Supporting Information Property focused structure-based optimization of small molecule inhibitors of the proteinprotein interaction between menin and Mixed Lineage Leukemia (MLL) Dmitry Borkin a, Jonathan Pollock a, Katarzyna Kempinska a, Trupta Purohit a, Xiaoqin Li b, Bo Wen b, Ting Zhao b, Hongzhi Miao a, Shirish Shukla a, Miao He b, Duxin Sun b, Tomasz Cierpicki a, Jolanta Grembecka a,* a Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA b College of Pharmacy, University of Michigan, Ann Arbor, MI, 48109, USA * Corresponding author; Jolanta Grembecka, PhD Assistant Professor, Department of Pathology, University of Michigan 1150 West Medical Center Dr, MSRB I, Room 4510D Ann Arbor, MI, 48108 e-mail: jolantag@umich.edu Tel. 734-615-9319 Table of Contents 1. Supplementary Figure 1..........S2 2. Supplementary Figure 2....S3 3. Supplementary Table 1. S6 4. Supplementary Table 2 S18 5. Supplementary Table 3....S20 6. Supplementary Table 4 S21 1
Supplementary Figure 1. Representative titration curves from the FP assay for selected menin-mll inhibitors. 2
Supplementary Figure 2. Compound 27 undergoes glucuronidation in blood plasma. a) Chemical structure of 27 (left) and the glucuronide (Gluc-27) derivative (right). b) Ratio of glucuronidated 27 over the parent compound is shown at different time points. c) LC-MS/MS Enhance Product Ion (EPI) scan of 27 and its fragments. d) LC-MS/MS EPI scan of Gluc-27 and its fragments. 3
c 4
d 5
Supplementary Table 1. Profiling of compound 1 on kinase panel. Profiling was performed with 231 kinases at two concentrations of compound 1 (1 µm and 10 µm). Kinase Conc. tested (µm) Compound 1 (% inhibition) ABL-T315I 1-1 ABL-T315I 10 12 ABL1 1 2 ABL1 10 17 AKT1 1 0 AKT1 10 1 AKT2 1 2 AKT2 10 10 AKT3 1 0 AKT3 10 0 ALK 1-5 ALK 10 1 AMP-A1B1G1 1 4 AMP-A1B1G1 10 1 AMP-A2B1G1 1 1 AMP-A2B1G1 10 5 ARG 1 1 ARG 10 15 ARK5 1 0 ARK5 10 8 AURORA-A 1-1 AURORA-A 10 16 AURORA-B 1-2 AURORA-B 10 7 AURORA-C 1-1 AURORA-C 10 17 AXL 1-2 AXL 10 20 BLK 1-9 BLK 10 2 BMX 1 4 BMX 10 0 BRAF 1-1 BRAF 10 4 BRK 1-7 BRK 10-67 6
BRSK1 1-1 BRSK1 10 11 BRSK2 1 2 BRSK2 10 6 BTK 1 7 BTK 10 20 CAMK1D 1 2 CAMK1D 10 9 CAMK2A 1 1 CAMK2A 10 5 CAMK2D 1-1 CAMK2D 10 16 CAMK4 1 2 CAMK4 10 11 CDK1 1 1 CDK1 10 4 CDK2 1 0 CDK2 10 22 CDK2-CYCLINE 1 3 CDK2-CYCLINE 10 0 CDK3-CYCLINE 1 6 CDK3-CYCLINE 10 0 CDK4-CYCLIND 1 4 CDK4-CYCLIND 10 0 CDK5 1 1 CDK5 10-2 CDK6-CYCLIND3 1-1 CDK6-CYCLIND3 10-2 CHEK1 1-5 CHEK1 10-4 CHEK2 1 1 CHEK2 10 20 CK1 1 1 CK1 10 66 CK1-GAMMA1 1 2 CK1-GAMMA1 10 30 CK1-GAMMA2 1 6 CK1-GAMMA2 10 17 CK1-GAMMA3 1 4 CK1-GAMMA3 10 22 CLK1 1 2 7
CLK1 10 8 CLK2 1-3 CLK2 10 3 CLK3 1 0 CLK3 10 2 CLK4 1 2 CLK4 10 31 CRAF 1 6 CRAF 10 1 CSK 1 0 CSK 10 11 DAPK1 1 1 DAPK1 10 3 DAPK3 1 0 DAPK3 10 7 DCAMKL2 1 2 DCAMKL2 10 2 DDR2 1-1 DDR2 10 9 DYRK1A 1 1 DYRK1A 10 7 DYRK1B 1-1 DYRK1B 10 7 DYRK2 1-3 DYRK2 10 4 DYRK3 1 2 DYRK3 10 24 DYRK4 1 1 DYRK4 10 0 EGFR 1-1 EGFR 10 0 EGFR-T790M 1 3 EGFR-T790M 10 3 EPH-A1 1-1 EPH-A1 10 3 EPH-A2 1-1 EPH-A2 10-2 EPH-A3 1 0 EPH-A3 10 0 EPH-A4 1 1 EPH-A4 10 11 8
EPH-A5 1 0 EPH-A5 10-82 EPH-A8 1 2 EPH-A8 10 10 EPH-B1 1 5 EPH-B1 10 1 EPH-B2 1-4 EPH-B2 10 25 EPH-B3 1 0 EPH-B3 10-38 EPH-B4 1 1 EPH-B4 10 13 ERB-B2 1 1 ERB-B2 10 5 ERB-B4 1-2 ERB-B4 10 0 FER 1-3 FER 10 4 FES 1 1 FES 10 6 FGFR1 1 9 FGFR1 10 3 FGFR2 1 3 FGFR2 10-5 FGFR3 1 0 FGFR3 10-2 FGFR4 1 0 FGFR4 10-1 FGR 1 0 FGR 10 17 FLT-1 1-2 FLT-1 10 1 FLT-3 1 0 FLT-3 10 6 FLT-4 1 5 FLT-4 10 0 FMS 1 0 FMS 10 5 FYN 1 2 FYN 10 6 GRK6 1 24 9
GRK6 10-14 GRK7 1-9 GRK7 10-8 GSK3-ALPHA 1 1 GSK3-ALPHA 10-2 GSK3-BETA 1 2 GSK3-BETA 10 1 HASPIN 1 5 HASPIN 10 78 HCK 1 4 HCK 10 19 HIPK1 1-3 HIPK1 10-1 HIPK2 1 1 HIPK2 10 2 HIPK3 1-1 HIPK3 10 0 HIPK4 1 5 HIPK4 10 82 IGF1R 1-2 IGF1R 10-2 IKK-ALPHA 1-1 IKK-ALPHA 10 0 IKK-BETA 1-1 IKK-BETA 10 1 IKK-EPSILON 1-1 IKK-EPSILON 10-1 INSR 1-5 INSR 10-5 IRAK4 1 2 IRAK4 10 1 IRR 1-1 IRR 10 2 ITK 1 4 ITK 10 10 JAK1 1 2 JAK1 10 1 JAK2 1-3 JAK2 10 3 JAK3 1 9 JAK3 10-1 10
JNK1 1-1 JNK1 10 2 JNK2 1 0 JNK2 10 0 JNK3 1-2 JNK3 10-3 KDR 1 3 KDR 10 1 KIT 1 1 KIT 10 25 LATS2 1 2 LATS2 10 2 LCK 1 2 LCK 10 0 LOK 1-3 LOK 10 4 LRRK2-G2019S 1 1 LRRK2-G2019S 10 4 LTK 1 0 LTK 10 2 LYNA 1 3 LYNA 10 14 LYNB 1 2 LYNB 10 13 MAP4K2 1-1 MAP4K2 10 8 MAP4K4 1 0 MAP4K4 10 4 MAP4K5 1-3 MAP4K5 10 7 MAPK1 1-4 MAPK1 10-2 MAPK3 1 2 MAPK3 10-3 MAPKAPK-2 1 3 MAPKAPK-2 10 3 MAPKAPK-3 1-2 MAPKAPK-3 10-3 MARK1 1 2 MARK1 10 1 MARK3 1 1 11
MARK3 10 8 MARK4 1 3 MARK4 10 3 MEK1 1 1 MEK1 10 0 MEK2 1-2 MEK2 10 4 MELK 1-1 MELK 10 26 MER 1 2 MER 10 13 MET 1 1 MET 10 2 MINK 1-1 MINK 10 2 MKNK1 1 1 MKNK1 10 6 MNK2 1 5 MNK2 10 11 MRCK-ALPHA 1 2 MRCK-ALPHA 10 3 MRCK-BETA 1 0 MRCK-BETA 10 0 MSK1 1-1 MSK1 10 13 MSK2 1-2 MSK2 10 16 MSSK1 1 2 MSSK1 10 3 MST1 1 0 MST1 10-4 MST2 1 3 MST2 10-2 MST3 1-1 MST3 10 4 MUSK 1 0 MUSK 10-3 NEK1 1 3 NEK1 10 8 NEK2 1 6 NEK2 10 4 12
NEK6 1 0 NEK6 10-4 NEK7 1-2 NEK7 10-5 NEK9 1-4 NEK9 10 2 P38-ALPHA 1-1 P38-ALPHA 10 12 P38-BETA 1-1 P38-BETA 10 2 P38-DELTA 1-1 P38-DELTA 10 0 P38-GAMMA 1-1 P38-GAMMA 10 0 P70S6K1 1-1 P70S6K1 10 6 P70S6K2 1 1 P70S6K2 10 1 PAK1 1-1 PAK1 10 2 PAK2 1-1 PAK2 10 2 PAK3 1-2 PAK3 10 5 PAK4 1-2 PAK4 10-11 PAK5 1 0 PAK5 10 0 PAK6 1 0 PAK6 10 0 PAR-1B-ALPHA 1 2 PAR-1B-ALPHA 10 7 PASK 1-1 PASK 10 5 PDGFR-ALPHA 1 1 PDGFR-ALPHA 10 33 PDGFR-ALPHA- D842V 1 0 PDGFR-ALPHA- D842V 10 10 PDGFR-BETA 1 4 PDGFR-BETA 10 39 13
PDK1 1-1 PDK1 10-1 PHK-GAMMA1 1 1 PHK-GAMMA1 10 33 PHK-GAMMA2 1-1 PHK-GAMMA2 10 28 PI3-KINASE- ALPHA 1 8 PI3-KINASE- ALPHA 10 52 PI4-K-BETA 1 5 PI4-K-BETA 10 19 PIM-1-KINASE 1 1 PIM-1-KINASE 10 1 PIM2 1 0 PIM2 10 0 PIM3 1-1 PIM3 10 6 PKA 1 0 PKA 10 3 PKACB 1 0 PKACB 10 5 PKC-ALPHA 1 1 PKC-ALPHA 10 6 PKC-BETA1 1 0 PKC-BETA1 10 4 PKC-ETA 1 2 PKC-ETA 10 3 PKC-GAMMA 1 2 PKC-GAMMA 10 7 PKC-IOTA 1 4 PKC-IOTA 10 4 PKC-THETA 1-1 PKC-THETA 10 3 PKC-ZETA 1-1 PKC-ZETA 10 4 PKCB 1 0 PKCB 10 7 PKN1 1 0 PKN1 10 6 PKN2 1 1 PKN2 10 1 14
PLK1 1-1 PLK1 10 1 PRAK 1 0 PRAK 10 3 PRKD1 1-4 PRKD1 10 18 PRKD2 1 0 PRKD2 10 11 PRKD3 1 0 PRKD3 10 13 PRKG1 1 1 PRKG1 10 7 PRKG2 1 2 PRKG2 10 16 PRKX 1 1 PRKX 10 1 PTK5 1 4 PTK5 10 1 PYK2 1-3 PYK2 10 4 RET 1 4 RET 10 6 ROCK1 1 0 ROCK1 10 10 ROCK2 1-1 ROCK2 10 6 RON 1 2 RON 10-9 ROS 1-1 ROS 10-2 RSK1 1 6 RSK1 10 10 RSK2 1 1 RSK2 10 10 RSK3 1 5 RSK3 10 7 RSK4 1 0 RSK4 10 4 SGK1 1 0 SGK1 10-4 SGK2 1 1 15
SGK2 10 3 SGK3 1-2 SGK3 10-3 SIK 1 6 SIK 10 8 SNF1LK2 1 0 SNF1LK2 10 34 SPHK1 1 3 SPHK1 10-1 SPHK2 1-9 SPHK2 10-8 SRC 1-5 SRC 10 20 SRMS 1 0 SRMS 10-3 SRPK1 1-1 SRPK1 10 2 SRPK2 1 0 SRPK2 10 6 SYK 1 9 SYK 10-6 TAOK2 1-1 TAOK2 10 4 TAOK3 1 0 TAOK3 10-3 TBK1 1 0 TBK1 10 0 TEC 1 26 TEC 10 47 TIE2 1 3 TIE2 10 5 TNIK 1-3 TNIK 10 8 TNK1 1 0 TNK1 10 7 TNK2 1 3 TNK2 10 6 TRKA 1 0 TRKA 10 3 TRKB 1-5 TRKB 10 0 16
TRKC 1 1 TRKC 10 5 TSSK1 1-2 TSSK1 10 5 TSSK2 1 1 TSSK2 10-5 TTK 1 0 TTK 10 6 TXK 1 43 TXK 10 73 TYK2 1 2 TYK2 10 5 TYRO3 1 2 TYRO3 10 14 YES 1 1 YES 10 19 ZAP70 1 5 ZAP70 10-21 17
Supplementary Table 2. Profiling of compound 1 on a panel of GPCRs. Profiling was performed at 10 µm concentration of compound 1. % inhibition is shown for duplicate samples (n1 and n2) together with the mean values calculated from two replicates. % inhibition n1 Compound 1 % inhibition n2 18 % inhibition Mean GPCR 5-HT1A -30.7-51.4-41 5-HT2A 16 21.6 18.8 5-HT2B 99.8 99.4 99.6 5-HT2C -2.7 13.5 5.4 A1-8.7 5-1.8 A3 4.6 5.5 5 ADRA1A 17.6 19.8 18.7 ADRA1D ( 2-79) 6.8 9.3 4.3 ADRA2A 2.1-1.6 0.3 ADRB1-2.7 3.6 0.5 ADRB2 3.8-3.1 0.3 AT1 1.2 8.9 5.1 BB2 7 1.5 4.3 BDKR2-5 -10.7-7.8 BLT1-1.6 4.2 1.3 C5aR 0.4-0.7-0.2 CB1 0.8 7 3.9 CB2 25.7 27.6 26.6 CCK2-2.5 11.8 4.7 CCR1-12.1-0.7-6.4 CCR2B 9.3 19.8 14.6 CGPR1 2.1 0 1.1 CRF1 6.4-3.6 1.4 CX3CR1-4.5 1.3-1.6 CXCR1 3.3 10.6 6.9 CysLT1-1.7-4.8-3.2 D1 64.3 60.8 62.6 D2L 18.3 20.7 19.5 D5-4.2-6.9-5.6 DP -7.7-8.7-8.2 EP2-0.4 4 1.8 EP3-3.6 1.6-1 ETA -3.4-4.6-4 ETB 9.3 3.8 6.6 FP 1 5.3 3.1 FPR1-3 2.2-0.4
GAL1 36.4 26.3 31.3 GnRH -40.7-33.6-37.1 GPR109A 48.5 55 51.7 GPR14 7.1 4.5 5.8 H1 21.9 16.9 19.4 H2 0.7-2 -0.6 H3 7.1 13.8 10.4 IP1 4.8-4.4 0.2 LPA1 3.6 12.9 8.3 LPA3-15.5-26.3-20.9 M1-2.3-4.9-3.6 M2 4.6 10.8 7.7 M3-14.2-0.8-7.5 MC5 3.3-11.3-4 Motilin 13.1 4 8.5 MRGX1 23.4 21.5 22.5 NK1 4.8-6.3-0.8 NK3-9.8-19.1-14.5 NMU1-14.7-9.4-12 NTR1 3.1 2.9 3 OPRD1 1.1-1.4-0.2 OPRM1 5.4 8.1 6.7 OT 3.6-1.9 0.8 OX1 17.1 1 9 P2RY1-11.9-25.2-18.5 PAC1-1.7 6.4 2.4 PAF 10 3.7 6.9 PK1 0.1-16.2-8.1 PRP -3.3-17.7-10.5 PTH1 4 4.2 4.1 S1P3 17.4 2.5 10 SST4-9.4-5.2-7.3 Thrombin-activated PARs -1.9 6.7 2.4 TP -32.8-18.2-25.5 TSH -22.6-18.4-20.5 V1A 12.6 4.3 8.5 V2 13.7 11.4 12.6 VPAC1 3.2 3.4 3.3 VPAC2 40.6 19.1 29.8 Y2 0.8-1.2-0.2 19
Supplementary Table 3. Data collection and refinement statistics for menin-inhibitor complexes. Compound 18 Compound 3- (RS) Compound 7 Compound 31 Data collection Space group P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 Cell dimensions a, b, c (Å) 48.32, 79.82, 124.78 48.97, 79.97, 124.48 49.09, 79.98, 124.43 48.89, 80.45, 125.09 α, β, γ ( ) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å) 50-1.86 (1.89-1.86) 50-1.50 (1.53-1.50) 50-1.54 (1.57-1.54) 50-1.71 (1.74-1.71) R sym or R merge 0.090 (0.652) 0.070 (0.507) 0.094 (0.658) 0.087 (0.589) I / σι 19.04 (2.11) 25.15 (2.33) 18.05 (2.05) 25.77 (2.44) Completeness (%) 99.5 (99.7) 94.8 (84.7) 99.7 (99.9) 98.5 (95.3) Redundancy 6.2 (5.6) 5.6 (5.0) 6.5 (6.1) 7.1 (6.1) Refinement Resolution (Å) 39.94-1.86 40.02-1.50 38.1-1.54 41.82-1.71 No. reflections 38,899 71,069 69,365 50,618 R work / R free 16.31 / 19.68 16.05/18.47 15.92/18.76 16.10/19.41 No. atoms Protein 3674 3657 3666 3636 Ligand/ion 102 95 94 107 Water 319 457 495 399 B-factors (Å 2 ) Protein 22.01 16.59 13.86 17.98 Ligand/ion 37.56 24.85 30.18 30.85 Water 32.45 29.46 27.27 30.75 R.m.s. deviations Bond lengths (Å) 0.015 0.017 0.017 0.015 Bond angles ( ) 1.63 1.75 1.78 1.68 All diffraction data were obtained from a single crystal. *Values in parentheses are for highest-resolution shell. 20
Supplementary Table 4. Caco-2 monolayer permeability for menin-mll inhibitors. Compounds were tested at 10 µm concentrations. P app apparent permeability rate coefficient; mean values of two replicates are shown; Efflux ratio: P app (B A) / P app (A B). IC 50 values from FP assay, GI 50 values from cell viability assay and clogp values are also provided for each compound. Compound IC 50 (nm) GI 50 (MLL-AF9, µm) Mean A B P app (10-6 cm * s -1 ) Mean B A P app (10-6 cm * s -1 ) Efflux ratio clogp 3-(RS) 37 3.4 0.41 22.7 55.4 3.1 7 25 0.78 0.26 23.5 90.4 3.0 4-R 40 0.36 5.0 12.9 2.6 3.9 27 21 0.08 0.5 18.0 33.5 3.7 21 40 0.1 2.2 54.0 24.3 4.7 25 27 0.47 0.3 75.3 283.2 3.4 20 27 0.22 0.83 6.7 8.1 4.1 21