHKG Pass Pass Pass 0.70 PV3853/ SSBK9686_ Km app PHKG Pass Pass Pass 0.66 PV4555/37321 SSBK9686_ Km app PIM Pass Pass Pass 0.71 PV3503/ SSBK9686_ Km app PIM Pass Pass Pass 0.64 PV3649/32930 SSBK9686_ Km app PKN1 (PRK1) Pass Pass Pass 0.60 PV3790/ SSBK9686_ Km app PLK Pass Pass Pass 0.84 PV3501/ SSBK9686_ Km app PLK Pass Pass Pass 0.86 PV4204/38798 SSBK9686_ Km app PLK Pass Pass Pass 0.64 PV3812/38812 SSBK9686_ Km app PRKACA (PKA) Pass Pass Pass 0.80 P2912/37377 SSBK9686_ Km app PRKCA (PKC alpha) Pass Pass Pass 0.78 P2232/38479 SSBK9686_ Km app PRKCB1 (PKC beta I) Pass Pass Pass 0.59 P2291/ SSBK9686_ Km app PRKCB2 (PKC beta II) Pass Pass Pass 0.56 P2254/ SSBK9686_ Km app PRKCD (PKC delta) Pass Pass Pass 0.55 P2293/39038 SSBK9686_ Km app PRKCE (PKC epsilon) Pass Pass Pass 0.60 P2292/ SSBK9686_ Km app PRKCG (PKC gamma) Pass Pass Pass 0.83 P2233/39126 SSBK9686_ Km app PRKCH (PKC eta) Pass Pass Pass 0.80 P2633/25587 SSBK9686_ Km app PRKCI (PKC iota) Pass Pass Pass 0.88 PV3183/28662 SSBK9686_ Km app PRKCN (PKD3) Pass Pass Pass 0.78 PV3692/ SSBK9686_ Km app PRKCQ (PKC theta) Pass Pass Pass 0.71 P2996/26231 SSBK9686_ Km app PRKCZ (PKC zeta) Pass Pass Pass 0.54 P2273/31602 SSBK9686_ Km app PRKD1 (PKC mu) Pass Pass Pass 0.78 PV3791/34226 SSBK9686_ Km app PRKD2 (PKD2) Pass Pass Pass 0.91 PV3758/34015 SSBK9686_ Km app PRKG Pass Pass Pass 0.81 PV4340/ SSBK9686_ Km app PRKG2 (PKG2) Pass Pass Pass 0.85 PV3973/ SSBK9686_ Km app PRKX Pass Pass Pass 0.78 PV3813/34283 SSBK9686_ Km app PTK2 (FAK) Pass Pass Pass 0.92 PV3832/ SSBK9686_ Km app PTK2B (FAK2) Pass Pass Pass 0.89 PV4567/ SSBK9686_ Km app PTK6 (Brk) Pass Pass Pass 0.90 PV3291/ SSBK9686_ RAF1 (craf) Y340D Y341D Pass Pass Pass 0.88 PV3805/ SSBK9686_ Km app RET V804L Pass Pass Pass 0.93 PV4397/36640 SSBK9686_ Km app RET Y791F Pass Pass Pass 0.90 PV4396/36639 SSBK9686_ Km app RET Pass Pass Pass 0.90 PV3819/ S5

6 SSBK9686_ Km app ROCK Pass Pass Pass 0.82 PV3691/37178 SSBK9686_ Km app ROCK Pass Pass Pass 0.79 PV3759/ SSBK9686_ Km app ROS Pass Pass Pass 0.92 PV3814/ SSBK9686_ Km app RPS6KA1 (RSK1) Pass Pass Pass 0.79 PV3680/ SSBK9686_ Km app RPS6KA2 (RSK3) Pass Pass Pass 0.84 PV3846/34468 SSBK9686_ Km app RPS6KA3 (RSK2) Pass Pass Pass 0.92 PV3323/ SSBK9686_ Km app RPS6KA4 (MSK2) Pass Pass Pass 0.91 PV3782/ SSBK9686_ Km app RPS6KA5 (MSK1) Pass Pass Pass 0.79 PV3681/ SSBK9686_ Km app RPS6KA6 (RSK4) Pass Pass Pass 0.88 PV4557/37496 SSBK9686_ Km app RPS6KB1 (p70s6k) Pass Pass Pass 0.78 PV3815/38944 SSBK9686_ Km app SGK (SGK1) Pass Pass Pass 0.83 PV3818/ SSBK9686_ Km app SGK Pass Pass Pass 0.82 PV3858/ SSBK9686_ Km app SGKL (SGK3) Pass Pass Pass 0.85 PV3859/38954 SSBK9686_ Km app SNF1LK Pass Pass Pass 0.88 PV4792/ SSBK9686_ Km app SRC N Pass Pass Pass 0.84 P2904/21068 SSBK9686_ Km app SRC Pass Pass Pass 0.89 P3044/ SSBK9686_ Km app SRMS (Srm) Pass Pass Pass 0.87 PV4214/ SSBK9686_ Km app SRPK Pass Pass Pass 0.89 PV4215/ SSBK9686_ Km app SRPK Pass Pass Pass 0.54 PV3829/ SSBK9686_ Km app STK22B (TSSK2) Pass Pass Pass 0.80 PV3622/32396 SSBK9686_ Km app STK22D (TSSK1) Pass Pass Pass 0.76 PV3505/ SSBK9686_ Km app STK23 (MSSK1) Pass Pass Pass 0.89 PV3880/ SSBK9686_ Km app STK24 (MST3) Pass Pass Pass 0.54 PV3650/32932 SSBK9686_ Km app STK25 (YSK1) Pass Pass Pass 0.84 PV3657/33163 SSBK9686_ Km app STK3 (MST2) Pass Pass Pass 0.80 PV4805/ SSBK9686_ Km app STK4 (MST1) Pass Pass Pass 0.62 PV3854/38994 SSBK9686_ Km app SYK Pass Pass Pass 0.93 PV3857/ SSBK9686_ Km app TAOK2 (TAO1) Pass Pass Pass 0.67 PV3760/ SSBK9686_ Km app TBK Pass Pass Pass 0.83 PV3504/ SSBK9686_ Km app TEK (Tie2) Pass Pass Pass 0.75 PV3628/34398 SSBK9686_ Km app TXK Pass Pass Pass 0.80 PV5860/ SSBK9686_ Km app TYK Pass Pass Pass 0.90 PV4790/ SSBK9686_ Km app TYRO3 (RSE) Pass Pass Pass 0.67 PV3828/ SSBK9686_ Km app YES Pass Pass Pass 0.84 A15557/ SSBK9686_ Km app ZAP Pass Pass Pass 0.96 P2782/ S6

7 Lantha Life Technologies' SelectScreen Profiling Service: Single Point Results SelectScreen Scientist: Derek Johnston Date: SSBK-LanthaScreen Binding (Paisley UK) Quality Assurance Review: Andrew Heuze Date: Legend Z' Determination Pass < 40% Inhibition 40% - 80% Inhibition 80% Inhibition Project # Compound No. Difference 1X Test Compound Between Data Concentration Kinase Tested % Displacement % Displacement Points Test Compound Interference (nm) Point 1 Point 2 mean Point 1 - Point 2 Donor Acceptor SSBK9686_ ABL1 H396P Pass Pass 0.79 PV6148/ SSBK9686_ ABL1 M351T Pass Pass 0.53 PV6151/ SSBK9686_ ABL1 Q252H Pass Pass 0.94 PV6154/ SSBK9686_ ACVR1 (ALK2) R206H Pass Pass 0.65 PV6232/ SSBK9686_ ACVR1 (ALK2) Pass Pass 0.89 PV4877/ SSBK9686_ ACVR2A Pass Pass 0.73 PV6124/ SSBK9686_ ACVR2B Pass Pass 0.76 PV6049/ SSBK9686_ ACVRL1 (ALK1) Pass Pass 0.58 PV4883/ SSBK9686_ ALK C1156Y Pass Pass 0.94 PV6157/ SSBK9686_ ALK F1174L Pass Pass 0.79 PV6160/ SSBK9686_ ALK L1196M Pass Pass 0.89 PV6166/ SSBK9686_ ALK R1275Q Pass Pass 0.74 PV6169/ SSBK9686_ AMPK (A1/B1/G2) Pass Pass 0.80 PV6238/ SSBK9686_ AMPK (A1/B1/G3) Pass Pass 0.88 PV6241/ SSBK9686_ AMPK (A1/B2/G1) Pass Pass 0.91 PV6244/ SSBK9686_ AMPK (A2/B2/G1) Pass Pass 0.95 PV6247/ SSBK9686_ AMPK (A2/B2/G2) Pass Pass 0.93 PV6250/ SSBK9686_ AXL R499C Pass Pass 0.82 PV6253/ SSBK9686_ BMPR1A (ALK3) Pass Pass 0.81 PV6038/ SSBK9686_ BMPR1B (ALK6) Pass Pass 0.60 PV6235/ SSBK9686_ BMPR Pass Pass 0.81 PV6256/ SSBK9686_ BRAF V599E Pass Pass 0.74 PV3849/ SSBK9686_ BRAF Pass Pass 0.92 PV3848/ SSBK9686_ BRSK Pass Pass 0.74 PV6259/ SSBK9686_ CAMK2G (CaMKII gamma) Pass Pass 0.95 PV6268/ SSBK9686_ CAMKK1 (CAMKKA) Pass Pass 0.79 PV4670/ SSBK9686_ CAMKK2 (CaMKK beta) Pass Pass 0.95 PV4206/35319 SSBK9686_ CASK Pass Pass 0.83 PV6271/ SSBK9686_ CDC7/DBF Pass Pass 0.81 PV6274/ SSBK9686_ CDK1/cyclin A Pass Pass 0.58 PV6280/ SSBK9686_ CDK11 (Inactive) Pass Pass 0.92 PV6283/ SSBK9686_ CDK14 (PFTK1)/cyclin Y Pass Pass 0.71 PV6382/ SSBK9686_ CDK16 (PCTK1)/cyclin Y Pass Pass 0.78 PV6379/ SSBK9686_ CDK2/cyclin A Pass Pass 0.72 PV6289/ SSBK9686_ CDK2/cyclin E Pass Pass 0.75 PV6295/ SSBK9686_ CDK2/cyclin O Pass Pass 0.72 PV6286/ SSBK9686_ CDK3/cyclin E Pass Pass 0.72 PV6298/ SSBK9686_ CDK5 (Inactive) Pass Pass 0.56 PV6301/ SSBK9686_ CDK8/cyclin C Pass Pass 0.91 PV4402/ SSBK9686_ CDK9 (Inactive) Pass Pass 0.73 PV6304/ SSBK9686_ CDK9/cyclin K Pass Pass 0.82 PV4335/35774 SSBK9686_ CLK Pass Pass 0.83 PV3839/ SSBK9686_ DAPK Pass Pass 0.75 PV3614/32159 SSBK9686_ DDR Pass Pass 0.93 PV6047/ SSBK9686_ DDR2 N456S Pass Pass 0.86 PV6172/ SSBK9686_ DDR2 T654M Pass Pass 0.58 PV6175/ SSBK9686_ DDR Pass Pass 0.90 PV3870/ SSBK9686_ DMPK Pass Pass 0.95 PV3784/ SSBK9686_ DYRK Pass Pass 0.57 PV6331/ SSBK9686_ EGFR (ErbB1) d Pass Pass 0.60 PV6178/ SSBK9686_ EIF2AK2 (PKR) Pass Pass 0.57 PV4821/ SSBK9686_ EPHA Pass Pass 0.87 PV3359/30916 SSBK9686_ EPHA Pass Pass 0.72 PV6337/ SSBK9686_ EPHA Pass Pass 0.84 PV3689/33790 SSBK9686_ FGFR1 V561M Pass Pass 0.93 PV6343/ SSBK9686_ FGFR3 G697C Pass Pass 0.57 PV6184/ SSBK9686_ FGFR3 K650M Pass Pass 0.76 PV6187/ SSBK9686_ FLT3 ITD Pass Pass 0.64 PV6190/ SSBK9686_ FYN A Pass Pass 0.80 PV6346/ SSBK9686_ GRK Pass Pass 0.92 PV6352/ SSBK9686_ ICK Pass Pass 0.87 PV6358/ SSBK9686_ KIT A829P Pass Pass 0.89 PV6193/ SSBK9686_ KIT D816H Pass Pass 0.57 PV6196/ SSBK9686_ KIT D816V Pass Pass 0.67 PV6199/ SSBK9686_ KIT D820E Pass Pass 0.89 PV6307/ SSBK9686_ KIT N822K Pass Pass 0.90 PV6310/ SSBK9686_ KIT T670E Pass Pass 0.94 PV6313/ SSBK9686_ KIT V559D T670I Pass Pass 0.93 PV6316/ SSBK9686_ KIT V654A Pass Pass 0.80 PV4132/35129 SSBK9686_ KIT Y823D Pass Pass 0.77 PV6322/ SSBK9686_ LATS Pass Pass 0.80 PV6361/ SSBK9686_ LATS Pass Pass 0.97 PV6364/ SSBK9686_ LIMK Pass Pass 0.89 PV4337/ SSBK9686_ LIMK Pass Pass 0.92 PV3860/ SSBK9686_ MAP2K1 (MEK1) S218D S222D Pass Pass 0.92 P3099/38541 SSBK9686_ MAP2K1 (MEK1) Pass Pass 0.93 PV3303/ SSBK9686_ MAP2K2 (MEK2) Pass Pass 0.95 PV3615/32519 SSBK9686_ MAP2K3 (MEK3) Pass Pass 0.76 PV3662/ SSBK9686_ MAP2K6 (MKK6) S207E T211E Pass Pass 0.85 PV3293/ SSBK9686_ MAP2K6 (MKK6) Pass Pass 0.81 PV3318/ SSBK9686_ MAP3K10 (MLK2) Pass Pass 0.94 PV3877/34554 SSBK9686_ MAP3K11 (MLK3) Pass Pass 0.93 PV3788/ SSBK9686_ MAP3K14 (NIK) Pass Pass 0.90 PV4902/ SSBK9686_ MAP3K2 (MEKK2) Pass Pass 0.58 PV3822/ SSBK9686_ MAP3K3 (MEKK3) Pass Pass 0.85 PV3876/ SSBK9686_ MAP3K5 (ASK1) Pass Pass 0.74 PV3809/ SSBK9686_ MAP3K7/MAP3K7IP1 (TAK1-TAB1) Pass Pass 0.72 PV4394/ SSBK9686_ MAP4K1 (HPK1) Pass Pass 0.79 PV6355/ SSBK9686_ MAP4K3 (GLK) Pass Pass 0.79 PV6349/ SSBK9686_ MAPK10 (JNK3) Pass Pass 0.90 PV4563/ SSBK9686_ MAPK15 (ERK7) Pass Pass 0.93 PV6181/ SSBK9686_ MAPK8 (JNK1) Pass Pass 0.80 PV3319/ SSBK9686_ MAPK9 (JNK2) Pass Pass 0.94 PV3620/32388 SSBK9686_ MERTK (cmer) A708S Pass Pass 0.91 PV6325/ SSBK9686_ MET D1228H Pass Pass 0.90 PV6208/ SSBK9686_ MKNK2 (MNK2) Pass Pass 0.68 PV5607/ SSBK9686_ MLCK (MLCK2) Pass Pass 0.95 PV3835/34028 SSBK9686_ MYLK (MLCK) Pass Pass 0.97 PV4339/36152 SSBK9686_ MYO3B (MYO3 beta) Pass Pass 0.89 PV6367/ SSBK9686_ NLK Pass Pass 0.93 PV4309/35323 SSBK9686_ NUAK Pass Pass 0.68 PV6376/ Z' Kinase Part# / Lot# S7

8 SSBK9686_ PKN2 (PRK2) Pass Pass 0.74 PV3879/ SSBK9686_ PLK Pass Pass 0.82 PV6394/ SSBK9686_ PRKACB (PRKAC beta) Pass Pass 0.79 PV6388/ SSBK9686_ PRKACG (PRKAC gamma) Pass Pass 0.88 PV6391/ SSBK9686_ RAF1 (craf) Y340D Y341D Pass Pass 0.88 PV3805/ SSBK9686_ RET G691S Pass Pass 0.60 PV6214/ SSBK9686_ RET M918T Pass Pass 0.80 PV6217/ SSBK9686_ RET V804M Pass Pass 0.65 PV6223/ SSBK9686_ RIPK Pass Pass 0.94 PV4213/35334 SSBK9686_ RIPK Pass Pass 0.71 PV6397/ SSBK9686_ SIK Pass Pass 0.94 PV6445/ SSBK9686_ SIK Pass Pass 0.70 PV6403/ SSBK9686_ SLK Pass Pass 0.91 PV3830/34390 SSBK9686_ STK16 (PKL12) Pass Pass 0.81 PV4311/36847 SSBK9686_ STK17A (DRAK1) Pass Pass 0.92 PV3783/ SSBK9686_ STK17B (DRAK2) Pass Pass 0.63 PV6328/ SSBK9686_ STK32B (YANK2) Pass Pass 0.93 PV6406/ SSBK9686_ STK32C (YANK3) Pass Pass 0.88 PV6409/ SSBK9686_ STK Pass Pass 0.89 PV4343/ SSBK9686_ STK38 (NDR) Pass Pass 0.54 PV6370/ SSBK9686_ STK38L (NDR2) Pass Pass 0.51 PV6373/ SSBK9686_ STK39 (STLK3) Pass Pass 0.58 PV6412/ SSBK9686_ TAOK Pass Pass 0.86 PV6415/ SSBK9686_ TAOK3 (JIK) Pass Pass 0.71 PV3652/32935 SSBK9686_ TEC Pass Pass 0.91 PV3269/ SSBK9686_ TEK (TIE2) R849W Pass Pass 0.90 PV6226/ SSBK9686_ TEK (TIE2) Y1108F Pass Pass 0.55 PV6229/ SSBK9686_ TESK Pass Pass 0.73 PV6418/ SSBK9686_ TGFBR1 (ALK5) Pass Pass 0.70 PV5837/ SSBK9686_ TGFBR Pass Pass 0.54 PV6122/ SSBK9686_ TLK Pass Pass 0.82 PV6421/ SSBK9686_ TLK Pass Pass 0.93 PV6424/ SSBK9686_ TNIK Pass Pass 0.91 PV6427/ SSBK9686_ TNK2 (ACK) Pass Pass 0.98 PV4807/ SSBK9686_ TTK Pass Pass 0.57 PV3792/ SSBK9686_ ULK Pass Pass 0.95 PV6430/ SSBK9686_ ULK Pass Pass 0.75 PV6433/ SSBK9686_ ULK Pass Pass 0.88 PV6436/ SSBK9686_ WEE Pass Pass 0.78 PV3817/ SSBK9686_ WNK Pass Pass 0.82 PV4341/35976 SSBK9686_ WNK Pass Pass 0.55 PV4342/36047 SSBK9686_ ZAK Pass Pass 0.87 PV3882/34603 S8

9 Adapta Life Technologies' SelectScreen Profiling Service: Single Point Results SelectScreen Scientist: Derek Johnston Date: SSBK-Adapta (Paisley UK) Quality Assurance Review: Catriona MacKay Date: Legend < 40% Inhibition % Conversion Pass 40% - 80% Inhibition Z' Determination Pass 80% Inhibition Project # Compound No. 1X Test Compound Concentration Difference [ATP] Between Data Tested Kinase Tested % Inhibition % Inhibition Points Test Compound Interference (nm) (μm) Point 1 Point 2 mean Point 1 - Point 2 Donor Acceptor SSBK9686_ CAMK1 (CaMK1) Pass Pass 0.51 PV4391/36046 SSBK9686_ Km app CDK7/cyclin H/MNAT Pass Pass 0.81 PV3868/ SSBK9686_ Km app CDK9/cyclin T Pass Pass 0.83 PV4131/ SSBK9686_ Km app CHUK (IKK alpha) Pass Pass 0.68 PV4310/ SSBK9686_ Km app DAPK Pass Pass 0.53 PV3969/32654 SSBK9686_ Km app GSG2 (Haspin) Pass Pass 0.80 PV5708/ SSBK9686_ Km app IRAK Pass Pass 0.62 PV4403/ SSBK9686_ Km app LRRK2 FL Pass Pass 0.82 A15197PT/ SSBK9686_ Km app LRRK2 G2019S FL Pass Pass 0.66 A15200PT/ SSBK9686_ Km app LRRK2 G2019S Pass Pass 0.71 PV4881/ SSBK9686_ Km app LRRK2 I2020T Pass Pass 0.67 PV5854/ SSBK9686_ Km app LRRK2 R1441C Pass Pass 0.78 PV5858/ SSBK9686_ Km app LRRK Pass Pass 0.54 PV4873/ SSBK9686_ Km app NUAK1 (ARK5) Pass Pass 0.78 PV4127/ SSBK9686_ PI4KA (PI4K alpha) Pass Pass 0.60 PV5689/ SSBK9686_ Km app PI4KB (PI4K beta) Pass Pass 0.51 PV5277/ SSBK9686_ Km app PIK3C2A (PI3K-C2 alpha) Pass Pass 0.79 PV5586/ SSBK9686_ PIK3C2B (PI3K-C2 beta) Pass Pass 0.61 PV5374/ SSBK9686_ Km app PIK3C3 (hvps34) Pass Pass 0.61 PV5126/ SSBK9686_ Km app PIK3CA/PIK3R1 (p110 alpha/p85 alpha) Pass Pass 0.55 PV4788/ SSBK9686_ Km app PIK3CB/PIK3R1 (p110 beta/p85 alpha) Pass Pass 0.64 PV6454/52685 SSBK9686_ Km app PIK3CD/PIK3R1 (p110 delta/p85 alpha) Pass Pass 0.60 PV5273/ SSBK9686_ Km app PIK3CG (p110 gamma) Pass Pass 0.80 PV4786/ SSBK9686_ Km app SPHK Pass Pass 0.67 PV5214/ SSBK9686_ SPHK Pass Pass 0.51 PV5216/ Z' Kinase Part# / Lot# S9

10 Supporting Information 3 (Time dependency for CDK12 inhibition) Supplementary Table 1. Time dependency for CDK12 inhibition Compound 0h 1h 2h 5h >10 >10 >10 >10 3 (THZ-531) Staurosporine IC50 values ( M) for each preincubation time were described. Evaluation of time-dependent inhibition of human CDK12 using the LANCE system. Various concentrations of inhibitors in DMSO (50 nl) were dispensed in the assay plate (product # , Greiner Bio-One, Frickenhausen, Germany) using an Access Echo555 liquid handler (Labsite, Sunnyvale, CA). The enzymatic reaction was performed in reaction buffer (25 mm HEPES (ph 7.5), 10 mm MgCl2, 0.01% Tween 20, 1 mm DTT). To evaluate effect of preincubation with inhibitors on CDK12 inhibitory activity, 2.5 μl of 60 nm CDK12 protein was dispensed into each well and incubated for various periods of time at room temperature. Subsequently, the enzymatic reaction was initiated by the addition of 2.5 μl substrate premix (20 μm ATP and 300 nm ULight-4E-BP1 (Thr37/Thr46)), and the plate was incubated for 60 min at room temperature. For the evaluation of CDK12 inhibitory activity of inhibitors without preincubation, 2.5 μl of substrate mixture was added to the plate, followed by the addition of 2.5 μl of 60 nm CDK12 protein and incubation for 60 min at room temperature. The final concentrations of CDK12, ATP, and peptide were 30 nm, 10 μm, and 150 nm, respectively. The enzymatic reaction was terminated by the addition of 5 μl LANCE detection buffer (20 mm S10

11 EDTA (final 10 mm) and 0.30 nm Eu-labeled anti-phospho-eif4e-binding protein 1 (Thr37/46) antibody (final 0.15 nm)). The plate was incubated for 60 min at room temperature and the time-resolved fluorescence was monitored by using an EnVision plate reader. The solution in each well was excited with a laser (λ = 337 nm) reflected by a dichroic mirror (D400/D630 (PerkinElmer)), and the fluorescence signals from Eu and ULight were detected by using two emission filters (Cy5 620 (PerkinElmer) for Eu and APC 665 (PerkinElmer) for ULight). The values for the 0% and 100% controls were the signals obtained in the presence and absence of CDK12 protein, respectively. The inhibition at each inhibitor concentration was determined in quadruplicate. The IC50 values were derived from the fitting a sigmoidal dose-response curve to a plot of the assay readout over the inhibitor concentration. All fits were computed by GraphPad Prism. S11

12 Supporting Information 4 (Co-crystal structure of 29 and CDK12) Protein expression and purification. The cdnas encoding human CDK12 and CycK (encompassing residues and 1 267, respectively) were amplified by the polymerase chain reaction and subcloned into the plasmid pfastbacdual (Invitrogen). The CDK12 coding sequence was inserted after the polyhedron promoter (PpH), whereas CycK was placed behind the p10 promoter (Pp10). To enable the purification of the complex, the coding sequences were each engineered with an N-terminal Glutathione-S-transferase (GST) affinity tag that includes a recognition sequence for the TEV protease for cleavage and removal of GST fusion partner protein. In addition, the cdna encoding S. cerevisiae CDK-activating kinase (CAK1p) encompassing residues was amplified and subcloned into the plasmid pfastbac. The CAK1p construct was engineered with an N-terminal 6X-His affinity tag and a TEV recognition sequence to facilitate purification in the event that the isolated CAK1p was required for in vitro experiments. After construction, the cloned inserts were verified by DNA sequence analysis. Recombinant viruses were generated using the Bac-to-Bac baculovirus expression system (Invitrogen). High-titer viruses encoding CDK12/CycK and CAK1p were mixed in a 1:1 ratio and Hi5 cells were infected to express the recombinant proteins. The CAK1p was included during expression as the phosphorylation of CDK12 was reported to facilitate crystallization. 1 The infected Hi5 cells were harvested 66 h after the addition of the viruses. A cell pellet generated from a 10 L culture of Hi5 cells was homogenized in 1 L of lysate buffer (25 mm HEPES (ph 7.6), 0.5 M NaCl, 0.25 mm TCEP, and 5% glycerol; all chemicals were purchased from Sigma-Aldrich) supplemented with protease inhibitors (Roche). The lysate was clarified by centrifugation and the resulting supernatant was affinity purified by using glutathione sepharose (GE Healthcare). The glutathione affinity column was washed with 50 volumes of lysate buffer S12

13 in the absence of protease inhibitors. To remove the GST-tag, the column was incubated with TEV protease overnight during an on-column cleavage step and the following day the CDK12/CycK protein complex was eluted with buffer (25 mm HEPES ph 7.6, 0.4 M NaCl, 0.5 mm TCEP, 5% glycerol). The complex was further purified in this buffer by passage through a size exclusion column (Superdex 200, GE Healthcare). The peak fractions from size exclusion chromatography that contained the CDK12/CycK heterodimeric complex were pooled and stored in aliquots at 80 C. Analysis of the purified complex by mass spectrometry demonstrated that CDK12 was phosphorylated (data not shown). Crystallography. Prior to setting up crystallization trials, a CDK12/CycK-compound ternary complex was generated by the addition of a macrocyclic ATP-competitive compound (the identity of this tool compound is not disclosed) followed by co-concentration using an Amicon concentrator (EMD-Millipore) to produce a final concentration of 12 mg/ml. Crystallization was performed using the hanging drop vapor diffusion method with a reservoir solution composed of 0.1 M HEPES (ph 6.5), 18% PEG 3350, 0.2 M MgCl2, and 1 mm sarcosine (the additive sarcosine and PEG3350 solution were purchased from Hampton Research). The crystals were grown at 25 C and reaching their maximum size within 1 month. The crystals of CDK12/CycK were backsoaked with mother liquor supplemented with 2 mm compound 29 by transferring the crystals into fresh compound/mother liquor solution once per day for five consecutive days. After the soaking regimen, the crystals were cryo-protected by rapid transfer into a reservoir solution supplemented with 25% ethylene glycol and rapidly flash-cooled in ALS-style pucks that were submerged in liquid nitrogen. The diffraction data were collected at Advanced Light Source beamline (Lawrence Berkeley s National Laboratory, Berkeley, CA) and processed using the HKL2000 software package. 2 The structure was solved by molecular replacement with S13

14 Phaser 3 using stepwise searches with the coordinates of CDK12 followed by CycK (both models derived from PDB code:4nst). The graphics program COOT 4 was used for model building and refinement was performed with REFMAC5. 5 Phaser and REFMAC5 are distributed as part of CCP4. 6 Structural validation was performed by using Molprobity. 7 Refinement statistics are reported in Supplementary Table 2 and an image of the omit electron density for the ligand is shown in Supplementary Figure 2. S14

15 Supplementary Table 2. X-ray data collection and refinement statistics. X-Ray Structure CDK12/CycK-29 /PDB CODE: 6CKX Data collection Resolution range (Å) ( ) Space group P Cell dimensions: a, b, c (Å) , , α, β, γ (º) , , Rsym <I / σi> Completeness (%) Redundancy Structure Refinement Resolution (Å) No. reflections No. reflections Rfree test set Rwork / Rfree No. atoms: Protein Ligand Mg Ions Waters Mean B Value (Å 2 ) Protein B Value (Å 2 ) Ligand B Value (Å 2 ) Mg Ions B Value (Å 2 ) Water B Value (Å 2 ) RMS Bond lengths (Å) RMS Bond angles (º) Ramachandran statistics: Favored (%) 10.7 (100) 13.9 (1.05) 99.9 (99.8) 6.9 (6.7) ,375 1, /25.1 8, S15

16 Outliers (%) 0.19 *Values in parentheses are for highest-resolution shell. Supplementary Figure 2. Omit electron density Fo-Fc map (rendered as mesh) was generated by omitting atoms corresponding to the compound (displayed as green sticks). Map is contoured at ± 3.0 σ. Figure was created using Pymol. S16

17 Supporting Information 5 (Single crystal X-ray structure analyses) All measurements were made on a Rigaku R-AXIS RAPID-191R or a Rigaku XtaLAB P200 diffractometer using graphite monochromated Cu-Kα radiation. The structure was solved by direct methods with SIR and was refined using full-matrix least-squares on F 2 with SHELXL All non-h atoms were refined with anisotropic displacement parameters. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via Supplementary Figure 3. ORTEP of 1, thermal ellipsoids are drawn at 50% probability. Crystal data for 1 (CCDC ): C21H22N6O2S, MW = ; crystal size, 0.30 x 0.08 x 0.06 mm; colorless, block; monoclinic, space group P21/n, a = (19) Å, b = (5) Å, c = (6) Å, α = γ = 90, β = (6), V = (10) Å 3, Z = 4, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.073, wr2 = S17

18 Supplementary Figure 4. ORTEP of 16, thermal ellipsoids are drawn at 50% probability. Crystal data for 16 (CCDC ): C22H22N6O, MW = ; crystal size, 0.12 x 0.10 x 0.04 mm; colorless, block; monoclinic, space group P21/n, a = (14) Å, b = (14) Å, c = (3) Å, α = γ = 90, β = (14), V = (5) Å 3, Z = 4, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.045, wr2 = Supplementary Figure 5. ORTEP of 21, thermal ellipsoids are drawn at 50% probability. Crystal data for 21 (CCDC ): C24H26N6O 0.4 H2O, MW = ; crystal size, 0.21 x 0.11 x 0.06 mm; colorless, platelet; triclinic, space group P-1, a = (4) Å, b = (5) S18

19 Å, c = (5) Å, α = (7), β = (8), γ = (8), V = (13) Å 3, Z = 2, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.130, wr2 = Supplementary Figure 6. ORTEP of 22, thermal ellipsoids are drawn at 50% probability. Crystal data for 22 (CCDC ): C23H26N6O2S, MW = ; crystal size, 0.34 x 0.23 x 0.06 mm; colorless, block; orthorhombic, space group P212121, a = (7) Å, b = (8) Å, c = (15) Å, α = β = γ = 90, V = (3) Å 3, Z = 4, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.026, wr2 = Supplementary Figure 7. ORTEP of 23, thermal ellipsoids are drawn at 50% probability. S19

20 Crystal data for 23 (CCDC ): C31H32N6O 1.0 C3H8O, MW = ; crystal size, 0.14 x 0.04 x 0.04 mm; colorless, block; triclinic, space group P-1, a = (3) Å, b = (3) Å, c = (5) Å, α = (2), β = (3), γ = (2), V = (8) Å 3, Z = 2, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.046, wr2 = Supplementary Figure 8. ORTEP of 25, thermal ellipsoids are drawn at 50% probability. Crystal data for 25 (CCDC ): C30H30N6O2 C3H8O, MW = ; crystal size, 0.18 x 0.11 x 0.05 mm; colorless, block; triclinic, space group P-1, a = (19) Å, b = (2) Å, c = (3) Å, α = (16), β = (16), γ = (17), V = (5) Å 3, Z = 2, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.041, wr2 = S20

21 Supplementary Figure 9. ORTEP of 27, thermal ellipsoids are drawn at 50% probability. Crystal data for 27 (CCDC ): C30H31N7O, MW = ; crystal size, 0.13 x 0.11 x 0.07 mm; colorless, platelet; triclinic, space group P-1, a = (2) Å, b = (3) Å, c = (3) Å, α = (6), β = (5), γ = (5), V = (8) Å 3, Z = 2, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.069, wr2 = Supplementary Figure 10. ORTEP of 33, thermal ellipsoids are drawn at 50% probability. Crystal data for 33 (CCDC ): C24H26N6O, MW = ; crystal size, 0.21 x 0.04 x 0.04 mm; colorless, needle; monoclinic, space group P21/c, a = (16) Å, b = S21

22 (12) Å, c = (2) Å, α = γ = 90, β = (12), V = (8) Å 3, Z = 8, Dx = g/cm 3, T = 100 K, μ = mm -1, λ = Å, R1 = 0.037, wr2 = S22

23 References 1. Bösken C. A., Farnung L., Hintermair C., Merzel Schachter M., Vogel-Bachmayr K., Blazek D., Anand K., Fisher RP., Eick D., Geyer M. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 2014, 5, Otwinowski, Z., Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, McCoy, A. J., Grosse-Kunstleve, R.W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. Phaser crystallographic software. J. Appl. Cryst. 2007, 40, Emsley, P., Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004, 60, Murshudov G. N., Vagin A. A., Dodson E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 1997, 53, Collaborative Computing Project The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 1994, 50, Chen V. B., Arendall W. B. 3rd, Headd J. J., Keedy D. A., Immormino R. M., Kapral G. J., Murray L. W., Richardson J. S., Richardson D. C. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Siliqi, D.; Spagna, R. IL MILIONE: a suite of computer programs for crystal structure solution of proteins. J. Appl. Crystallogr. 2007, 40, Sheldrick, G. A short history of SHELX. Acta Crystallogr. Sect. A 2008, 64, S23