SETDB1/KMT2G
SETDB1-TTD-IN-1 ((R,R)-59) targets SETDB1 triple tudor domain (TTD/3TD) with KD = 0.088 ± 0.045 μM (ITC assay) and KD = 0.106 ± 0.002 μM (SPR assay) [1].
In a TR-FRET competition assay, it displaced H3K9me2K14ac peptide from SETDB1 3TD with IC50 = 1.7 ± 0.47 μM [2].
It showed no binding to the inactive enantiomer (S,S)-59 [1][2].

SETDB1-TTD-IN-1 exhibited some action against JMJD2A and 53BP1 with Kd of 4.3 μM and 86 μM, respectively. In tests with 16 tudor domains (Kd>100 μM), SETDB1-TTD-IN-1 did not exhibit any action. (1). To stabilize SETDB1-TTD protein in HEK293T cells, SETDB1-TTD-IN-1 (2.5-40 μM) exhibits potent and dose-dependent effects [1]. In human acute monocytic leukemia THP-1 cells, SETDB1-TTD-IN-1 (2.5–40 μM; 24 h) modifies 72 genes' expression considerably [1].

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SETDB1-TTD-IN-1 ((R,R)-59) inhibited the binding of endogenous H3 peptides (H3K9me/K14ac, H3K9me2/K14ac, H3K9me3/K14ac) to SETDB1-TTD in ITC competition experiments, completely preventing association after preincubation [1].
In HTRF assays, it inhibited TTD-H3K9me2/K14ac interaction with IC50 = 0.93 μM and TTD-H3K9me3/K14ac with IC50 = 0.75 μM [1].
In contrast, it promoted SETDB1 methyltransferase activity toward Akt1-K64 peptide in a dose-dependent manner in vitro, with up to 50% increase at 100 μM and EC50 = 19 μM for methylation increase [2].
Kinetic analysis showed increased kcat (from 0.29 to 0.50 h⁻¹) with constant SAM KM (≈0.9-1.0 μM), indicating enhanced catalytic efficiency (kcat/KM from 0.32 to 0.49 μM⁻¹ h⁻¹) [2].
In cells (HEK293T transfected with HA-Akt1), treatment with SETDB1-TTD-IN-1 ((R,R)-59) increased Akt1 trimethylation and T308 phosphorylation in dose-dependent (0-10 μM, 24 h) and time-dependent (0-24 h, 10 μM) manners, as shown by immunoblot [2].
Similar effects were observed in DLD1 cells stably expressing HA-Akt1 [2].
In MDA-MB-231 breast cancer cells, low doses (2.5 and 5 μM) of SETDB1-TTD-IN-1 ((R,R)-59) promoted cell proliferation (72 h treatment), while higher doses (>5 μM) showed no effect; the enantiomer (S,S)-59 had no effect [2].
In HEK293T cells expressing Flag-SETDB1-TTD, CETSA showed that SETDB1-TTD-IN-1 ((R,R)-59) at ≥5 μM dose-dependently stabilized the TTD protein, whereas (S,S)-59 did not [1].
FRAP assay with EGFP-tagged SETDB1-TTD (WT) in 293T cells treated with 6 μM SETDB1-TTD-IN-1 ((R,R)-59) for 18 h showed significantly decreased normalized half-life recovery time (t₁/₂ = 1.05 ± 0.33 s) compared to DMSO (1.45 ± 0.34 s) or (S,S)-59 (1.50 ± 0.31 s), indicating increased protein mobility and target engagement [1].
RNA-seq in THP-1 cells treated with 10 μM SETDB1-TTD-IN-1 ((R,R)-59) for 24 h affected expression of 72 genes (>4-fold), with 49 genes uniquely affected compared to (S,S)-59 or DMSO [1].

In vitro methyltransferase activity assay: Active recombinant full-length SETDB1 (SETDB1-FL) was incubated with biotinylated Akt1-K64 peptide (10 μM) and [³H]-SAM (5 μM) in buffer (20 mM Tris pH 8.5, 5 mM DTT, 0.01% Triton X-100) at 23°C for 60 min. Reactions were stopped by adding 7.5 M guanidine hydrochloride, and incorporated radioactivity was captured on SAM2 biotin capture membranes and quantified by liquid scintillation counting. To test compound effect, 0.5 μM SETDB1-FL was used with varying concentrations of SETDB1-TTD-IN-1 ((R,R)-59) (0-100 μM) [2].
ITC assay: Isothermal titration calorimetry was performed to determine binding affinity of SETDB1-TTD-IN-1 ((R,R)-59) to SETDB1-TTD protein. The KD value was 0.088 ± 0.045 μM [1].
SPR assay: Surface plasmon resonance was carried out at 25°C on a Biacore 8K system. Biotinylated SETDB1-TTD was immobilized on a SA sensor chip. Varying concentrations of SETDB1-TTD-IN-1 ((R,R)-59) were flowed over immobilized protein (contact 120 s, dissociation 300 s, flow rate 30 μL/min). Data were analyzed using Biacore Evaluation Software, giving KD = 0.106 ± 0.002 μM [1]. For isolated 3TD, SPR yielded KD = 4.8 ± 1.6 μM [2].
DSF assay: Differential scanning fluorimetry was performed using a real-time PCR system. SETDB1-TTD (10-20 μM) was incubated with SETDB1-TTD-IN-1 ((R,R)-59) (100-200 μM) and Sypro Orange dye. Temperature gradient from 25 to 90°C at 1°C/min. ΔTm values: 5.79°C [1] and 2.9 ± 0.20°C [2].
HTRF competition assay: Time-resolved fluorescence energy transfer assay used 6×His-tagged SETDB1 (40 nM), biotinylated H3K9me2K14ac peptide (40 nM), Lance Eu-streptavidin (2 nM), and ULight-anti-6×His antibody (10 nM) in PBS pH 7.0, 0.005% Tween 20, 2 mM DTT. Compounds were added in 10-point 3-fold serial dilutions. After 1 h equilibration, plates were read with excitation at 320 nm and emission at 615 and 655 nm. IC50 for SETDB1-TTD-IN-1 ((R,R)-59) was 1.7 ± 0.47 μM [2].
TR-FRET competition for H3 peptide displacement: Similar assay using GST-SETDB1-TTD and biotinylated H3 peptides (H3K9me2/K14ac or H3K9me3/K14ac) gave IC50 values of 0.93 μM and 0.75 μM respectively [1].
CETSA: HEK293T cells stably transfected with Flag-SETDB1-TTD were treated with SETDB1-TTD-IN-1 ((R,R)-59) or (S,S)-59 at various concentrations for 6 h, then heated at 50°C and lysed by freeze-thaw cycles. Soluble protein content was analyzed by western blot. Compound at ≥5 μM dose-dependently stabilized TTD protein [1].
FRAP: 293T cells transfected with EGFP-tagged SETDB1-TTD (wild-type or Y268A mutant) were treated with 6 μM SETDB1-TTD-IN-1 ((R,R)-59), (S,S)-59 or DMSO for 18 h. Fluorescence recovery after photobleaching was measured, and normalized half-life recovery times (t₁/₂) were calculated. For WT with (R,R)-59: 1.05 ± 0.33 s; with (S,S)-59: 1.50 ± 0.31 s; DMSO: 1.45 ± 0.34 s [1].
RNA-seq: THP-1 cells were treated with 10 μM SETDB1-TTD-IN-1 ((R,R)-59), (S,S)-59, or DMSO for 24 h. Cells were collected and RNA-sequencing performed. Treatment affected 72 genes (>4-fold change, p<0.05), with 49 genes uniquely affected by (R,R)-59 [1].

Cell culture and transfection: HEK293T, DLD1, and MDA-MB-231 cells were cultured in DMEM with 10% FBS, penicillin and streptomycin. Transfections were performed using polyethyleneimine. For Akt1 experiments, cells were transfected with HA-Akt1 plasmid or stably expressing lenti-viral HA-Akt1 [2].
Immunoblot and immunoprecipitation: Cells lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) or Triton X-100 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100) with protease and phosphatase inhibitors. Protein concentrations measured, and equal amounts loaded for SDS-PAGE. For immunoprecipitation, 1 mg total lysate incubated with anti-HA agarose beads for 3-4 h at 4°C. Washed with NETN buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40). Antibodies used: anti-HA (1:1000), anti-trimethyl lysine motif (1:1000), anti-phospho-Akt-Thr308 (1:1000) [2].
Cell proliferation assay: MDA-MB-231 cells (5000 cells/well in 96-well plates) were treated with indicated doses of SETDB1-TTD-IN-1 ((R,R)-59) or (S,S)-59 for 72 h. Cell proliferation was analyzed by Cell Titer-Glo Kit measuring luminescence [2].
CETSA: HEK293T cells stably transfected with pLVX-mCherry-N1-SETDB1-TTD plasmid were treated with compound at 0, 0.625, 1.25, 2.5, 5, 10, 20 μM for 6 h. Cells were then heated at 50°C and lysed by repeated freeze-thaw cycles with liquid nitrogen. Soluble fraction protein content determined by western blot [1].
FRAP: 293T cells transfected with EGFP-tagged SETDB1-TTD wild-type or Y268A mutant plasmids. Cells treated with 6 μM compound or DMSO for 18 h. Fluorescence recovery after photobleaching measured. Eighteen cells per group, one-way ANOVA with Dunnett's correction (p<0.05) [1].
RNA-seq: Human acute monocytic leukemia THP-1 cells treated with 10 μM SETDB1-TTD-IN-1 ((R,R)-59), (S,S)-59, or DMSO for 24 h, then collected for RNA-seq analysis. Genes with >4-fold change and p<0.05 considered significant [1].

[1]. Structure-Guided Discovery of a Potent and Selective Cell-Active Inhibitor of SETDB1 Tudor Domain. Angew Chem Int Ed Engl. 2021 Apr 12;60(16):8760-8765.

[2]. SETDB1 Triple Tudor Domain Ligand, (R, R)-59, Promotes Methylation of Akt1 in Cells. ACS Chem Biol. 2023 Aug 18;18(8):1846-1853.

SETDB1-TTD-IN-1 ((R,R)-59) is a first-in-class potent and selective small molecule inhibitor of SETDB1 triple tudor domain discovered through structure-guided optimization from hit compound Cpd1 (KD 4.4 μM) [1].
It binds to the region between tudor domain 2 and tudor domain 3 of SETDB1-TTD, forming key interactions including hydrogen bond with Y268 and electrostatic interaction with D299 [1].
The cocrystal structure (PDB entry 7CJT) reveals an α²-π interaction between Y268 phenolic hydroxyl and the propenyl C=C double bond [1].
The enantiomer (S,S)-59 is completely inactive in all assays, serving as a negative control [1][2].
Unexpectedly, SETDB1-TTD-IN-1 ((R,R)-59) acts as a positive allosteric modulator of SETDB1 methyltransferase activity, promoting Akt1 K64 methylation and subsequent Akt1 T308 phosphorylation, leading to increased cell proliferation in breast cancer cells where SETDB1 is overexpressed [2].
This compound is the first reported small-molecule activator of SETDB1 methyltransferase activity [2].
The 3TD of SETDB1 is required for efficient methylation of Akt1-K64, as a construct lacking 3TD (SETDB1-S) showed no significant methylation [2].

C28H31N5O2

469.58

469.247

2755823-12-0

SETDB1-TTD-IN-1 TFA

155884456

White to off-white solid powder

3.9

2

4

8

35

758

2

C1(N[C@@H]2C[C@H](C3=CC=C(OCC4=CC=CC=C4)C=C3)CN(C)C2)N(CC=C)C(=O)C2NC=CC=2N=1

NBDJDKAXIGWAIM-XZOQPEGZSA-N

InChI=1S/C28H31N5O2/c1-3-15-33-27(34)26-25(13-14-29-26)31-28(33)30-23-16-22(17-32(2)18-23)21-9-11-24(12-10-21)35-19-20-7-5-4-6-8-20/h3-14,22-23,29H,1,15-19H2,2H3,(H,30,31)/t22-,23+/m0/s1

2-[[(3R,5R)-1-methyl-5-(4-phenylmethoxyphenyl)piperidin-3-yl]amino]-3-prop-2-enyl-5H-pyrrolo[3,2-d]pyrimidin-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : 125 mg/mL (266.20 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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