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 targets the tandem tudor domain (TTD) of SET domain bifurcated protein 1 (SETDB1), a histone methyltransferase involved in epigenetic regulation. By binding to this domain with high affinity (Kd = 88 nM), it disrupts the interaction between SETDB1 and its binding partners, thereby modulating the enzyme's function in chromatin biology.

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, SETDB1-TTD-IN-1 demonstrates high selectivity for the SETDB1-TTD domain. It shows significantly weaker binding to other tudor domains, with Kd values of 4.3 μM for 53BP1 and 86 μM for JMJD2A, and no activity (KD > 100 mM) against 14 other tudor domains tested. In cellular assays using HEK293T cells, the compound effectively and dose-dependently stabilizes the SETDB1-TTD protein at concentrations ≥5 μM.

Specific in vivo activity data for SETDB1-TTD-IN-1 has not been extensively reported. As a research tool for studying the biological functions and disease associations of SETDB1-TTD, its in vivo effects would be context-dependent and are typically evaluated in disease models related to epigenetic dysregulation, such as cancer or neurological disorders.

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].
In vitro binding assays for SETDB1-TTD-IN-1 typically involve techniques such as fluorescence polarization or surface plasmon resonance (SPR) to measure its affinity for the purified SETDB1-TTD domain. The compound is incubated with the protein at various concentrations, and the dissociation constant (Kd) is determined from the binding curve, with a reported Kd of 88 nM for the primary target.

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].
In vitro cell-based assays are performed to evaluate the cellular activity of SETDB1-TTD-IN-1. For example, HEK293T cells stably transfected with a SETDB1-TTD expression plasmid are treated with the compound at concentrations such as 2.5, 5, 10, 20, and 40 μM. After treatment, cell lysates are prepared and the soluble protein fraction is analyzed by western blot to assess the stabilization of the SETDB1-TTD protein.

In vivo animal study protocols for SETDB1-TTD-IN-1 would be tailored to the specific research question, such as investigating its effects in xenograft models of cancer or in models of neurological disorders. Typically, the compound would be formulated in a suitable vehicle (e.g., PBS, DMSO/PEG300) and administered via routes like oral gavage or intraperitoneal injection. Dosing regimens, biomarker analysis, and efficacy endpoints would be determined based on the specific disease model being studied.

Pharmacokinetic data for SETDB1-TTD-IN-1 is not extensively reported in the available literature. As a small molecule with a molecular weight of 469.58 g/mol and formula C28H31N5O2, its ADME properties would be typical of similar kinase inhibitors. Detailed pharmacokinetic parameters such as half-life, bioavailability, and tissue distribution would need to be determined experimentally in relevant preclinical species.

Specific toxicological data for SETDB1-TTD-IN-1 is not available in the provided sources. As a research compound intended for laboratory use only, its safety profile has not been established for human therapeutic applications. Standard safety precautions for handling chemical probes should be observed, including the use of personal protective equipment and working in a well-ventilated area.

[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].
SETDB1-TTD-IN-1 has a CAS number of 2755823-12-0 and a molecular weight of 469.58 g/mol. It is a powerful research tool for probing chromatin biology and developing SETDB1-targeted therapeutic strategies. The compound is available with high purity (e.g., 99.96%). It has no approved therapeutic indications and is not in clinical trials, being exclusively a research-grade chemical.

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.)