Bicyclic helical peptides as dual inhibitors selective. for Bcl2A1 and Mcl-1 proteins

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1 Supporting Inormation Bicyclic helical peptides as dual inhibitors selective or Bcl2A1 and Mcl-1 proteins de Araujo A.D.; Lim, J.; Wu, K-C.; Xiang, Y.; Good, A. C.; Skerlj, R.; Fairlie, D. P.* Contents Page Figure S1. Synthetic solid phase route or preparing synthetic cyclic peptides S2-3 Figure S2. Inhibition o A1/FBid complex measured by FP assay. Figure S3. Denaturing SDS-PAGE analysis or covalent adduct ormation with A1. Figure S4. Mass spectra o A1-peptide conjugates. S4 S4 S5 Figure S5. MS/MS analysis o trypsin-digested electrophoresis gel band containing protein ragment bearing Cys55 covalently bonded to peptide 5. Figure S6. Stability o BIM, 1, 5 and 8 in rat plasma. Figure S7. Stability o BIM, 1 and 5 with trypsin or chymotrypsin. S6 S7 S7 Figure S8. Western blot showing covalent bonding o intracellular A1 to peptides 5 and 10 in live SKMel28 cells ater 24h incubation. Figure S9. SKMel28 cell viability ater incubation with BIM or BimSAB A or 48 h Figure S10. Analytical PLC showing retention times o puriied peptides 5 and 8. Figure S11. UV spectra or puriied peptides 2-10 measured by UPLC. Figure S12. ESI-MS mass spectra or puriied peptides Table S1. IC 50 and Ki values or inhibition o Bcl2 protein/fbid complexes. Table S2. Structure and MS analysis o peptides synthesized in this study. S8 S8 S9 S10 S11 S12 S13 S1

2 A Step Reagents and conditions B SPPS a,d a b c d e SPPS: a) 30% piperidine in DMF (2 x 3 min); b) Fmoc-AA- (4 equiv), CTU (4 equiv), DIPEA (8 equiv), 2 x 30 min 10 mm Grubbs catalyst 1 st generation in dry DCE, 2 x 2 h 2% TFA in DCM, 10 x 1 min a) 30% piperidine in DMF (2 x 3 min); b) 2 (1 M): DIPEA (0.4M), 2 x 5 min acrylic acid (4 equiv), ATU (4 equiv), DIPEA (4 equiv) in DMF, 15 min I A Q E L R S 5 I G D S 5 F * b Mtt Mtt c,e g h TFA:TIS: 2 (95:2.5:2.5), 2 h 30 % piperidine in DMF, 2 x 3 min PyBP (4 equiv), DIPEA (8 equiv) in DMF, 4-6 h * i j k acrylic acid (1.2 equiv), ATU (1.2 equiv), DIPEA (2.4 equiv) in DMF, 30 min propiolic acid (1.2 equiv), ATU (1.2 equiv), DIPEA (2.4 equiv) in DMF, 30 min FITC (2 equiv), DIPEA (4 equiv) in DMF, overnight 2 Cycle 2 C SPPS a D SPPS a,d Mtt Pip Fmoc Fmoc E L R S 5 I G D S 5 F b E L R S 5 I G D S 5 F a,d SI 5 X I A SQ 5 E L R I G D F Mtt b Mtt * * c,e * K I A QD E L R S 5 I G D S 5 F Boc c,h I A Q E L R S 5 I G D S 5 F Boc b Boc 2 Bicycle i 2 Bicycle 4 S2

3 E Fmoc I A Q E L R K I G D D F c,h Fmoc I A SQ 5 E L R I G D F a,d SI 5 X I A SQ 5 E L R I G D F Mtt b Mtt c,e 2 SPPS a S 5 Mtt Pip Bicycle 5 c,j 2 Bicycle 7 F SPPS a I A Q E L R * * Boc I A Q E L R Mtt Pip Fmoc E L R K I G D D F c,h Fmoc E L R I G D F a,d Mtt Pip K I A QD E L R I G D F Boc c,h 2 i I A Q E L R Bicycle 6 I G D * F I G D F I G D F 2 2 G SPPS a Fmoc I A Q E L R K I G D D F c,h Fmoc I A SQ 5 E L R I G D F a Fmoc-βAla SI 5 X I A SQ 5 E L R I G D F Mtt b Fmoc-βAla Mtt c,j Fmoc-βAla g,k FITC-βAla FITC-βAla 2 S 5 Mtt Pip Bicycle 9 Figure S1. Synthetic solid phase route or preparing cyclic truncated peptides A. Reaction conditions and reagents used or each synthetic step depicted in B (peptide 2), C (peptide 3), D (peptide 4), E (peptides 5 and 7, similar route or peptide 8 and 10), F (peptide 6) and G (peptide 9). Asterisks denote that a standard protecting group was used or the amino acid side chain. S3

4 IC50 (nm) BIM time (min) Figure S2. Inhibition o A1/FBid complex measured by FP assay. Time-course o changing IC 50 or inhibition o the FBid/A1 complex measured by competitive FP assay. Assay conditions: FBid (5 nm) was combined with A1 (15 nm) or 30 min in running buer (50 mm Tris, 150mM acl, 1 mm EDTA, 1 mm TCEP, 0.005% Tween-20, p 7.2.) and combined with a serial dilution o the peptide (ranging rom 8 µm to 0.1 nm). Ater 15, 30, 60, 90 and 120 min incubation at rt, luorescence polarization was measured and IC 50 values were determined by nonlinear regression analysis using Prism sotware 7.0. A Buer BIM kda Figure S3. Denaturing SDS-PAGE analysis or covalent adduct ormation with A1. rylamide-containing peptides (1-6) covalently bind to A1 (showing an upper band shit or the protein ater incubation with peptide), whereas those without an electrophile do not orm a covalent adduct with A1 (BIM and 7). S4

5 20492 A A A A A m/z Figure S4. Mass spectra o A1-peptide conjugates. Incubation o A1 (1 µm, MW: 18756) with peptides 2-6 (10 µm) or 2-6 h in buer p 7.2, showing the expected molecular weight (within ±1 Da o calculated mass). o evidence o multiple addition o the peptide to the protein was ound. Conditions: A1 and peptides were combined in buer 50 mm Tris, 150mM acl, 1 mm EDTA, 2 mm TCEP, p 7.2 to a inal concentration o 1 µm A1 and 10 µm peptide in a total o 20 µl reaction volume. The reaction was analyzed by LC-MS on a Shimadzu exera uplc system coupled to a Triple TF 5600 mass spectrometer (ABSCIEX) using Zorbax C18 column (Agilent). S5

6 Bicycle 5/Bl-1 covalent conjugate in gel trypsin digestion LK I A Q E L R S SC 55 LDVVVSLDTAR TLF I G D F Intensity, cps Data Set ame: 3000 MS/MS or ragment [M+3] +3 R LR ELR SLDTAR VSLDTAR VVSLDTAR I A Q E L R 2500 S TAR VVSLDTAR LDVVVSLDTAR LDTAR DVVVSLDTAR DTAR TF Product (880.1): Exp 3, [M+3]+3 min rom Sample 1 (Aline AR VVVSLDTAR 0.1% o tallest A 47 D) peak o Aline A 47 D.wi m/z, Da Scan or combined ELR and LDVVVSLDTAR sequences S SCLDVVVSLDTAR only ragment containing sequence ELR +TF Product (880.1): Exp 3, min rom Sample 1 (Aline A 47 D) o Aline A 47 D.wi General Precursor MW: Precursor Charge: 3 Mass tolerance: 0.06 Da -terminal Modiication: (none) Cysteine Modiication: (none) C-terminal Modiication: (none) Methionine Modiication: (none) Digest Agent: Trypsin Present AAs: (none speciied) Missing AAs: (none speciied) Fragment Ions Data Mass Set Type: ame: Monoisotopic m/z Tolerance: 0.06 Da Minimum Intensity: General Peptide Precursor Sequence MW: Results: (none) Precursor Charge: 3 Mass tolerance: 0.06 Da -terminal Modiication: (none) Partial Cysteine Sequence Modiication: Tag Results: (none) (none) C-terminal Modiication: (none) Methionine Modiication: (none) Digest Agent: Trypsin Matches: Present AAs: 28 (none speciied) Missing AAs: (none speciied) ypertag: MS/MS Fragment Ions or (none) ragment ELR [M+3] +3 (none) : matching MS ingerprint or sequence ELR Ion Charge: Mass Type: 1 Monoisotopic m/z Tolerance: 0.06 Da Fragments: Minimum Intensity: 0.1% o tallest peak Residue Mass Immo a a-3 b b-2 b-3 b-3 b- c x y y-2 y-3 y-3 z E, Glu Peptide Sequence Results: (none) L, Leu R, Arg Partial Sequence Tag Results: (none) Matches: 131 ypertag: (none) LDVVVSLDTAR (none) Ion Charge: 1 Fragments: Residue Mass Immo a a-3 b b-2 b-3 b-3 b- c x y y-2 y-3 y-3 z L, Leu D, Asp , Asn V, Val , Asn V, Val V, Val S, Ser L, Leu D, Asp T, Thr A, Ala R, Arg MS/MS or ragment [M+3] +3 : matching MS ingerprint or sequence LDVVVSLDTAR Figure S5. MS/MS analysis o trypsin-digested electrophoresis gel band containing protein ragment bearing Cys55 covalently bonded to peptide 5. The MS/MS ingerprint or m/z ion revealed the expected y ion ragmentation pattern or the A1 derived-sequence LDVVVSVDTAR and the peptide 5 derived-sequence ELR. S6

7 Figure S6. Stability o BIM, 1, 5 and 8 in rat plasma. Time-course degradation o BIM and peptides 1, 5 and 8 (10 µm) in resh rat plasma (containing 1% DMS) at 37 C over 24 h (n = 3). TRYPSI CYMTRYPSI XIC o +TF MS: 400 to 1800 Da rom Sample 1 (J51A) o J51A.wi (DuoSpray ()) Max. 4.1e7 cps. 1.7e7 1.6e I W I A Q E L R R I G D E F A Y Y A R R 1.4e7 I W I A Q E L R R I G D E F A Y Y A R R 1.2e7 BIM Intensity, cps 1.0e7 8.0e6 6.0e6 BIM e6 2.0e6 1.08e7 1.00e XIC o +TF MS: to Da rom Sample (J51B) 4.0 o J51B.wi 5.0 (DuoSpray 6.0()) Max. 3.7e cps. Time, min e7 3.0e7 Intensity, cps Intensity, cps 9.00e6 8.00e6 7.00e6 6.00e6 5.00e6 4.00e6 3.00e6 2.00e6 1.00e XIC 0.00 o +TF MS: to Da rom 3.0 Sample 4.01 (Aline S F 6) o Aline Max e7 cps Time, min e7 1.8e7 1.6e7 1.4e7 1.2e7 1.0e7 8.0e6 A Y Y A R R 1 5 Intensity, cps Intensity, cps 2.5e7 2.0e7 1.5e7 1.0e7 5.0e6 0.0 XIC o +TF MS: to Da rom Sample (J51E) 4.0 o J51E.wi 5.0 (DuoSpray 6.0()) Max. 3.4e7 cps Time, min e7 3.0e7 2.5e7 2.0e7 1.5e7 A Y Y A R R e e7 4.0e6 2.0e e Time, min Time, min Figure S7. Stability o BIM, 1 and 5 with trypsin or chymotrypsin. Stability o BIM, 1 and 5 (10 µm) in the presence o trypsin (1:150, protease:peptide ratio) or chymotrypsin (1:50, protease:peptide ratio) in 50 mm ammonium bicarbonate buer ater 1h incubation at 37 0 C. The integrity o the peptides in the presence o the proteases was analysed by LC-MS, and ragmentation determined by MS. Yellow arrows pointed to retention time o intact peptide. Dotted red lines indicated site o proteolytic cleavage. S7

8 Bicycle 5 (µm) Bicycle 10 (µm) Bl GAPD Figure S8. Western blot showing covalent bonding o intracellular A1 to peptides 5 and 10 in live SKMel28 cells ater 24 h incubation. 125 % cell viability BimSAB A BIM log [peptide] (M) Figure S9. SKMel28 cell viability ater incubation with BIM or BimSAB A or 48 h. BIM is not active up to 50 µm while BimSAB A displayed a dose-dependent activity with a IC 50 o 15.7 µm. S8

9 mau 120 bicycle min mau bicycle min Figure S10. Analytical PLC showing the retention times o puriied peptides 5 and 8. Conditions: Phenomenex Luna C18 5 um (250 x 4.60 mm) column eluting at a low rate o 1 ml/min and a gradient o 30 to 100 % buer B (90% C 3 C/10% 2 /0.1% TFA in buer A, 0.1% TFA in water) over 20 min. S9

10 Figure S11. UV spectra or puriied peptides 2-10 measured by UPLC. Conditions: Shimadzu UPLC system connected to LCMS-2020 single quadrupole mass spectrometer using an Agilent Zorbax R-DS III column (Fig.S9) and and a gradient o 20 to 80 % buer B (90% C 3 C/10% 2 /0.1% ormic acid in buer A, 0.1% ormic acid in water) over 6 min. For peptide 9, gradient was 30 to 100% B over 6 min. S10

11 9 Figure S12. ESI-UPLC-MS mass spectra or puriied peptides Calculated expected mass ound in Table S2. S11

12 Table S1. IC 50 and Ki values or inhibition o Bcl2 protein/fbid complexes measured by competitive FP assays ater 2h incubation with peptide. Ki values were calculated rom the IC 50 values accordingly to reerence 30. (nd = not determined). IC 50 and K i or A1/FBid inhibition o these peptides is time-dependent, here are the calculated values ater 2h incubation. IC 50 (nm) Bcl-w Bcl-xL Bcl-2 Mcl-1 A1 BIM 38.1 ± ± ± ± ± ± ± ± ± ± 0.7* ± ± ± ± ± 4.2* 3 > 3200 >32000 > ± ± 1.5* 4 > ± ± 1.1* ± ± ± ± ± 0.2* ± ± ± ± ± 0.3* ± ± ± ± ± ± ± ± ± ± nd nd nd 63.0 ± ± 0.2* * IC 50 or A1/FBid inhibition o these peptides is time-dependent. ere is IC 50 ater 2h incubation. Ki (nm) Bcl-w Bcl-xL Bcl-2 Mcl-1 A1 BIM < ± 0.3 < 1 < ± < ± ± 0.6 < 1 < 1* ± ± ± ± ± 1.2* 3 > 1000 > 1000 > 1000 < 1 < 1* 4 > ± ± 24 < ± 0.3* ± ± ± 2.0 < 1 < 1* ± ± ± 4.0 < 1 < 1* ± ± ± 6 < 1 < ± ± ± 7 < ± nd nd nd < 1 < 1* * K i or A1/FBid inhibition o these peptides is time-dependent. ere is K i ater 2h incubation. S12

13 Table S2. Structure and MS analysis o synthetic peptides synthesized in this study. Cycle 1 was synthesized as described (re 22). Compound Sequence Expected mass bserved mass I A Q L L R I G D F FITC-βAla I A Q L L R I G D F I A Q L L R I G D F BIM -IWIAQELRRIGDEFAYYARR S13