Supplemental Information. The Hedgehog Pathway Effector Smoothened. Exhibits Signaling Competency in the Absence. of Ciliary Accumulation

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1 Chemistry & Biology, Volume 21 Supplemental Information The edgehog Pathway Effector Exhibits Signaling Competency in the Absence of Ciliary Accumulation Chih-Wei Fan, Baozhi Chen, Irene Franco, Jianming Lu, eping Shi, Shuguang Wei, Changguang Wang, Xiaofeng Wu, Wei Tang, Michael G. Roth, oelle S. Williams, Emilio irsch, Chuo Chen, and Lawrence Lum

2 Figure S1 A B C IR-1 oramlized h pathway response h pathway response (3T3-ShhFL cells) Wnt pathway response (L-Wnt-STF cells) IC50 (μm) h: FL < -6x SD -1x SD < RL < 1x SD 1600 inhibitors Dose-response test (0.8, 2.5, μm) FL < -6x SD -1x SD < RL < 1x SD 94 inhibitors Wnt pathway test (2.5μM) FL/RL > 80% control 81 inhibitors otch pathway test (2.5μM) FL/RL > 80% control IR-1 Br ovel h pathway antagonists h pathway test (2.5μM) DMS h: cda IR-2 200K compounds 3T3-ShhFL cells 2.5μM compound Acetylated tubulin (I3T3 cells) DMS Cmpd [μm] D IR-1 IR-3 Br h: IR-2 IR-4 h: Br IR-3 IR-4 S IR-3 h: inhibitors Br IR-2 IR-5 Exogenous h test (2.5μM) FL/RL < -5x SD 38 inhibitors of h response (IR) IC50 test IR-6 - h: 0.16 IR-4 IR-5 h IC50 < 0.2μM Wnt IC50 > 10μM IR-7 7 IRs h: 0.20 Br IR-5 IR-6 Cyclopamine oramlized h pathway response Known Smo antagonists IC50 (μm) h: 0.27 IR-6 Cmpd [μm] IR-7 h: SAT1 IR-7 SAT1

3 Figure S1 (continued) E Alkaline phosphatase activity Shh CM: - Cmpd: IR DMS DMS SAT1 C310T1/2 cells F Frizzled4 Bodipy-cycloapmine DMS DMS Cyclopamine SAT1 IWR-1 0% Bodipy 100% Bodipy Bodipy Bodipy 0% Bodipy 0% Bodipy Bodipy 108% IR-1 IR-2 IR-3 IR-4 IR Bodipy Bodipy Bodipy 1% Bodipy Bodipy 1% 1% 1% 9% IR-6 IR-7 G Frizzled 4 DMS DMS SAG 2% 1% 0% Bodipy 100% Bodipy 0% Bodipy Figure S1, related to Figure 1. (A) The UTSW chemical library was screened in a cell line that reports cell-autonomous, ligand-mediated Shh pathway response with a pathway responsive firefly luciferase (FL) reporter (GliBS reporter). A dose-response test was performed to identify potent compounds and to eliminate those with cytotoxicity [indicated by change in activity of a control Renilla luciferase (RL) reporter]. Compounds with specific h pathway activity were revealed following testing in cells that report Wnt and otch signaling. Compounds with activity against h pathway response were separated from those that potentially block Shh protein production by testing their activity against exogenously provided Shh protein in I3T3 cells transfected with the GliBS and control Renilla luciferase reporters. Seven inhibitors (IR-1 to -7) with IC50s against h pathway response less than 0.2μM were further evaluated. Concentration of compounds used in each assay is indicated on the left and criteria used for selecting compounds of interest are indicated to the right of the flow chart. Standard deviation (SD) calculations are mean centered. (B) IR compounds are specific for h response. IC50 plots for IR compounds in h and Wnt pathway response. h pathway response with compounds was measured in 3T3-ShhFL. Wnt pathway activity was measured in L-Wnt-STF cells. Cyclopamine and SAT1 are known Smo antagonists. (C) IR compounds do not affect ciliogenesis. I3T3 cells were treated with IR compounds and immunostained for acetylated tubulin to visualize primary cilia. (D) expression induced by Shh is inhibited by the IR compounds. cda generated from 3T3-ShhFL cells treated with IR compounds or the Smo antagonist SAT1 was used to determine relative abundance of mra. The assay is linear across a broad range of cda concentrations used as template. (E) IR compounds inhibit h-induced expression of alkaline phosphatase. Lysate from C310T1/2 cells treated with conditioned medium containing Shh protein (Shh CM) in the absence or presence of various IR compounds or the Smo antagonist SAT1 for 5 days were analyzed for alkaline phosphatase activity levels. Data are meansem from three measurements. (F) Flow cytometric quantification of labeled cells in the presence of various Smo antagonists. Cells transfected with Smo or Frizzled4 (control) cda were labeled with in the presence of DMS, known Smo antagonists, IR compounds, or IWR1 (inhibitor of Wnt/β-catenin pathway activity). Results are normalized to Bodipy signal from Smo-transfected cells treated with. G) Flow cytometric quantification of labeled cells in the presence of SAG. Same experiment as in F except with SAG.

4 A Shh CM: - Gli3 Gli3R α-tubulin Smo-/- MEFs Shh CM B Cmpd (2.5μM): Shh CM: DMS DMS SAT1 Vismodegib IR1 - Gli2-FLAG Gli2R-FLAG α-tubulin C ormalized h pathway response PTC1-/- MEFs Cyclopamine IC50: 0.079μM Cmpd [log μm] SAT1 IC50: μM Vismodegib IC50: 0.027μM IR-1 IC50: 0.011μM Figure S2, related to Figure 1. (A) h induced suppression of Gli proteolytic processing is dependent upon Smo. (B) IR-1 blocks Smo-mediated disengagement of Gli2 proteolytic processing. I3T3 cells stably expressing Gli2-FLAG stimulated with Shh CM were additionally treated with indicated known Smo antagonists (SAT1, Vismodegib) or IR-1. (C) IC50 of IR-1 in PTC1-/- MEFs. Known Smo antagonists (cyclopamine, SAT1, Vismodegib) or IR-1 were dosed in PTC1-/- MEFs transfected with GliBS reporter and a control reporter used to normalize for transfection

5 A ormalized response to Shh protein D IC50: 0.1μM IR-Cy3 [log μm] ormalized h pathway response B % Smo ciliary localization Shh CM: Compound (2.5μM): Vismodegib IR-cy3 C % Smo ciliary localization ortho-ir-1 (/A) meta-ir-1 (/A) IR-1 (0.01μM) nM SAG: Compound (2.5μM): Vismodegib IR-cy3 E Fzd4 Smo Smo IR-1 Smo meta-ir-1 Smo ortho-ir Cmpd [log μm] % cells labeled with IR-Cy Figure S3, related to Figures 1 and 2. (A) IR-Cy3 is active. 3T3-ShhFL cells were treated with increasing amounts of IR-Cy3 and h pathway response determined by luciferase activity. (B) IR-Cy3 blocks Shh CM induced Smo accumulation in (C) IR-Cy3 blocks SAG induced Smo accumulation in the primary cilia. % of cells with Smo localized to the primary (D) IR-1 activity is supported by a para substitution pattern centered on the central aromatic ring. IC50 of IR-1, meta-ir-1, and ortho-ir-1 for h pathway response was determined in 3T3-ShhFL cells. Data are meansem from three measurements. (E) and not Fzd4. Furthermore, this binding can be competed away with IR-1 and not meta-ir-1 or ortho-ir-1 molecules (all tested at 10μM). (F) SAG is able to achieve maximal activity in the presence of IR-1. Assays were perfromed in I- 3T3 cells transfected with the GliBS reporter. Data are meansem from three meaasurements. F h pathway response (RLU) SAG EC50: 0.04μM SAG0.1μM IR-1 EC50: 0.37μM SAG [log μm]

6 A Frizzled4 DA Smo DA SSC- 0% Bodipy Bodipy Bodipy-Cyclopamine B DA: SSC % Bodipy-Cyclopamine Smo - Endo: - SmoM SSC- 0.7% Bodipy Bodipy-Cyclopamine IR-Ac Endo insensitive Endo sensitive tubulin C Cmpd (10μM): DA: total lysate IR-Ac IR-1 - Smo SmoM2 Post medial Golgi Smo Pre medial Golgi Smo tubulin streptavidin pull down biotinylated Smo D SmoM2 DA: - IR-Ac (10μM): chase time (hrs): Post medial Golgi Smo Pre medial Golgi Smo Figure S4, related to Figure 2. (A) The ability of IR-Ac to compete with the binding of to Smo was tested as described in Figure S1F. (5nM) and IR-Ac (1μM). (B) Assignment of Smo subcellular distributions was achieved using Endo sensitivity. Lysate from cells transiently transfected with Smo or SmoM2 DA were treated with medial Golgi. (C) IR-Ac but not IR-1 is able to promote SmoM2 exit from the ER. Cos7 cells transfected with either Smo-myc or SmoM2-myc DA were subjected to cell surface biotinylation using a cell membrane impermeable biotinylation reagent. Biotinylated Smo protein was isolated using streptavidin cross-linked agarose beads and detected with an anti-myc antibody. (D) IR-Ac induces accumulation of mature SmoM2 by facilitating SmoM2 exit from the ER. Cos-7 cells transiently transfected with SmoM2 DA were pulse-labeled with S35-methionine and -cysteine. Immature SmoM2 (pre-medial Golgi) was then chased in the presence or absence of IR-Ac.

7 total lysate Sufu IP Sufu: / -/ - / -/ - 50kD 37kD Sufu IgG heavy chain Figure S5, related to Figure 3. Control for Sufu immunoprecipitation. Immunoprecipitated material from Sufu K MEFs using an anti-sufu antibody show no reactivity to protein detected by an anti-sufu antibody in wt MEF-derived samples.

8 A Cell line: Shh CM: Cmpd (10μM): I3T3 DMS SmoM2-I3T DMS DMS Vismodegib IR-1 IR-Ac Gli3 B DAPI, Ac-tubulin, Gpr ± 4% 26 ± 2% 3T3 DMS SmoM2 DMS 29 ± 4% 92 ± 3% Gli3R tubulin SmoM2 50μM IR-1 SmoM2 50μM IR-Ac Figure S6, related to Figure 5. (A) The IR-1 derivative IR-Ac has improved capacity as compared to IR1 for inhibiting SmoM2-induced disruption of Gli3 proteolytic processing. Lysate from I3T3 (with or without Shh conditioned medium) or I3T3 cells stably expressing SmoM2 treated with IR1 or IR-Ac were analyzed by Western blotting for Gli3 and Gli3 repressor (Gli3R). (B) IR-Ac but not IR-1 blocks SmoM2-induced depletion of Gpr161 from the primary cilium. % of IMCD3 cells with Gpr161 localized to the primary cilium (labeled with an acetylated tubulin antibody) is indicated for each chemical condition (=100). Data are meansem of three

9 Supplemental Experimental Procedures All reactions were performed in glassware under a positive pressure of argon. TLC analyses were performed on EMD 250 µm Silica Gel 60 F 254 plates and visualized by quenching of UV fluorescence (λ max = 254 nm) or by staining with ceric ammonium molybdate.. 1 and spectra were recorded on a Varian Inova-400 instrument. Chemical shifts for 1 spectra are reported in ppm (δ) relative to the residual 1 signals of the solvent (CD 3 D: δ 3.31; DMS-d 6 : δ 2.50) and the multiplicities are presented as follows: s = singlet, d = doublet, t = triplet, m = multiplet. To a suspension of acid 1 (1.91 g, 10.0 mmol) in DCM (20 ml) and DMF (0.020 ml) was added oxalyl chloride (0.875 ml, 10 mmol) at 0 C under Ar. After h stirring at room temperature, a solution of 2 (4 g, 5.0 mmol) in DCM (25 ml) and Et 3 (3.5 ml, 25 mmol) was added. The reaction was stirred overnight and concentrated. After washing the resulted solid by EtAc (2 ml 2), compound 3 was collected as white solid (1.93 g, 85%). To a suspension of acid 1 (98 mg, mmol) in DCM (2 ml) and DMF (0.020 ml) was added oxalyl chloride (0.044 ml, mmol) at 0 C under Ar. After 10 min stirring at room temperature, a solution of 4 (0.104 g, mmol) in DCM (1 ml) and Et 3 (0.35 ml, 2.5 mmol) was added. After 3h at room temperature, the solvent was removed by evaporation. The residue was dissolved in 20% TFA/DCM ( ml). After 1 h at room temperature, the reaction was concentrated and gave crude 5 (138 mg) which was used without further purification. To a suspension of acid 6 (136 mg, mmol) in DCM (2 ml) and DMF (0.020 ml) was added oxalyl chloride (0.044 ml, mmol) at 0 C under Ar. After 20 min stirring at room

10 temperature, a solution of crude 5 above in DCM (1 ml) and Et 3 (0.35 ml, 2.5 mmol) was added. After 3h at room temperature, the reaction was concentrated. The resulted solid was washed by EtAc ( ml 2) and gave 7 as a white solid (200 mg, 73%). 1 MR (400 Mz, DMS-d6) δ 16 (s, 1), (s, 1), 9.67 (s, 1), (m, 10), 1.46 (s, 9). Compound 7 (70 mg, 0.13 mmol) was dissolved in 20% TFA/DCM ( ml). After 20 min, the solvent was removed by evaporation. The deprotected product was used without further purification. To a suspension of acid 8 (32 mg, 0.13 mmol) in DCM (2 ml) and DMF (0.010 ml) was added oxalyl chloride (0.013 ml, 0.14 mmol) at 0 C under Ar. After 10 min stirring at room temperature, a solution of deproteded product of 7 above in DCM (1 ml) and Et 3 (0.07 ml, mmol) was added. After 5h at room temperature, the reaction was concentrated. The resulted solid was washed by EtAc ( ml 2) and gave 9 as a white solid (46 mg, 40%). 1 MR (400 Mz, DMS-d6) δ 16 (s, 1), (s, 1), (s, 1), (m, 10), 6.76 (t, 1, J = 5.6 z), (m, 2), (m, 2), 2.30 (t, 2, J = 7.2 z), (m, 2), 1.35 (s, 9).

11 Compound 9 (20 mg, mmol) was dissolved in 20% TFA/DCM ( ml). After 20 min, the reaction was concentrated. The deprotected product was used without further purification. To a solution of Cy 3 -Su (27 mg, 0.03 mmol) in DMF (0.40 ml) was added a solution of deprotected product of 9 above in Py. (0.30 ml) under Ar. After stirring overnight at 40 C, the product was purified by RP-TLC (30% TFA- 2 ) and gave red solid 10 (6.0 mg, 15%). IR-Cy 3 : 1 MR (400 Mz, CD 3 D) δ 8.56 (t, 1, J = 13.2 z), (m, 2), (m, 3), 7.68 (m, 3), (m, 5), (m, 1), (m, 2), (m, 3), 6.52 (t, 2, J = 13.2 z), (m, 4), (m, 1), (m, 2), 2.38 (t, 2, J = 7.2 z), 2.24 (t, 1, J = 7.6 z), 2.18 (t, 2, J = 7.2 z), 1.78 (s, 6), 1.77 (s, 6), (m, 3). Compound 7 (44 mg, mmol) was dissolved in 20% TFA/DCM ( ml). After 30 min, the solvent was removed by evaporation. The residue was dissolved in Py. (1 ml) which was

12 followed by the addition of Ac (0.009 ml, 0.13 mmol). After stirring at room temperature overnight, the reaction was concentrated and the residue was washed by EtAc ( ml 2). Compound 11 was collected as white solid (34 mg, 88%). 1 MR (400 Mz, DMS-d6) δ 16 (s, 1), 10 (s, 1), (s, 1), (m, 10), 5 (s, 3).

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