Table of contents 1. Thermal denaturation assay with BRD4(BD2) incubated with the covalent inhibitors vs. their. non-covalent controls.

Transcription

1 Supporting Information DESIGN AND CHARACTERIZATION OF NOVEL COVALENT BROMODOMAIN AND EXTRA-TERMINAL DOMAIN (BET) INHIBITORS TARGETING A METHIONINE Olesya A. Kharenko a *, Reena G. Patel a, S. David Brown a, Cyrus Calosing a, Andre White b, Damodharan Lakshminarasimhan b, Robert K. Suto b, Bryan C. Duffy c, Douglas B. Kitchen c, Kevin G. McLure a, Henrik C. Hansen a, Edward H van der Horst a, Peter R. Young a a Zenith Epigenetics, Suite 300, 4820 Richard Road SW, Calgary, Alberta, T3E 6L1, Canada b Xtal BioStructures, Inc., 12 Michigan Dr., Natick, MA 01760, USA c AMRI, 26 Corporate Circle, PO Box 15098, Albany, NY , USA To whom correspondence should be addressed: Olesya@zenithepigenetics.com Zenith Epigenetics, Suite 300, 4820 Richard Road SW, Calgary, Alberta, T3E 6L1, Canada Table of contents 1. Thermal denaturation assay with BRD4(BD2) incubated with the covalent inhibitors vs. their non-covalent controls. S2 2. Thermal denaturation assay with non-bet bromodomain proteins incubated with the covalent inhibitors vs. their non-covalent controls. S3 3. Thermal denaturation assay demonstrating stability of the covalent 1A/BRD4(BD1) complex at various ph and temperature conditions S4 4. Thermodenaturation assay with BRD4(BD1) and covalent inhibitor in the presence of the increasing concentrations of methionine or cysteine amino acids S5 5. Determination of the half-life of BRD4 post cycloheximide treatment in OCI-AML3 cell line. S6 6. Effect of covalent inhibitors on durability of proliferation in MV4-11 cells S7 7. Effect of the covalent compound vs. non-covalent control on the viability of PBMCs. S7 8. HPLC traces of tested compounds to demonstrate purity S8 9. Molecular Formula Strings S X-ray data reduction statistics and crystal parameters. S Crystallographic refinement statistics. S References S15 S1

2 Figure S1. Thermal denaturation assay with BRD4(BD2) incubated with the covalent inhibitors vs. their non-covalent controls at 30 min and 4 h. S2

3 Figure S2. Thermal denaturation assay with non-bet bromodomain proteins incubated with the covalent inhibitors vs. their non-covalent controls. S3

4 Figure S3. Thermal denaturation assay demonstrating stability of the covalent 1A/BRD4(BD1) complex in various ph and temperature conditions. A. 1A was incubated with BRD4(BD1) for 4 h at ph=5.4, 7.4, and 9 at room temperature. B. 1A/BRD4(BD1) complex was incubated for 4 h at room temperature and 37 0 C. C. 1A/BRD4(BD1) complex was incubated for 4 h followed by 3 freeze-thaw cycles. Thermoshifts was measured as described in the Materials and Method section. S4

5 Figure S4. Thermodenaturation assay with BRD4(BD1) and covalent inhibitor 1A in the presence of increasing concentrations of methionine or cysteine amino acids. S5

6 Figure S5. Determination of the half-life of BRD4 post cycloheximide treatment in OCI-AML3 cell line. S6

7 % Proliferation Relative to DMSO DMSO 3 3A 2 2A Figure S6. Effect of covalent inhibitors on durability of proliferation in MV4-11 cells S7

8 Figure S7. Effect of the covalent compound vs. non-covalent control on the viability of PBMCs. S8

9 Figure S8. HPLC traces of tested compounds to demonstrate purity 2 (ZEN-2906) 1 (ZEN-2759) S9

10 3 (ZEN-3212) S10

11 2A (ZEN-3862) 3A (ZEN-3411) S11

12 1A (ZEN-3219) S12

13 Table S1. Molecular Formula Strings Compound name 1 (ZEN-2759) CC1=C(C(C)=NO1)C1=CN(CC2=CC=CC=C2)C(=O)C=C1 2 (ZEN-2906) CC1=C(C(C)=NO1)C1=CN(CC2=CC=CC(F)=C2)C(=O)C=C1 3 (ZEN-3212) CC1=C(C(C)=NO1)C1=CC(N)=C2N=CN(CC3=CC=CC=C3)C2=C1 1A (ZEN-3219) CC1=C(C(C)=NO1)C1=CN(CC2=CC=C(C=C2)C2CO2)C(=O)C=C1 2A (ZEN-3411) CC1=C(C(C)=NO1)C1=CC(N)=C2N=CN(CC3=CC=C(C=C3)C3CO3)C2=C1 3A (ZEN-3862) CC1=C(C(C)=NO1)C1=CN(CC2=CC=C(C3CO3)C(F)=C2)C(=O)C=C1 S13

14 Table S2. X-ray data reduction statistics and crystal parameters. 1 (ZEN-2759) 1A (ZEN-3219) Space group P P Unit cell a= Å, b= Å, c= Å, α=β=γ=90 a= Å, b= Å, c= Å, α=β=γ=90 Resolution range (Å) ( ) ( ) Number of measurements 74, ,794 Number of unique reflections 11,124 22,125 R sym (%) a (0.444) (0.483) Completeness (%) a 99.8 (100) 99.9 (100) I/σ a 24.4 (5.8) 12.7 (4.2) Redundancy a 6.7 (6.9) 6.7 (6.6) a Numbers in the parentheses are for the highest resolution shell. Table S3. Crystallographic refinement statistics. 1 (ZEN-2759) 1A (ZEN-3219) Resolutionrange (Å) R cryst (%) R free (%) Number of ligand; waters 1; 176 1; 218 Rmsd bond lengths (Å) a Rmsd bond angles ( ) a Average B-factors (Å 2 ) Main chain atoms Side chain atoms Ligands Waters Ramachandran Plot (%) Favored 96% 100% Allowed 4% 0% Disallowed 0% 0% a Root-mean square deviation (rmsd) from the standard stereochemistry (Engh and Huber, 1991). S14

15 References: Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and Development of COOT Acta Cryst D. 2010, 66, Engh, R.A.; Huber, R. Accurate bond and angle parameters for X-ray protein structure refinement Acta Cryst. 1991, A47, Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W.B.; Fedorov, O.; Morse, E.M.; Keates, T.; Hickman, T.T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M.R.; Wang, Y.; Christie, A.L.; West, N.; Cameron, M.J.; Schwartz, B.; Heightman, T.D.; La Thangue, N.; French, C.A.; Wiest, O.; Kung, A.L.; Knapp, S.; Bradner, J.E.; Selective inhibition of BET bromodomains. Nature 2010, 468, McLure, K.G.; Gesner, E.M.; Tsujikawa, L.; Kharenko, O.A.; Atwell, S.; Campeau, E.; Wasiak, S.; Stein, A.; White, A.; Fontano, E.; Suto, R.K.; Wong, N.C.W.; Wagner, G.S.; Hansen, H.C.; Young, P.R. RVX-208, an inducer of ApoA-I in humans, is a BET Bromodomain antagonist. PLoS ONE, 2013, 8, Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology, 1997, 276: S15