Probe		Negative control		Alternative control
A-196		SGC2043		A-197

• SMILES:
• A-196: [H]N(C1=NN=C(C2=CC=NC=C2)C3=C1C=C(Cl)C(Cl)=C3)C4CCCC4
• A-197: C1CCC(C1)Nc1c2cc(c(cc2c(C(N2CCC(CC2)O)=O)nn1)[Cl])[Cl]
• InChI:
• A-196:InChI=1S/C18H16Cl2N4/c19-15-9-13-14(10-16(15)20)18(22-12-3-1-2-4-12)24-23-17(13)11-5-7-21-8-6-11/h5-10,12H,1-4H2,(H,22,24)
• A-197: InChI=1S/C19H22Cl2N4O2/c20-15-9-13-14(10-16(15)21)18(22-11-3-1-2-4-11)24-23-17(13)19(27)25-7-5-12(26)6-8-25/h9-12,26H,1-8H2,(H,22,24)
• InChIKey:
• A-196: ABGOSOMRWSYAOB-UHFFFAOYSA-N
• A-197: NGGSLWXNDXYNJL-UHFFFAOYSA-N

Selectivity of A-196 is selective against 29 methyltransferases

Selectivity of A-196 against epigenetic reader domains

Cell Assay

[1] Jorgensen S, Schotta G, Sorenson CS. (2013) Histone H4 Lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Research 41: 2797-806.

[2] Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, et al. (2004) A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18:1251–1262.

[3] Sanders SL, Portoso M, Mata J, Bahler J, Allshire RC, et al. (2004) Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage. Cell; 119:603–614.

[4] Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, et al. (2005) Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37:391-400.

[5] Bromberg KD, Mitchell TR, Upadhyay AK, Jakob CG, Jhala MA, et al. (2017 Jan 23) The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol. 

Probe

NVS-CECR2-1

• SMILES:
• NVS-CECR2-1: CC1(CC(CC(C)(N1)C)N2C=CC3=C2C=CC(C4=CC(NC5CC5)=NC(S(=O)(CCC)=O)=N4)=C3)C
• NVS-CECR2-C: CC1(CC(CC(C)(N1)C)N2C=CC3=C2C=CC(C4=NC(NC5CC5)=CC(S(=O)(CCC)=O)=N4)=C3)C
• InChI:
• NVS-CECR2-1: InChI=1S/C27H37N5O2S/c1-6-13-35(33,34)25-29-22(15-24(30-25)28-20-8-9-20)18-7-10-23-19(14-18)11-12-32(23)21-16-26(2,3)31-27(4,5)17-21/h7,10-12,14-15,20-21,31H,6,8-9,13,16-17H2,1-5H3,(H,28,29,30)
• InChIKey:
• NVS-CECR2-1: XVECNLUKQDKOST-UHFFFAOYSA-N
• NVS-CECR2-C: MUEOODFBWCTDNL-UHFFFAOYSA-N

Isothermal Titration Calorimetry

ITC measurements of the NVS-CECR2-1 with CECR2.

Temperature Shift Assay

Selectivity screening of chemical probe NVS-CECR2-1 determined by temperature shift assay. The temperature shifts were mapped onto the phylogenetic tree using red circles corresponding to ΔTm as indicated in the figure.

NanoBRET

Dose-dependent displacement of CECR2 from histone H3.3 following treatment with NVS-CECR2-1.

Fluorescence Recovery After Photobleaching

Half-times of fluorescence recovery (t1/2) after photo bleaching measured for CECR2 after treatment with NVS-CECR2-1 at different concentrations with or without SAHA.

1. Footz, TK., Brinkman-Mills, P et al.  Analysis of the cat eye syndrome critical region in humans and the region of conserved synteny in mice: a search for candidate genes at or near the human chromosome 22 pericentromere. Genome Res, 2001,11: 1053–1070. 
2. Banting GS Barak O et al.  CECR2, a protein involved in neurulation, forms a novel chromatin remodeling complex with SNF2L. Hum Mol Genet, 2005, 14(4):513-24.
3. Lee SK, Park EJ et al. Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein. Mol Cells, 2012, 34(1):85-91
4. Filippakopoulos P. et al., Histone recognition and largescale structural analysis of the human bromodomain family. Cell. 2012, 149, 214-231.
5. Philpott M., et al., Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery. Mol Biosyst. 2011, 10, 2899-908.

Isothermal Titration Calorimetry (ITC)

All calorimetric experiments were performed on a VP-ITC micro-calorimeter (MicroCalTM, LLC Northampton, MA). Protein solutions were buffer exchanged by gel filtration or dialysis into buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). All measurements were carried out at 288.15 K. All injections were performed using an initial injection of 2 µL followed by injections of 8 µL. The data were analysed with the MicroCal ORIGIN software package employing a single binding site model. The first data point was excluded from the analysis. 

Temperature shift assay

Thermal melting experiments were carried out using a Stratagene Mx3005p Real Time PCR machine (Agilent Technologies). NVS-CECR2-1 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000 as described (6).

AlphaScreen Assay

Assays were performed as described previously with minor modifications (7). All reagents were diluted in 25 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. An 11-point 1:2.0 serial dilutions of the ligands was prepared on lowvolume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), using LabCyte Eco liquid handler. Plates filled with 5 µL of the assay buffer followed by 7 µL of peptide H4K5acK8acK12acK16ac. Plates were sealed and incubated for a further 30 minutes, before the addition of 8 µM of the mixture of streptavidin-coated donor beads (12.5 µg/mL) and nickel chelate acceptor beads (12.5 µg/mL) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. IC₅₀ values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls.

Fluorescence Recovery After Photobleaching (FRAP) Assay

FRAP studies were performed using U20S cells expressing full-length CECR2. Six hours after transfection 2.5 µM SAHA (to increase global histone acetylation) was added and cells were treated with 1 µM or 5 µM of NVS-CECR2-1 1 hour before imaging and half recovery times from the fluorescence signal of the bleached U2OS nuclei were plotted.

NanoBRET

U2OS cells were co-transfected with Histone H3.3-HaloTag and NanoLuc-CECR2. Twenty hours post-transfection cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then treated with an increasing dose of NVS-CECR2-1. Five minutes prior to reading, NanoBRET furimazine substrate (Promega) was added to both control and experimental samples and plates were read on a CLARIOstar (BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5 sec reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by and the emission at 610 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000. Relative IC₅₀ values were estimated by non-linear regression analysis of (log) concentration of each inhibitor versus milliBRET ratios (GraphPad Prism).

Probe

MS049

• SMILES:
• MS049: CNCCN1CCC(OCC2=CC=CC=C2)CC1
• MS049N: COCCN1CCC(OCC2=CC=CC=C2)CC1
• InChI:
• MS049: InChI=1S/C15H24N2O/c1-16-9-12-17-10-7-15(8-11-17)18-13-14-5-3-2-4-6-14/h2-6,15-16H,7-13H2,1H3
• InChIKey:
• MS049: HBOJWAYLSJLULG-UHFFFAOYSA-N
• MS049N: MDKBNFXGBVKIHT-UHFFFAOYSA-N

1. Discovery of a Potent, Selective and Cell-active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6, Shen Y, Szewczyk MM, Eram MS, Smil D, Kaniskan HÜ, de Freitas RF, Senisterra G, Li F, Schapira M, Brown PJ, Arrowsmith CH, Barsyte-Lovejoy D, Liu J,Vedadi M, Jin J., J Med Chem. 2016 Sep 1.

Potency against Target

Time-resolved FRET BRD9 Binding Assay

I-BRD9 is a potent and selective binder to (full length) BRD9 with a pIC₅₀ in a Time-Resolved FRET assay of 7.3. BRD4 was used as a representative member of the BET family for initial selectivity screening, I-BRD9 displayed a pIC₅₀ of 5.3 against this protein. 

Selectivity 

I-BRD9 was profiled by BROMOscan™, a novel bromodomain inhibitor binding platform that measures the interactions between test compounds and a panel of bromodomain assays (http://www.discoverx.com), against 34 bromodomains. The results from this screen indicated nanomolar affinity binding at BRD9, with pK_d = 8.7. I-BRD9 showed >700-fold selectivity over the BET family of bromodomains and 200-fold over the highly homologous BRD7 bromodomain (pK_d = 6.4). I-BRD9 displayed >70-fold selectivity against every other bromodomain tested.

Click to enlarge
 

Selectivity Beyond Target Family

I-BRD9 was found to be inactive against a panel of 49 human receptors, ion channels, kinases and other enzymes. Cellular target engagement of BRD9 and disruption of chromatin binding was demonstrated through a NanoBRET assay; and CLEC1, DUSP6, FES, and SAMSN1 genes are selectively regulated in the presence of I-BRD9

Engagement of BRD9 in a cell assay

In a chemoproteomic competition binding assay in HUT-78 cell lysate followed by Western blot analysis, the binding of I-BRD9 to endogenous BRD9 displayed >625-fold selectivity against BET family member BRD3. These data confirm potency at BRD9, and selectivity over the BET family is maintained with endogenous proteins. 

Cellular target engagement of BRD9 and disruption of chromatin binding was demonstrated through a NanoBRET assay measuring displacement of NanoLuc-tagged BRD9 bromodomain from Halotagged histone H3.3

qPCR validation of CLEC1, DUSP6, FES, and SAMSN1 genes selectively regulated by compound I-BRD9 (10 μM), but not by I-BET151 (1 μM). Genes were previously identified by full gene transcriptomics in Kasumi-1 cells

1. Bromodomains as therapeutic targets
   Susanne Muller, Panagis Filippakopoulos, Stephan Knapp, Expert Reviews in Molecular Medicine 2011; 13: e29
    
2. The Discovery of I-BRD9, a Selective Cell Active Chemical Probe for Bromodomain Containing Protein 9 Inhibition,
   Natalie H Theodoulou, Paul Bamborough, Andrew J Bannister, Isabelle Becher, Rino A Bit, Ka Hing Che, Chun-Wa Chung, Antje Dittmann, Gerard Drewes, David H. Drewry, Laurie Gordon, Paola Grandi, Melanie Leveridge, Matthew Lindon, Anne-Marie Michon, Judit Molnar, Samuel C. Robson, Nicholas Charles Oliver Tomkinson, Tony Kouzarides, Rab K. Prinjha, and Philip G Humphreys. Journal of Medicinal Chemistry 2015, DOI: 10.1021/acs.jmedchem.5b00256

The co-crystal structure of I-BRD9 with BRD9 has been solved with a resolution of 1.73 Å (PDB id: 4UIW). The key features are:

- I-BRD9 makes H-bond interactions to the Asn100 side chain, the backbone carbonyl of Ile53, and the backbone NH of Arg101.

- The N-ethyl thienopyridinone Kac mimetic is comfortably accommodated in the hydrophobic binding pocket with a slight movement of Phe45, having little effect on the conformation of the remainder of the site.

- Other interactions are illustrated in the diagram below

Click to enlarge
 

TR-FRET BRD9 Binding Assay BRD9 TR-FRET Assay

A proprietary bromodomain binding small molecule containing a pendant primary amine was tagged with Alexa Fluor 647 (GSK2833930A). All assay components were dissolved in buffer (50 mM HEPES pH7.4, 50 mM NaCl, 5% Glycerol, 1 mM DTT and 1 mM CHAPS). The final concentration of BRD9 protein was 10 nM and the Alexa Fluor647 ligand was at Kd (~100 nM for BRD9). 5 uL of this reaction mixture was added to all wells containing 50 nl of various concentrations of test compound or DMSO vehicle (0.5% DMSO final) in 384 well microtitre plates and incubated in dark for 30 min at room temperature. Eu-W1024 Anti-6xHis Antibody (AD0111 PerkinElmer) at 1.5 nM FAC was used as a detection reagent. The plates were read on the Envision reader and the donor and acceptor counts were determined and the ratio of acceptor/donor was calculated.

Chemoproteomic Profiling

Nuclear extract was produced from fresh HuT78 cells. Affinity profiling assays were performed by derivatising sepharose beads with 2.0 mM of a proprietary bromodomain binding small molecule containing a pendant primary amine (GSK2893910A). I-BRD9 was spiked into HuT78 mixed nuclear and chromatin extracts and incubated for 45 minutes at 4 °C. Derivatised sepharose beads (35 μl beads per sample) were equilibrated in lysis buffer and incubated with cell extract pre-incubated with compound. Beads were washed with lysis buffer containing 0.2 % NP-40 and eluted with 2x SDS sample buffer supplemented with DTT. Aliquots of the eluates from chemoproteomic assays were separated on 4–12 % gel (NuPAGE, Invitrogen) and this was used for Western Blot analysis with antI-BRD9 (Abcam, ab-66443) and anti-BRD3 (Santa Cruz, SC-81202) antibodies.

NanoBRET Assay

HEK293 cells were plated in each well of a 6-well plate and co-transfected with Histone H3.3-HaloTag (NM_002107) and NanoLuc-BRD9 (Q9H8M2) BD domain amino acids 120-240. 20 hrs post-transfection the cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100nM NanoBRET 618 fluorescent ligand (Promega). Cell density was adjusted, re-plated in a 96-well plate and inhibitor added directly to media at final concentrations between 0-33 μM and the plates were incubated for 18 hours at 37 °C in the presence of 5% CO₂. NanoBRET furimazine substrate (Promega) was added to both control and experimental samples at a final concentration of 10 μM and readings were performed within 5 minutes using the CLARIOstar (BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5sec reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by and the emission at 610 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand). 

BAY-598 is a potent SMYD2 inhibitor and acts via a peptide competitive mechanism of action.

Selectivity

Selectivity of BAY-598 within methyltransferase family

Cellular activity

BAY-598 is a potent inhibitor of p53 K370 methylation in cells

1.Brown MA, Sims RJ 3rd, Gottlieb PD, Tucker PW (2006) Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex. Mol Cancer 5: 26.

2.Komatsu S, Imoto I, Tsuda H, Kozaki KI, Muramatsu T, et al. (2009) Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous cell carcinoma. Carcinogenesis 30: 1139-1146.

3.Cho HS, Hayami S, Toyokawa G, Maejima K, Yamane Y, et al. (2012) RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation. Neoplasia 14: 476-486.

4.Eggert E, Hillig RC, Köhr S, Stöckigt D, Weiske J, Barak N,, et al. (2016) Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2  J Med Chem

5.Nguyen H, Allali-Hassani A, Antonysamy S, Chang S, Chen LH, et al. (2015) LLY-507, a Cell-Active, Potent and Selective Inhibitor of Protein Lysine Methyltransferase SMYD2. 290: 13641-53.

6.Ferguson AD, Larsen NA, Howard T, Pollard H, Green I, et al. (2011) Structural Basis of Substrate Methylation and Inhibition of SMYD2. Structure 19: 1262-73.

7.Sweis RF, Wang Z, Algire M, Arrowsmith CH, Brown PJ, et al. (2015) Discovery of A-893, A New Cell-Active Benzoxazinone Inhibitor of Lysine Methyltransferase SMYD2. ACS Med Chem Lett. 6: 695-700.

Main features

• SMYD2 structure with peptide and SAM bound
• Showing how BAY-598 binds in the lysine side-chain pocket
• BAY-598 key hydrogen bonds with substrate pocket
• SMYD2 structure showing BAY-598 and SAM

4-(4-((dimethylamino)methyl)-2,5-dimethoxyphenyl)-2-methyl-2,7-naphthyridin-1(2H)-one

• SMILES:
• CN(CC1=CC(OC)=C(C=C1OC)C(C2=C3C=NC=C2)=CN(C3=O)C)C
• InChI:
• InChI=1S/C20H23N3O3/c1-22(2)11-13-8-19(26-5)15(9-18(13)25-4)17-12-23(3)20(24)16-10-21-7-6-14(16)17/h6-10,12H,11H2,1-5H3
• InChIKey:
• BJFSUDWKXGMUKA-UHFFFAOYSA-N

Temperature Shift Assay

Selectivity profile of BI-9564 using temperature shift assay at 10µM.

Isothermal Titration Calorimetry (ITC)

Binding affinity of BI-9564 towards BRD 7 and BRD 9 measured by ITC.

Fluorescence Recovery After Photobleaching (FRAP) Assay

Half-times of fluorescence recovery (t_1/2) after photo-bleaching measured for BRD9, BRD7 and CECR2 treated either with or without SAHA and BI-9564 at indicated concentrations.

Work on this probe has been published in ‘Structure-based design of an in vivo active selective BRD9 inhibitor’.

1. Muller S. et al., Bromodomains as therapeutic targets. Exp Rev Mol Med. 2011, 13, e29.
2. Filippakopoulos P. et al., Histone recognition and largescale structural analysis of the human bromodomain family. Cell. 2012, 149, 214-231.
3. Whitehouse I. et al., Nucleosome mobilization catalysed by the yeast SWI/SNF complex. Nature 1999, 400, 784–787.

Isothermal Titration Calorimetry (ITC)

All calorimetric experiments were performed on a VP-ITC micro-calorimeter (MicroCalTM, LLC Northampton, MA). Protein solutions were buffer exchanged by gel filtration or dialysis into buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). All measurements were carried out at 288.15 K. All injections were performed using an initial injection of 2 µL followed by injections of 8 µL. The data were analysed with the MicroCal ORIGIN software package employing a single binding site model. The first data point was excluded from the analysis. 

Temperature shift assay

Thermal melting experiments were carried out using a Stratagene Mx3005p Real Time PCR machine (Agilent Technologies). BI-9564 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000 as described (2).

Fluorescence Recovery After Photobleaching (FRAP) Assay

U2OS cells were transfected (Fugene HD; Roche) with mammalian over- expression constructs encoding GFP fused to the N-terminus of full length BRD9, Brd7 or CECR2, respectively. Mutant proteins mutating the conserved Asn to Phe or Ala were generated as described in (3). The following mutations were introduced: N140A for human CECR2, N211F for mouse Brd7 and N163F for human BRD9. The imaging systemconsisted of a Zeiss LSM 710 laser-scanning and control system (Zeiss) coupled to an inverted Zeiss Axio Observer.Z1 microscope equipped with a high-numerical-aperture (N. A. 1.3) 40 x oil immersion objective (Zeiss). Samples were placed in an incubator chamber in order to maintaining temperature and humidity. FRAP and GFP fluorescence imaging were both carried out with an argon-ion laser (488 nm) and with a PMT detector set to detect fluorescence between 500-550 nm. Once an initial scan had been taken, a region of interest corresponding to approximately 50 % of the entire GFP positive nucleus was empirically selected for bleaching. A time lapse series was then taken to record GFP recovery using 1% of the power used for bleaching. The image datasets and fluorescence recovery data were exported from ZEN 2009, the microscope control software, into Origin to determine the average half-time for full recovery for 10-20 cells per treatment point.