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
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, 0.127 mmol) in DMF (2 ml) was added Cs 2C 3 (83.0 mg, 0.255 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 216-218 C. H MR (400 MHz, DMS-d o 1 6) d 10.63 (s, 1 H), 8.65 (s, 1 H), 8.08-8.02 (m, 5 H), 7.68-7.60 (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 392.2. 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
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 248-250 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), 7.90-7.85 (m, 3 H), 7.78 (d, J = 8.0 Hz, 2 H), 7.74-7.70 (m, 1 H), 7.62-7.58 (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, 0.123 mmol) in dry DMF (3 ml) was added BP (70.4 mg, 0.159 mmol) and DBU (27.6 µl, 0.185 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 196-198 C. H MR (400 MHz, DMS-d o 1 6) d 11.84 (s, 1 H), 10.68 (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), 7.80-7.77 (m, 2 H), 7.74-7.70 (m, 1 H), 7.62-7.58 (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 408.2. S3
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, 0.423 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, 0.346 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
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 11.83 (s, 1 H), 10.63 (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 409.9. S5
S6
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
LBL1 (µm) input pulldown IP: streptavidin 20 10 5 1 0 20 10 5 1 0 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
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
A input pulldown IP: streptavidin fragment - 1-664 1-427 428-664 - 1-664 1-427 428-664 IB: anti-flag B input pulldown IP: streptavidin fragment 1-387 10-387 20-387 30-387 78-387 1-387 10-387 20-387 30-387 78-387 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
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
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
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
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), 1153-5. S14
Protein ID SpectraCounts (w/ LBL1) SpectraCounts (w/o LBL1) LMA_HUMA 147 218 LMB1_HUMA 118 189 PYC_HUMA 177 165 K2C1_HUMA 137 122 ACTB_HUMA 96 110 CATD_HUMA 70 100 PCCA_HUMA 88 90 TBB5_HUMA 49 68 K2C5_HUMA 73 66 DHCR7_HUMA 54 65 ACTA_HUMA 54 65 K2C7_HUMA 78 65 K1C9_HUMA 69 63 K22E_HUMA 74 63 K1C14_HUMA 64 61 TBA1B_HUMA 41 55 CH60_HUMA 54 51 TBA1C_HUMA 35 50 TBB4B_HUMA 37 49 TBB2A_HUMA 39 48 TBA1A_HUMA 36 48 EF1A1_HUMA 32 43 K2C6C_HUMA 40 43 AT1A1_HUMA 47 40 ADT2_HUMA 50 39 ACTBL_HUMA 32 39 K2C4_HUMA 28 36 RA2_HUMA 30 35 ERG7_HUMA 32 35 HRPL_HUMA 26 34 HS90B_HUMA 22 34 AL3A2_HUMA 40 33 MCCA_HUMA 33 33 ECHA_HUMA 27 31 HRPM_HUMA 34 31 LMB2_HUMA 19 31 ATPA_HUMA 26 30 VDAC2_HUMA 26 30 GRP78_HUMA 23 30 AXA2_HUMA 28 28 VDAC1_HUMA 31 27 HSP7C_HUMA 20 27 TBA8_HUMA 18 27 DHX9_HUMA 25 26 HRPK_HUMA 16 25 CLH1_HUMA 20 25 HRPU_HUMA 24 25 EPL_HUMA 25 25
ADT3_HUMA 33 25 ADT1_HUMA 33 25 HRPC_HUMA 22 24 HS90A_HUMA 13 24 PM_HUMA 24 23 FLA_HUMA 16 23 G3P_HUMA 13 22 KPYM_HUMA 15 22 PPIA_HUMA 16 22 ACT4_HUMA 16 22 K2C75_HUMA 26 22 K2C79_HUMA 26 22 EA_HUMA 14 21 GRP75_HUMA 19 21 H4_HUMA 15 21 K1C13_HUMA 18 21 SCRB2_HUMA 10 20 HRPF_HUMA 17 20 HSP71_HUMA 17 20 PDIA3_HUMA 18 19 HRH1_HUMA 17 19 H2B1J_HUMA 15 19 ASAH1_HUMA 16 18 PDIA6_HUMA 22 18 SGMR1_HUMA 13 18 DDX17_HUMA 15 18 LPPRC_HUMA 17 17 PPT1_HUMA 15 17 H2B1K_HUMA 15 17 UCL_HUMA 15 16 MPCP_HUMA 18 16 PTBP1_HUMA 17 15 GLYM_HUMA 14 15 MDHM_HUMA 19 15 P5CS_HUMA 18 15 ILF2_HUMA 11 15 ILF3_HUMA 16 15 LAC2_HUMA 6 15 HS71L_HUMA 13 15 K22_HUMA 18 15 HYEP_HUMA 20 14 AT2A2_HUMA 24 14 TIF1B_HUMA 14 14 PLEC_HUMA 18 14 DX39B_HUMA 14 14 EGFR_HUMA 18 13 DHC24_HUMA 17 13 CTL2_HUMA 23 13 RAB1B_HUMA 19 13
H2A1_HUMA 15 13 AT_HUMA 13 12 MUTA_HUMA 12 12 CALX_HUMA 24 12 ST48_HUMA 10 12 ACADV_HUMA 8 12 TCPQ_HUMA 8 12 GBLP_HUMA 6 12 VDAC3_HUMA 15 12 DDX3X_HUMA 9 12 RAB1A_HUMA 15 12 DDX5_HUMA 14 12 1C03_HUMA 13 12 ALDA_HUMA 8 11 ATPB_HUMA 12 11 DPB_HUMA 9 11 EF2_HUMA 6 11 ABCD3_HUMA 10 11 RAB7A_HUMA 14 11 SYRC_HUMA 7 11 TERA_HUMA 10 11 GAAB_HUMA 9 11 SF3B3_HUMA 15 11 SQRD_HUMA 10 11 HRPR_HUMA 7 11 H2A1B_HUMA 12 11 HRH2_HUMA 8 11 1C02_HUMA 12 11 1C12_HUMA 13 11 K2C72_HUMA 13 11 K2C73_HUMA 11 11 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 11 10 TM205_HUMA 14 10 TECR_HUMA 9 10 MTCH2_HUMA 5 10 DX39A_HUMA 10 10 ACT1_HUMA 8 10