Anticancer pyrroloquinazoline LBL1 targets nuclear lamins

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1 Supporting Information Anticancer pyrroloquinazoline LBL1 targets nuclear lamins Bingbing X. Li, #,* Jingjin Chen, # Bo Chao, # Larry L. David, & Xiangshu Xiao #,* # Program in Chemical Biology, Department of Physiology and Pharmacology, & Department of Biochemistry and Molecular Biology, regon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, regon 97239, USA Synthetic procedures......s2 Figure S1... S6 Figure S2... S8 Figure S3... S9 Figure S4. S10 Figure S5. S11 Figure S6. S12 References.. S13 S1

2 H H 2 H Cs 2 C 3, propargyl bromide H H 2 LBL1 1 -(1-amino-7-(prop-2-yn-1-yl)-7H-pyrrolo[3,2-f]quinazolin-3-yl)-2-naphthamide (1). To a solution of LBL1 (45 mg, mmol) in DMF (2 ml) was added Cs 2C 3 (83.0 mg, mmol) at rt. After stirring for 30 min, propargyl bromide (12 µl, 0.22 mmol) was added and the resulting mixture was stirred for an additional 4 h. The solid was filtered off and the filtrate was concentrated. The residue was purified by column chromatography on silica gel, eluting with 20:1 EtAc:THF containing 1% DIPEA to give the a gray solid that was further treated with Et 2 (3 ml) and collected by filtration to give the desired compound 1 (8.0 mg, 16%) as a gray solid: mp C. H MR (400 MHz, DMS-d o 1 6) d (s, 1 H), 8.65 (s, 1 H), (m, 5 H), (m, 3 H), 7.45 (d, J = 9.2 Hz, 1 H), 7.34 (d, J = 3.2 Hz, 1 H), 7.28 (brs, 2 H), 5.28 (d, J = 2.4 Hz, 2 H), 3.50 (t, J = 2.4 Hz, 1 H); ESIMS, calcd 392.1, found H 2 H S1 H + S2 H S3 H H 4-Benzoyl--(1-hydroxy-7H-pyrrolo[3,2-f]quinazolin-3-yl)benzamide (S3). A mixture of S2 1 (363 mg, 1.12 mmol) and S1 (250 mg, 0.75 mmol) in dry DMF (5 ml) was stirred at 90 o C for 1 h. Then the solvent was removed and the residue was purified by column chromatography on silica S2

3 gel, eluting with 3:1 EtAc:DCM containing 1% DIPEA to give compound S3, which was washed with DCM (3 ml) to give the desired compound as a yellow solid (80 mg, 26% yields): mp C. H MR (400 MHz, DMS-d o 1 6) d 12.6 (brs, 2 H), 11.7 (s, 1 H), 8.28 (d, J = 8.0 Hz, 2 H), (m, 3 H), 7.78 (d, J = 8.0 Hz, 2 H), (m, 1 H), (m, 3 H), 7.33 (d, J = 8.4 Hz, 1 H), 7.24 (s, 1 H). H H H i) BP, DBU ii) H 3 /MeH H H 2 H S3 2 -(1-amino-7H-pyrrolo[3,2-f]quinazolin-3-yl)-4-benzoylbenzamide (2). To a stirred solution of S3 (50.0 mg, mmol) in dry DMF (3 ml) was added BP (70.4 mg, mmol) and DBU (27.6 µl, mmol). The resultant reaction mixture was stirred for 4 h at 25 C. Then o H 3 (7 in MeH, 0.70 ml, 4.90 mmol) was added. The reaction mixture was stirred at 25 C o for 16 h. The solvent was removed and the residue was purified by column chromatography on silica gel, eluting with 4:1 EtAc:THF containing 1% DIPEA to give a yellow solid, which was further treated with DCM (2 ml) and collected by filtration to give the desired compound 2 (12.0 mg, 25%) as a yellow solid: mp C. H MR (400 MHz, DMS-d o 1 6) d (s, 1 H), (s, 1 H), 8.12 (d, J = 5.2 Hz, 2 H), 7.89 (d, J = 8.8 Hz, 1 H), 7.82 (d, J = 8.0 Hz, 2 H), (m, 2 H), (m, 1 H), (m, 3 H), 7.35 (d, J = 8.4 Hz, 1 H), 7.31 (brs, 1 H), 7.23 (brs, 2 H); ESIMS, calcd 408.1, found S3

4 H 2 H S1 H + F 3 C 8 F 3 C H S4 H H F 3 C H H H i) BP, DBU ii) H 3 /MeH F 3 C H H 2 H S4 3 -(1-hydroxy-7H-pyrrolo[3,2-f]quinazolin-3-yl)-4-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzamide (S4). A mixture of 8 (180 mg, 0.55 mmol) and S1 (84 mg, mmol) in dry DMF (2 ml) was stirred at 90 C for 3 h. Then the solvent was removed and the residue was purified by o column chromatography on silica gel, eluting with 3:1 EtAc:DCM containing 1% DIPEA to give compound S4, which was washed with DCM (3 ml) to give the desired compound as a yellow solid (100 mg, 57% yield): mp >160 C (dec). H MR (400 MHz, DMS-d o 1 6) d 12.6 (brs, 2 H), 11.7 (s, 1 H), 8.23 (d, J = 8.4 Hz, 2 H), 7.87 (d, J = 8.8 Hz, 1 H), 7.61 (t, J = 2.4 Hz, 1 H), 7.42 (d, J = 8.4 Hz, 2 H), 7.31 (d, J = 8.8 Hz, 1 H), 7.22 (brs, 1 H). -(1-Amino-7H-pyrrolo[3,2-f]quinazolin-3-yl)-4-(3-(trifluoromethyl)-3H-diazirin-3- yl)benzamide (3). To a stirred solution of S4 (95.0 mg, 0.23 mmol) in dry DMF (4 ml) was added BP (153 mg, mmol) and DBU (67 µl, 0.46 mmol). The resultant reaction mixture was stirred for 6 h at 25 C. Then H o 3 (7 in MeH, 1.3 ml, 9.1 mmol) was added. The reaction mixture was stirred at 25 C for 16 h. The solvent was removed and the residue was o purified by column chromatography on silica gel, eluting with 20:1 to 4:1 EtAc:THF S4

5 containing 1% DIPEA to give the desired compound 3, which was treated with DCM (2 ml) and collected by filtration to give the desired compound 3 (19.0 mg, 20% yield) as a gray solid (23.0 mg starting material 10 was recovered): mp >200 C (dec). H MR (400 MHz, DMS-d o 1 6) d (s, 1 H), (s, 1 H), 8.08 (d, J = 8.0 Hz, 2 H), 7.89 (d, J = 8.8 Hz, 1 H), 7.60 (s, 1 H), 7.39 (d, J = 8.0 Hz, 2 H), 7.34 (d, J = 8.8 Hz, 1 H), 7.29 (s, 1 H), 7.20 (brs, 2 H); ESIMS, calcd 410.1, found S5

6 S6

7 Figure S1. In vitro cancer cell growth inhibitory activities of LBL1 in the CI-60 cancer cell panel. The GI 50 values (Log 10 transformed) were obtained after incubating the cells with LBL1 for 48 h using sulforhodamine B assay. S7

8 LBL1 (µm) input pulldown IP: streptavidin LA LC LB1 GAPDH Figure S2. Dose-dependent competition labeling experiment of Figure 3A. The experimental design is exactly the same as that in Figure 3A except that different concentrations of LBL1 were used. S8

9 LA 1 METPS--QRRATR SGAQASSTPLSPTRITRLQEKEDLQELDRLAVYIDRVRSLETEAGLRL 61 LB1 1 MAT ATPVPprmgSRAGGPTTPLSPTRLSRLQEKEELRELDRLAVYIDKVRSLETESALQL 62 LB2 1 MSPPSpgRRREQRrpraaatmATPLP----GRAGGPATPLSPTRLSRLQEKEELRELDRLAHYIDRVRALELEDRLLL 76 LA 62 RITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARTKKEGDLIAAQARLKDLEALL 141 LB1 63 QVTEREEVRGRELTGLKALYETELADARRALDDTARERAKLQIELGKCKAEHDQLLLYAKKESDLGAQIKLREYEAAL 142 LB2 77 KISEKEEVTTREVSGIKALYESELADARRVLDETARERARLQIEIGKLRAELDEVKSAKKREGELTVAQGRVKDLESLF 156 LA 142 SKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAERLQTMKEELDFQKIYSEELRETKRR 221 LB1 143 SKDAALATALGDKKSLEGDLEDLKDQIAQLEASLAAAKKQLADETLLKVDLERCQSLTEDLEFRKSMYEEEIETRRK 222 LB2 157 HRSEVELAAALSDKRGLESDVAELRAQLAKAEDGHAVAKKQLEKETLMRVDLERCQSLQEELDFRKSVFEEEVRETRRR 236 LA 222 HETRLVEIDGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDARQSAERSLVGAAHEELQQSRIRIDS 301 LB1 223 HETRLVEVDSGRQIEYEYKLAQALHEMREQHDAQVRLYKEELEQTYHAKLEARLSSEMTSTVSAREELMESRMRIES 302 LB2 237 HERRLVEVDSSRQQEYDFKMAQALEELRSQHDEQVRLYKLELEQTYQAKLDSAKLSSDQDKAASAAREELKEARMRLES 316 LA 302 LSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLE 381 LB1 303 LSSQLSLQKESRACLERIQELEDLLAKEKDSRRMLTDKEREMAEIRDQMQQQLDYEQLLDVKLALDMEISAYRKLLE 382 LB2 317 LSYQLSGLQKQASAAEDRIRELEEAMAGERDKFRKMLDAKEQEMTEMRDVMQQQLAEYQELLDVKLALDMEIAYRKLLE 396 LA 382 GEEERL 387 LB1 383 GEEERL 388 LB2 397 GEEERL 402 Identities 60% 59% Figure S3. Sequence alignment of human LA(1-387), LB1(1-388) and LB2(1-402). The identical sequences are highlighted in green. S9

10 A input pulldown IP: streptavidin fragment IB: anti-flag B input pulldown IP: streptavidin fragment IB: anti-flag Figure S4. Representative blots to show that LBL1-P labeled LA(1-387). HEK293T cells were transfected with indicated FLAG-tagged LA fragments. Then the cells were treated with LBL1- P followed by click reaction with a biotin- 3. The lysates were then precipitated with streptavidin-agarose beads. The bound proteins were then analyzed by SDS-PAGE followed by anti-flag blotting analysis. S10

11 LA(1-387) LA(78-387) Coomassie gel Figure S5. LA(1-387) (left) and LA(78-387) were purified to homogeneity from E. coli. The purified proteins were run a 10% SDS-PAGE and then the gels were stained with coomassie blue. S11

12 Figure S6. LBL1 did not change the melting profile of GST. GST (2 µm) was incubated with LBL1 (0 or 5 µm). Then the melting of the samples was analyzed in the same way as in Figure 4E. S12

13 Figure S7. LBL1 induced DSB formation and apoptosis. (A) LBL1 induced phosphorylation of H2AX. MDA-MB-231 cells were treated with LBL1 or doxorubicin (Dox, 0.5 µm) for 24 h. Then the cells were harvested and the cell lysates were prepared for Western blot analysis with indicated antibodies. Dox was used as a positive control. (B-C) LBL1 induced apoptosis in MDA-MB-231 cells. The cells were treated with LBL1 for 5 days. Then the cells were analyzed by flow cytometry after the cells were stained with annexin V and propidium iodide (PI). Quantification of the apoptotic cells (Q1) was presented in (C). S13

14 References: (1) livo, H. F.; Perez-Hernandez,.; Liu, D.; Iruthayanathan, M.; 'Leary, B.; Homan, L. L.; Dillon, J. S., Synthesis and application of a photoaffinity analog of dehydroepiandrosterone (DHEA). Bioorg. Med. Chem. Lett. 2010, 20 (3), S14

15 Protein ID SpectraCounts (w/ LBL1) SpectraCounts (w/o LBL1) LMA_HUMA LMB1_HUMA PYC_HUMA K2C1_HUMA ACTB_HUMA CATD_HUMA PCCA_HUMA TBB5_HUMA K2C5_HUMA DHCR7_HUMA ACTA_HUMA K2C7_HUMA K1C9_HUMA K22E_HUMA K1C14_HUMA TBA1B_HUMA CH60_HUMA TBA1C_HUMA TBB4B_HUMA TBB2A_HUMA TBA1A_HUMA EF1A1_HUMA K2C6C_HUMA AT1A1_HUMA ADT2_HUMA ACTBL_HUMA K2C4_HUMA RA2_HUMA ERG7_HUMA HRPL_HUMA HS90B_HUMA AL3A2_HUMA MCCA_HUMA ECHA_HUMA HRPM_HUMA LMB2_HUMA ATPA_HUMA VDAC2_HUMA GRP78_HUMA AXA2_HUMA VDAC1_HUMA HSP7C_HUMA TBA8_HUMA DHX9_HUMA HRPK_HUMA CLH1_HUMA HRPU_HUMA EPL_HUMA 25 25

16 ADT3_HUMA ADT1_HUMA HRPC_HUMA HS90A_HUMA PM_HUMA FLA_HUMA G3P_HUMA KPYM_HUMA PPIA_HUMA ACT4_HUMA K2C75_HUMA K2C79_HUMA EA_HUMA GRP75_HUMA H4_HUMA K1C13_HUMA SCRB2_HUMA HRPF_HUMA HSP71_HUMA PDIA3_HUMA HRH1_HUMA H2B1J_HUMA ASAH1_HUMA PDIA6_HUMA SGMR1_HUMA DDX17_HUMA LPPRC_HUMA PPT1_HUMA H2B1K_HUMA UCL_HUMA MPCP_HUMA PTBP1_HUMA GLYM_HUMA MDHM_HUMA P5CS_HUMA ILF2_HUMA ILF3_HUMA LAC2_HUMA 6 15 HS71L_HUMA K22_HUMA HYEP_HUMA AT2A2_HUMA TIF1B_HUMA PLEC_HUMA DX39B_HUMA EGFR_HUMA DHC24_HUMA CTL2_HUMA RAB1B_HUMA 19 13

17 H2A1_HUMA AT_HUMA MUTA_HUMA CALX_HUMA ST48_HUMA ACADV_HUMA 8 12 TCPQ_HUMA 8 12 GBLP_HUMA 6 12 VDAC3_HUMA DDX3X_HUMA 9 12 RAB1A_HUMA DDX5_HUMA C03_HUMA ALDA_HUMA 8 11 ATPB_HUMA DPB_HUMA 9 11 EF2_HUMA 6 11 ABCD3_HUMA RAB7A_HUMA SYRC_HUMA 7 11 TERA_HUMA GAAB_HUMA 9 11 SF3B3_HUMA SQRD_HUMA HRPR_HUMA 7 11 H2A1B_HUMA HRH2_HUMA C02_HUMA C12_HUMA K2C72_HUMA K2C73_HUMA AATM_HUMA 8 10 PDIA1_HUMA 9 10 DLDH_HUMA 4 10 H14_HUMA 5 10 PLAK_HUMA 9 10 RL4_HUMA 4 10 _HUMA 6 10 SDHL_HUMA TM205_HUMA TECR_HUMA 9 10 MTCH2_HUMA 5 10 DX39A_HUMA ACT1_HUMA 8 10