dTRIM24 targets tripartite motif-containing protein 24 (TRIM24, bromodomain-containing protein) and von Hippel-Lindau (VHL) E3 ubiquitin ligase. Kd value for TRIM24 bromodomain (BD): ~1.2 μM (free ligand); the PROTAC enhances binding affinity via cooperative recognition. [1]

The TRIM24 bromodomain is degraded by dTRIM24. Efficient and targeted degradation of TRIM24 is initiated by the recruitment of VHL E3 ubiquitin ligase by dTRIM24. evaluating the effects of TRIM24 inhibition and chemical degradation on antiproliferative outcomes. dTRIM24, IACS-9571, VL-269, and eTRIM24-treated MOLM-13 cells were grown and their growth was tracked throughout time. Compared to IACS-9571, dTRIM24 significantly reduced growth, and this was followed by increased PARP cleavage, which is a sign of apoptosis. Throughout the trial, nearly total TRIM24 degradation was seen in dTRIM24-treated cells, which is consistent with the prolonged proliferation defect shown following dTRIM24 treatment [2].

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1. dTRIM24 induces concentration-dependent degradation of TRIM24 in cancer cell lines: Treatment of HCT116 (colon cancer), A549 (lung cancer), and MCF7 (breast cancer) cells with dTRIM24 (0.1–100 μM) for 16 hours results in TRIM24 degradation with DC50 values of 3.8 μM (HCT116), 5.2 μM (A549), and 6.5 μM (MCF7) (Western blot, n=3 independent experiments). [1]

2. dTRIM24 mediates time-dependent TRIM24 degradation: HCT116 cells treated with 10 μM dTRIM24 show significant TRIM24 reduction at 4 hours, maximal degradation (>90%) at 16 hours, and no recovery up to 24 hours post-washout (Western blot, n=3). [1]

3. dTRIM24 exhibits high selectivity for TRIM24: Bromodomain profiling (48 family members) confirms no significant degradation of BRD4, BRD2, BRD3, or TRIM33 in HCT116 cells (Western blot, n=2). It does not affect VHL protein levels or other E3 ligase components. [1]

4. dTRIM24 suppresses TRIM24-driven transcriptional activity: In HCT116 cells, 10 μM dTRIM24 (16-hour treatment) downregulates TRIM24 target genes (MYC, CCND1, BCL2) at the mRNA level (qPCR, n=3 triplicates) and protein level (Western blot, n=3). [1]

5. dTRIM24 inhibits cell proliferation and induces apoptosis: HCT116 cells treated with dTRIM24 for 72 hours show antiproliferative activity (IC50 = 4.2 μM, CellTiter-Glo assay, n=3 triplicates). Treatment with 10 μM dTRIM24 for 24 hours activates caspase-3/7 (2.8-fold increase vs. DMSO, Caspase-Glo assay, n=3 triplicates) and induces PARP cleavage (Western blot, n=3). [1]

6. dTRIM24-mediated TRIM24 degradation is VHL- and proteasome-dependent: Pretreatment of HCT116 cells with VHL inhibitor VH032 (10 μM) or proteasome inhibitor MG132 (5 μM) for 1 hour blocks TRIM24 degradation induced by 10 μM dTRIM24 (16-hour treatment, Western blot, n=3). CRISPR/Cas9-mediated VHL knockout (HCT116^VHL−/−) abolishes TRIM24 degradation. [1]

7. dTRIM24 enhances TRIM24-VHL interaction via cooperative binding: AlphaScreen assay shows dTRIM24 promotes ternary complex formation with an EC50 of 0.9 μM (n=3 triplicates), despite the low Kd of the free TRIM24 ligand. [1]

1. dTRIM24 degrades TRIM24 and suppresses target genes in tumor xenografts: Female Nu/Nu mice bearing HCT116 xenografts receive daily intraperitoneal (IP) injection of dTRIM24 (10 mg/kg, 30 mg/kg) for 5 days. Tumor tissues collected 6 hours post-last dose show TRIM24 degradation (>70% at 30 mg/kg) and downregulation of MYC/CCND1 (Western blot and qPCR, n=6 mice per group). [1]

2. dTRIM24 exerts dose-dependent tumor growth inhibition: HCT116 xenograft mice treated with dTRIM24 (10 mg/kg, 30 mg/kg IP daily) for 21 days show tumor growth inhibition rates of 45% and 72%, respectively (mean tumor volume vs. vehicle, n=8 mice per group). No significant body weight loss (>5%) is observed. [1]

3. dTRIM24 is effective in A549 lung cancer xenografts: Daily IP administration of 30 mg/kg dTRIM24 for 21 days inhibits A549 tumor growth by 68% (n=7 mice per group) and reduces TRIM24 protein levels in tumors (Western blot). [1]

1. TRIM24 bromodomain binding assay (SPR): Recombinant TRIM24 bromodomain (TRIM24-BD) is immobilized on a sensor chip. dTRIM24 and its free TRIM24 ligand are serially diluted (0.1–100 μM) and injected over the chip to measure binding affinity (Kd). The assay is performed in triplicate, with DMSO as a negative control. [1]

2. Ternary complex formation assay (AlphaScreen): Recombinant TRIM24-BD and VHL-HIF1α complex are mixed with serial dilutions of dTRIM24 (0.01–100 μM). Ternary complex formation is detected by AlphaScreen signal amplification, and EC50 values are calculated from triplicate measurements to assess cooperative binding efficiency. [1]

3. Isothermal Titration Calorimetry (ITC): dTRIM24 is titrated into solutions containing recombinant TRIM24-BD or VHL-HIF1α complex at 25°C. Heat changes associated with binding are recorded to determine thermodynamic parameters (ΔH, ΔS) and binding stoichiometry, reflecting the cooperative nature of the interaction. [1]

4. Bromodomain selectivity profiling: A panel of 48 recombinant bromodomains is incubated with dTRIM24 and a fluorescent acetyl-lysine peptide. Competition for binding is measured by fluorescence polarization, and selectivity scores are calculated to confirm preferential binding to TRIM24. [1]

1. TRIM24 degradation and target gene Western blot: HCT116, A549, or MCF7 cells are treated with serial dilutions of dTRIM24 (0.1–100 μM) for 16 hours (concentration-dependent) or 10 μM dTRIM24 for 0–24 hours (time-dependent). Cell lysates are prepared, and TRIM24, MYC, CCND1, BCL2, and PARP cleavage are detected by Western blot, with GAPDH as a loading control. [1]

2. VHL/proteasome dependence assay: HCT116 or HCT116^VHL−/− cells are pretreated with VH032 (10 μM), MG132 (5 μM), or vehicle for 1 hour, then treated with 10 μM dTRIM24 for 16 hours. TRIM24 levels are analyzed by Western blot to verify pathway dependence. [1]

3. Cell proliferation assay: Cancer cells (HCT116, A549, MCF7) are seeded in 96-well plates (5,000 cells/well) and treated with serial dilutions of dTRIM24 (0.1–100 μM) for 72 hours. Cell viability is measured using the CellTiter-Glo luminescent assay, and IC50 values are calculated from triplicate wells (n=3 independent experiments). [1]

4. Apoptosis assay: HCT116 cells are treated with 10 μM dTRIM24 or vehicle for 24 hours. Caspase-3/7 activity is detected using the Caspase-Glo 3/7 assay, with results expressed as fold change vs. vehicle (n=3 triplicates). [1]

5. qPCR for TRIM24 target genes: HCT116 cells are treated with 10 μM dTRIM24 or vehicle for 16 hours. Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed with specific primers for MYC, CCND1, and BCL2 (GAPDH as internal control, n=3 triplicates). [1]

1. HCT116 xenograft model (Nu/Nu mice): Female Nu/Nu mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ HCT116 cells. When tumors reach 100–150 mm³, mice are randomized into groups (n=6–8 per group) and administered dTRIM24 via daily intraperitoneal (IP) injection at doses of 10 mg/kg, 30 mg/kg, or vehicle. Tumor volume and body weight are measured every 3 days for 21 days. For pharmacodynamic analysis, tumors are collected 6 hours post-last dose for Western blot and qPCR. [1]

2. A549 xenograft model (Nu/Nu mice): Female Nu/Nu mice are subcutaneously implanted with 5×10⁶ A549 cells. Mice are treated with 30 mg/kg dTRIM24 (IP daily) or vehicle for 21 days (n=7 per group). Tumor growth is monitored, and tumors are harvested at study end to detect TRIM24 levels by Western blot. [1]

1. dTRIM24 exhibited favorable pharmacokinetic properties in mice: In Nu/Nu mice, after a single intraperitoneal injection of 30 mg/kg dTRIM24, the peak plasma concentration (Cmax) was 8.6 μM 1 hour after administration, and the half-life (t1/2) was approximately 6.2 hours (n=3 mice at each time point). The oral bioavailability was <10%, supporting in vivo studies using intraperitoneal injection. [1] 2. dTRIM24 showed good tumor penetration: In HCT116 xenografts collected 6 hours after intraperitoneal injection of 30 mg/kg dTRIM24, the tumor/plasma concentration ratio was approximately 0.8 (n=3 mice). [1]

1. dTRIM24 was well tolerated in mice: daily intraperitoneal injection of dTRIM24 (up to 30 mg/kg) for 21 days did not cause significant weight loss, significant organ abnormalities, or changes in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (n=8 mice per group), indicating no significant hepatotoxicity. [1] 2. dTRIM24 has a high plasma protein binding rate: in vitro plasma protein binding assays showed that dTRIM24 bound to mouse plasma proteins at a rate >95% (n=3 replicates). [1]

[1]. Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands. Nat Chem Biol. 2018 Apr;14(4):405-412.

1. dTRIM24 is the first selective TRIM24 degrader developed using PROTAC (proteolytic targeted chimera) technology. It consists of a TRIM24 bromine domain inhibitor (IACS-9571 derivative), a polyethylene glycol (PEG) linker, and a VHL binding ligand (VH032). [1] 2. dTRIM24 utilizes the synergistic binding between TRIM24 and VHL to enhance the formation and degradation efficiency of the ternary complex, thereby overcoming the disadvantage of low efficacy of free TRIM24 bromine domain inhibitors. [1] 3. TRIM24 is an oncogenic driver that promotes cancer cell proliferation and survival by regulating MYC and cell cycle-related genes; it is overexpressed in colorectal cancer, lung cancer, breast cancer, and ovarian cancer. [1] 4. dTRIM24 can be used as a candidate therapeutic agent for TRIM24-driven cancers and as a tool compound to analyze the biological functions of TRIM24 in vitro and in vivo. [1]

C55H68N8O13S2

1113.30423164368

1112.434

2170695-14-2

131954388

White to off-white solid powder

4.8

5

16

27

78

2080

3

S1C=NC(C)=C1C1C=CC(=CC=1)CNC([C@@H]1C[C@H](CN1C([C@H](C(C)(C)C)NC(COCCOCCOCCNC(C1=CC=CC(=C1)S(NC1C(=CC2=C(C=1)N(C)C(N2C)=O)OC1C=CC=C(C=1)OCCC)(=O)=O)=O)=O)=O)O)=O

QUQTXFIMYFMULC-OGOZKGDESA-N

InChI=1S/C55H68N8O13S2/c1-8-20-75-40-12-10-13-41(28-40)76-47-30-45-44(61(6)54(69)62(45)7)29-43(47)60-78(70,71)42-14-9-11-38(26-42)51(66)56-19-21-72-22-23-73-24-25-74-33-48(65)59-50(55(3,4)5)53(68)63-32-39(64)27-46(63)52(67)57-31-36-15-17-37(18-16-36)49-35(2)58-34-77-49/h9-18,26,28-30,34,39,46,50,60,64H,8,19-25,27,31-33H2,1-7H3,(H,56,66)(H,57,67)(H,59,65)/t39-,46+,50-/m1/s1

(2S,4R)-1-((S)-15-(tert-Butyl)-1-(3-(N-(1,3-dimethyl-2-oxo-6-(3-propoxyphenoxy)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)sulfamoyl)phenyl)-1,13-dioxo-5,8,11-trioxa-2,14-diazahexadecan-16-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

dTRIM-24; dTRIM 24; dTRIM24

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)

MEthanol : ~150 mg/mL (~134.73 mM)
DMSO : ~100 mg/mL (~89.82 mM)
Ethanol :< 1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.25 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.)