TAF1(2) ( Kd = 1.8 μM ); TAF1 ( IC50 = 0.9 μM )
TAF1 (second bromodomain, TAF1(2)) - dissociation constant (Kd) = 1.8 μM (determined by isothermal titration calorimetry); IC50 = 0.9 μM (determined by FRET displacement of tetraacetylated H4 peptide) [1]
CREBBP (bromodomain) [1]
EP300 (bromodomain) [1]
BRD9 (bromodomain) [1]

CeMMEC1, a BRD4 inhibitor, exhibits a high affinity for TAF1, as evidenced by its IC50 of 0.9 μM and Kd of 1.8 μM (2) additionally demonstrates a strong affinity for the BRD9, EP300, and CREBBP bromodomains. CeMMEC1, at doses of 1, 10, and 20 μM, reduces the quantity of THP1 cells in the S phase. Moreover, CeMMEC1 triggers apoptosis. When CeMMEC1 and (S)-JQ1 are combined, treatment alone significantly reduces cell viability[1].

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CeMMEC1 (10 μM) did not bind the first or second bromodomain of BRD4 in AlphaLISA assays; it showed high affinity for bromodomains of CREBBP, EP300, BRD9 and TAF1(2) in BromoScan profiling [1].
CeMMEC1 dose‑dependently reduced MYC mRNA expression in wild‑type KBM7 cells to a level comparable to (S)‑JQ1 (Fig. 2c; exact values not provided) [1].
In THP1 human acute monocytic leukemia cells, CeMMEC1 caused a clear and dose‑dependent decrease in the number of cells in S phase (indicative of G1 arrest) after 48 h, and induced apoptosis (Annexin V staining) after 72 h; potency was lower than (S)‑JQ1 but higher than CeMMEC2 at equivalent concentrations (exact percentages not given) [1].
Knockdown of TAF1 in REDS3 cells increased RFP‑positive nuclei, whereas knockdown of CREBBP, EP300 or BRD9 did not, confirming that TAF1 is the main mediator of CeMMEC1’s reporter‑activating effect [1].
CeMMEC1 did not inhibit any of 97 representative kinases by more than 60% at 10 μM, indicating bromodomain specificity [1].

The EPIgeneous Binding Domain kit B is used for TAF1 binding assays. The displacement of an acetylated biotin peptide from a GST-tagged TAF1 protein using HTRF in conjunction with a streptavidin-conjugated acceptor and an Eu³⁺-conjugated GST antibody donor indicates binding. Using an Echo 525 Liquid Handler, compounds (CeMMEC1) are dispensed into ProxiPlate-384 Plus assay plates. 5 nM TAF1-GST, 50 nM peptide (SGRGK (ac)GGK (ac)GGAK (ac)RHRK (biotin)-acid), 6.25 nM Streptavidin-XL665, 1:200 Anti-GST-Eu³⁺ cryptate, and 0.1% DMSO are used in binding assays, which are carried out in a final volume of 20 μL. Utilizing a Multidrop combi, assay reagents are applied to plates and allowed to sit at room temperature for three hours. A PHERAstar microplate reader with the HTRF module and dual emission protocol is used to measure fluorescence (A = 320 nm excitation, 665 nm emission, and B = 320 nm excitation, 620 nm emission). HTRF ratio (channel A/B × 10,000) is obtained by processing raw data and is utilized in the creation of IC50 curves[1].

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AlphaLISA immunoassay for BRD4 bromodomains: biotinylated histone peptide substrate captured by streptavidin‑donor beads; GST‑tagged bromodomain bound by anti‑GST‑acceptor beads. Excitation at 680 nm triggers singlet oxygen transfer, producing light emission at 615 nm only when donor and acceptor are within 200 nm. Compound (10 μM) that blocks histone docking reduces emission. Assay performed with BRD4(BD1) and BRD4(BD2) inhibitor screening kits following manufacturer’s protocol. For full‑length GST‑BRD4, a mixture of acetylated substrates was used. IC50 values determined from two‑fold serial dilutions (12 points, 50 μM to 0.02 μM) [1].
BromoScan profiling: binding of CeMMEC1 (10 μM) to representative bromodomain proteins measured as percentage inhibition of binding to an acetylated substrate [1].
Isothermal titration calorimetry (ITC) for TAF1(2): experiments performed on a VP‑ITC micro‑calorimeter at 293.15 K with stirring at 286 r.p.m. Protein solution (295 μM) in syringe, compound solution (25 μM) in cell (2 ml). Initial injection of 2 μl followed by 34 injections of 8 μl (duration 16 s per injection, spacing 240 s). Data analyzed with single binding site model using MicroCal ORIGIN software; first data point excluded. Kd = 1.8 μM [1].
FRET‑based TAF1 binding assay (EPlgeneous Binding Domain kit B): displacement of acetylated biotin peptide from GST‑tagged TAF1 measured by HTRF with Eu³⁺‑conjugated GST antibody donor and streptavidin‑conjugated acceptor. Final volume 20 μl containing 5 nM TAF1‑GST, 50 nM peptide (SGRGK(ac)GGK(ac)GLGK(ac)GGAK(ac)RHKR(biotin)‑acid), 6.25 nM Streptavidin‑XL665, 1:200 anti‑GST‑Eu³⁺ cryptate and 0.1% DMSO. Incubated at room temperature for 3 h; fluorescence measured with PHERAstar microplate reader (excitation 320 nm, emission 665 nm and 620 nm). HTRF ratio (665/620 × 10,000) used to generate IC50 curve (0.9 μM) [1].
Protein expression and purification of TAF1(2) bromodomain (residues 1501‑1634): cloned into pNIC28‑Bsa4, expressed in E. coli BL21(DE3)‑R3‑pRARE2 cells grown in Terrific broth with kanamycin and chloramphenicol. Induced with 0.2 mM IPTG at 18 °C for 16 h. Cells lysed in 20 mM Hepes pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 0.2 mM TCEP; cleared by centrifugation. Supernatant loaded onto nickel column, eluted with imidazole gradient; tag removed by TEV protease. Untagged protein further purified by size‑exclusion chromatography (HiLoad 16/60 Superdex 75). GST‑tagged TAF1(2) purified using glutathione Sepharose column and eluted with 50 mM Tris pH 8, 10 mM reduced glutathione; final gel filtration on Superdex 200. Mass and purity confirmed by LC/MSD TOF [1].

Target genes like the MYC oncogene are transcriptionally activated by proteins of the BET family that contain a bromodomain and can detect acetylation of histone lysine. BET domain pharmacological inhibitors offer potential treatment advantages for a range of malignancies. A high-diversity chemical compound screen was conducted to identify agents that have the ability to alter the BRD4-dependent heterochromatization of a generic reporter in human cells. We found small molecules that mimic BRD4 inhibition without direct engagement in addition to new and known compounds that target BRD4. One such substance was a strong inhibitor of TAF1's second bromodomain. By using this inhibitor, we were able to ascertain that TAF1 functions in concert with BRD4 to regulate the growth of cancer cells, rendering TAF1 a desirable epigenetic target in MYC-driven cancers.

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REDS3 cell assay (reporter): REDS3 cells (KBM7‑derived with RFP integrated at heterochromatic locus) were seeded in clear flat‑bottom 96‑ or 384‑well plates and treated with compounds for 24 h. Live‑cell imaging performed with Operetta High Content Screening System (20× objective, nonconfocal mode). RFP‑positive nuclei detected and quantified using Harmony software (nucleus diameter ~13 μm for KBM7). CeMMEC1 induced dose‑dependent increase in RFP‑positive cells (Supplementary Fig. 3d,e) [1].
Cell cycle assay: THP1 cells treated with CeMMEC1 for 48 h, fixed with 70% ethanol for 24 h, washed with PBS/0.1% Tween, incubated with RNase for 20 min, stained with 5 μg/ml propidium iodide for 10 min, and analyzed by FACS (BD FACSCalibur). Decrease in S‑phase cells observed [1].
Apoptosis assay: THP1 cells treated with CeMMEC1 for 72 h, stained with Annexin V‑FITC apoptosis detection kit, quantified with Harmony software for nuclei and cytoplasm detection [1].
qPCR for MYC expression: RNA extracted with TRIzol, reverse transcribed with High Capacity cDNA Kit. qPCR using Power SYBR Green Master mix with MYC primers (forward: GAAGGTGATCGACAGTCTFACCT, reverse: CTTCTCTCCGTCCTCGAGTTCT). Normalized to actin or GAPDH, relative quantification by ΔΔCT method. CeMMEC1 reduced MYC mRNA dose‑dependently in KBM7 cells [1].
Knockdown experiments: REDS3 cells transduced with shRNAs against TAF1, CREBBP, EP300, BRD9. Knockdown confirmed by western blot (antibodies: Taf1 sc‑735, BRD9 ab49313). RFP‑positive nuclei quantified by live‑cell imaging (≥1,500 cells per condition). TAF1 knockdown increased RFP‑positive cells, while CREBBP, EP300 or BRD9 knockdown did not [1].

NA NA

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[1]. Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk. Nat Chem Biol. 2016 Jul;12(7):504-10.

CeMMEC1 is an N‑methylisoquinolinone derivative. A set of 28 analogs (13‑40) were synthesized and tested for RFP activation and binding to BRD4(1), CREBBP and TAF1(2). Substitutions on the dihydrobenzodiazepinone core affected activity, consistent with the molecular docking model (Supplementary Fig. 6a) [1].
Molecular docking of CeMMEC1 into TAF1(2) crystal structure (PDB 3uv4) using template from ATAD2 bromodomain (PDB 4qst) predicted hydrogen bond between lactam carbonyl and N1604, and with Y1561 through a conserved water molecule [1].
CeMMEC1 is structurally distinct from (S)‑JQ1 and other BRD4 inhibitors; it was identified as a functional BRD4 inhibitor that does not directly engage BRD4 but acts via TAF1 [1].
Combination treatments: simultaneous inhibition of BRD4 (with (S)‑JQ1) and TAF1 (with CeMMEC1 or its analogs) showed synergy in reducing cell viability in H23 lung cancer cells and increasing RFP‑positive cells in REDS3 reporter (Fig. 4e,f) [1].

C19H16N2O4

336.341344833374

336.11

C, 67.85; H, 4.80; N, 8.33; O, 19.03

440662-09-9

440662-09-9

24152379

White to off-white solid powder

1.4±0.1 g/cm3

590.1±50.0 °C at 760 mmHg

310.7±30.1 °C

0.0±1.7 mmHg at 25°C

1.673

2.69

1

4

2

25

576

0

O1CCOC2C=CC(=CC1=2)NC(C1=CN(C)C(C2C=CC=CC1=2)=O)=O

PEOQAZBGLOADFJ-UHFFFAOYSA-N

InChI=1S/C19H16N2O4/c1-21-11-15(13-4-2-3-5-14(13)19(21)23)18(22)20-12-6-7-16-17(10-12)25-9-8-24-16/h2-7,10-11H,8-9H2,1H3,(H,20,22)

N-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-1-oxoisoquinoline-4-carboxamide

Ce MMEC1; Ce-MMEC1; CeMMEC1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.43 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)