Supporting Information. Property focused structure-based optimization of small molecule inhibitors of the proteinprotein

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1 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, jolantag@umich.edu Tel Table of Contents 1. Supplementary Figure 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

2 Supplementary Figure 1. Representative titration curves from the FP assay for selected menin-mll inhibitors. 2

3 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

4 c 4

5 d 5

6 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 ABL1 1 2 ABL AKT1 1 0 AKT AKT2 1 2 AKT AKT3 1 0 AKT ALK 1-5 ALK 10 1 AMP-A1B1G1 1 4 AMP-A1B1G AMP-A2B1G1 1 1 AMP-A2B1G ARG 1 1 ARG ARK5 1 0 ARK AURORA-A 1-1 AURORA-A AURORA-B 1-2 AURORA-B 10 7 AURORA-C 1-1 AURORA-C AXL 1-2 AXL BLK 1-9 BLK 10 2 BMX 1 4 BMX 10 0 BRAF 1-1 BRAF 10 4 BRK 1-7 BRK

7 BRSK1 1-1 BRSK BRSK2 1 2 BRSK BTK 1 7 BTK CAMK1D 1 2 CAMK1D 10 9 CAMK2A 1 1 CAMK2A 10 5 CAMK2D 1-1 CAMK2D CAMK4 1 2 CAMK CDK1 1 1 CDK CDK2 1 0 CDK 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 CDK CDK6-CYCLIND3 1-1 CDK6-CYCLIND CHEK1 1-5 CHEK CHEK2 1 1 CHEK CK1 1 1 CK CK1-GAMMA1 1 2 CK1-GAMMA CK1-GAMMA2 1 6 CK1-GAMMA CK1-GAMMA3 1 4 CK1-GAMMA CLK

8 CLK CLK2 1-3 CLK CLK3 1 0 CLK CLK4 1 2 CLK CRAF 1 6 CRAF 10 1 CSK 1 0 CSK DAPK1 1 1 DAPK DAPK3 1 0 DAPK DCAMKL2 1 2 DCAMKL DDR2 1-1 DDR DYRK1A 1 1 DYRK1A 10 7 DYRK1B 1-1 DYRK1B 10 7 DYRK2 1-3 DYRK DYRK3 1 2 DYRK DYRK4 1 1 DYRK EGFR 1-1 EGFR 10 0 EGFR-T790M 1 3 EGFR-T790M 10 3 EPH-A1 1-1 EPH-A EPH-A2 1-1 EPH-A EPH-A3 1 0 EPH-A EPH-A4 1 1 EPH-A

9 EPH-A5 1 0 EPH-A EPH-A8 1 2 EPH-A EPH-B1 1 5 EPH-B EPH-B2 1-4 EPH-B EPH-B3 1 0 EPH-B EPH-B4 1 1 EPH-B ERB-B2 1 1 ERB-B ERB-B4 1-2 ERB-B FER 1-3 FER 10 4 FES 1 1 FES 10 6 FGFR1 1 9 FGFR FGFR2 1 3 FGFR FGFR3 1 0 FGFR FGFR4 1 0 FGFR FGR 1 0 FGR FLT FLT FLT FLT FLT FLT FMS 1 0 FMS 10 5 FYN 1 2 FYN 10 6 GRK

10 GRK GRK7 1-9 GRK GSK3-ALPHA 1 1 GSK3-ALPHA 10-2 GSK3-BETA 1 2 GSK3-BETA 10 1 HASPIN 1 5 HASPIN HCK 1 4 HCK HIPK1 1-3 HIPK HIPK2 1 1 HIPK HIPK3 1-1 HIPK HIPK4 1 5 HIPK 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 IRAK IRR 1-1 IRR 10 2 ITK 1 4 ITK JAK1 1 2 JAK JAK2 1-3 JAK JAK3 1 9 JAK

11 JNK1 1-1 JNK JNK2 1 0 JNK JNK3 1-2 JNK KDR 1 3 KDR 10 1 KIT 1 1 KIT LATS2 1 2 LATS 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 LYNB 1 2 LYNB MAP4K2 1-1 MAP4K MAP4K4 1 0 MAP4K MAP4K5 1-3 MAP4K MAPK1 1-4 MAPK MAPK3 1 2 MAPK MAPKAPK MAPKAPK MAPKAPK MAPKAPK MARK1 1 2 MARK MARK

12 MARK MARK4 1 3 MARK MEK1 1 1 MEK MEK2 1-2 MEK MELK 1-1 MELK MER 1 2 MER MET 1 1 MET 10 2 MINK 1-1 MINK 10 2 MKNK1 1 1 MKNK MNK2 1 5 MNK MRCK-ALPHA 1 2 MRCK-ALPHA 10 3 MRCK-BETA 1 0 MRCK-BETA 10 0 MSK1 1-1 MSK MSK2 1-2 MSK MSSK1 1 2 MSSK MST1 1 0 MST MST2 1 3 MST MST3 1-1 MST MUSK 1 0 MUSK 10-3 NEK1 1 3 NEK NEK2 1 6 NEK

13 NEK6 1 0 NEK NEK7 1-2 NEK NEK9 1-4 NEK P38-ALPHA 1-1 P38-ALPHA 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 P70S6K P70S6K2 1 1 P70S6K PAK1 1-1 PAK PAK2 1-1 PAK PAK3 1-2 PAK PAK4 1-2 PAK PAK5 1 0 PAK PAK6 1 0 PAK PAR-1B-ALPHA 1 2 PAR-1B-ALPHA 10 7 PASK 1-1 PASK 10 5 PDGFR-ALPHA 1 1 PDGFR-ALPHA PDGFR-ALPHA- D842V 1 0 PDGFR-ALPHA- D842V PDGFR-BETA 1 4 PDGFR-BETA

14 PDK1 1-1 PDK PHK-GAMMA1 1 1 PHK-GAMMA PHK-GAMMA2 1-1 PHK-GAMMA PI3-KINASE- ALPHA 1 8 PI3-KINASE- ALPHA PI4-K-BETA 1 5 PI4-K-BETA PIM-1-KINASE 1 1 PIM-1-KINASE 10 1 PIM2 1 0 PIM PIM3 1-1 PIM 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-BETA 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 PKN PKN2 1 1 PKN

15 PLK1 1-1 PLK PRAK 1 0 PRAK 10 3 PRKD1 1-4 PRKD PRKD2 1 0 PRKD PRKD3 1 0 PRKD PRKG1 1 1 PRKG PRKG2 1 2 PRKG PRKX 1 1 PRKX 10 1 PTK5 1 4 PTK PYK2 1-3 PYK RET 1 4 RET 10 6 ROCK1 1 0 ROCK ROCK2 1-1 ROCK RON 1 2 RON 10-9 ROS 1-1 ROS 10-2 RSK1 1 6 RSK RSK2 1 1 RSK RSK3 1 5 RSK RSK4 1 0 RSK SGK1 1 0 SGK SGK

16 SGK SGK3 1-2 SGK SIK 1 6 SIK 10 8 SNF1LK2 1 0 SNF1LK SPHK1 1 3 SPHK SPHK2 1-9 SPHK SRC 1-5 SRC SRMS 1 0 SRMS 10-3 SRPK1 1-1 SRPK SRPK2 1 0 SRPK SYK 1 9 SYK 10-6 TAOK2 1-1 TAOK TAOK3 1 0 TAOK TBK1 1 0 TBK TEC 1 26 TEC TIE2 1 3 TIE TNIK 1-3 TNIK 10 8 TNK1 1 0 TNK TNK2 1 3 TNK TRKA 1 0 TRKA 10 3 TRKB 1-5 TRKB

17 TRKC 1 1 TRKC 10 5 TSSK1 1-2 TSSK TSSK2 1 1 TSSK TTK 1 0 TTK 10 6 TXK 1 43 TXK TYK2 1 2 TYK TYRO3 1 2 TYRO YES 1 1 YES ZAP ZAP

18 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 HT2A HT2B HT2C A A ADRA1A ADRA1D ( 2-79) ADRA2A ADRB ADRB AT BB BDKR BLT C5aR CB CB CCK CCR CCR2B CGPR CRF CX3CR CXCR CysLT D D2L D DP EP EP ETA ETB FP FPR

19 GAL GnRH GPR109A GPR H H H IP LPA LPA M M M MC Motilin MRGX NK NK NMU NTR OPRD OPRM OT OX P2RY PAC PAF PK PRP PTH S1P SST Thrombin-activated PARs TP TSH V1A V VPAC VPAC Y

20 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 P P P P Cell dimensions a, b, c (Å) 48.32, 79.82, , 79.97, , 79.98, , 80.45, α, β, γ ( ) 90.0, 90.0, , 90.0, , 90.0, , 90.0, 90.0 Resolution (Å) ( ) ( ) ( ) ( ) R sym or R merge (0.652) (0.507) (0.658) (0.589) I / σι (2.11) (2.33) (2.05) (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) No. reflections 38,899 71,069 69,365 50,618 R work / R free / / / /19.41 No. atoms Protein Ligand/ion Water B-factors (Å 2 ) Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) All diffraction data were obtained from a single crystal. *Values in parentheses are for highest-resolution shell. 20

21 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) R

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