TRIM21 (E3 ubiquitin ligase) and NUP98 (nucleoporin) [1]

- TRIM21-Dependent NPC Degradation: In IFNγ-prestimulated A549 cells, (S)-ACE-OH (10 µM) treatment for 8 hours triggered the degradation of nuclear pore proteins NUP35, SMPD4, and GLE1, whereas the inactive enantiomer (R)-ACE-OH did not. This degradation was blocked by the proteasome inhibitor bortezomib and the E1 ubiquitin-activating enzyme inhibitor TAK-243 [1].
- Stereo-selective Cytotoxicity: (S)-ACE-OH displayed IFN-enhanced toxicity in A549 and DLD-1 cells. In contrast, (R)-ACE-OH was inactive in either cell line. The IC50 for (S)-ACE-OH in A549 cells with IFNγ was 6.2 µM (95% CI: 5.2-7.4 µM) [1].

- Isothermal Titration Calorimetry (ITC) for Binding Affinity: ITC was performed at 25°C using a MicroCal PEAQ-ITC. The purified PRYSPRY domain of TRIM21D355A was dialyzed in a buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, pH 8.0, 1% DMSO) and loaded into the sample cell at 20-25 µM. (S)-ACE-OH was diluted from a 100 mM DMSO stock to 300-500 µM in the same buffer (final DMSO 1%). The titration consisted of an initial injection of 0.4 µL followed by 18 injections of 2 µL with a stirring speed of 1000 rpm. The binding affinity (Kd) of (S)-ACE-OH to the PRYSPRY domain was determined to be 17.9 µM. No detectable binding was observed when (S)-ACE-OH was titrated into NUP98APD [1].
- ITC for Ternary Complex Formation: To probe ternary complex formation, NUP98APD (200 µM in the syringe) was titrated into the cell containing full-length His-Lipoyl-TRIM21D355A (50 µM) pre-incubated with (S)-ACE-OH (100 µM). The experiment showed that NUP98APD exhibited a strong binding affinity to the preformed TRIM21D355A;(S)-ACE-OH complex, with a dissociation constant (Kd) of 3.38 µM (CI: 2.06-4.70 µM). No interaction was detected in the absence of the ligand [1].
- GST Pull-Down Assay: His-Lipoyl-TRIM21 (WT or D355A) and GST-NUP98APD were mixed at a 1:1 molar ratio in pull-down assay buffer. The protein mix was supplemented with DMSO, (S)-ACE-OH, or (R)-ACE-OH. After incubation on ice, the mixture was incubated with glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. The assay showed that GST-NUP98APD effectively pulled down His-Lipoyl-TRIM21 in the presence of (S)-ACE-OH but not (R)-ACE-OH [1].
- CEBIT (Condensate-aided Enrichment of Biomolecular Interactions in Test Tubes) Assay: SmF-EGFP-SH3-NUP98APD and SmF-EGFP-PRM condensates were formed. Cell lysates containing TRIM21D355A-3xFLAG or purified TRIM21D355A-3xFLAG protein were incubated with varying concentrations of (S)-ACE-OH or (R)-ACE-OH. The condensates were pelleted by centrifugation and analyzed by western blotting. The assay demonstrated dose-dependent enrichment of TRIM21 in the condensates upon treatment with (S)-ACE-OH, whereas (R)-ACE-OH did not exhibit such an effect, showing the specificity of the interaction [1].
- In Vitro Ubiquitination Assay: An in vitro ubiquitination reaction was performed by mixing purified TRIM21D355A-3xFLAG, SmF-EGFP-PRM, SmF-EGFP-SH3-NUP98APD, UBA1 (E1), a mixture of seven E2 enzymes, and ubiquitin in an ATP-containing buffer. (S)-ACE-OH or (R)-ACE-OH was added 30 minutes prior to the enzymes. The results showed that (S)-ACE-OH, but not (R)-ACE-OH, induced the polyubiquitination of both SmF-EGFP-SH3-NUP98APD and SmF-EGFP-PRM, as detected by anti-ubiquitin and anti-GFP antibodies [1].

- Cell Viability Assay: For the concentration-response curves, cells (e.g., A549, DLD-1) were seeded in 96-well plates and dosed with a serial dilution of (S)-ACE-OH or (R)-ACE-OH using a digital dispenser. Cell survival was measured three days later using a luminescent cell viability assay kit according to the manufacturer's instructions. The IC50 was determined using GraphPad Prism [1].
- Western Blotting for Protein Degradation: A549 cells were pre-treated with IFNγ and then treated with (S)-ACE-OH or (R)-ACE-OH for 8 hours. Cells were lysed in SDS lysis buffer containing benzonase and protease inhibitors. Protein lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies against target proteins (e.g., NUP35, SMPD4, GLE1) and a loading control (β-actin). This confirmed that (S)-ACE-OH, but not (R)-ACE-OH, induced the degradation of specific nuclear pore proteins [1].
- Immunofluorescence and Confocal Imaging: A549 cells were treated with ACE (the parent compound) for indicated times. Cells on coverslips were fixed with paraformaldehyde, permeabilized with Triton X-100, and blocked with serum. They were then incubated with primary antibodies (e.g., anti-NPC, anti-NUP98, anti-SMPD4) followed by fluorescent secondary antibodies and DAPI. Images were captured using a confocal microscope to visualize the impact on the nuclear pore complex [1].

- Metabolic Activation: The parent drug Acepromazine (ACE) is a prodrug. It is converted by the aldo-keto reductase enzymes AKR1C1, AKR1C2, and AKR1C3 into the active metabolite (S)-ACE-OH in sensitive cancer cell lines. This conversion was observed in A549 and HeLa cells but not in DLD-1 or HCT-116 cells. The conversion was confirmed by thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS), which showed a species with a mass increase of 2 daltons, corresponding to the addition of two hydrogen atoms [1].

[1]. Selective degradation of multimeric proteins by TRIM21-based molecular glue and PROTAC degradersJ. Cell, 2024 Dec 12;187(25):7126-7142.e20.

- Stereospecificity of Activity: The chiral center created by the reduction of Acepromazine results in two enantiomers. The (S)-enantiomer is the active metabolite responsible for the interferon-enhanced cytotoxicity and protein degradation activity, while the (R)-enantiomer is inactive. The absolute conformations were determined by X-ray diffraction after chemical derivation [1].
- Structural Insights: Co-crystal structures of the TRIM21 PRYSPRY domain with (S)-ACE-OH revealed that the tricyclic phenothiazine ring occupies a hydrophobic pocket. The aliphatic chain of (S)-ACE-OH orients the amine group to form polar interactions with side chains of E389 and Q395, a conformation distinct from the inactive (R)-ACE-OH, suggesting this is critical for molecular glue activity [1].

C19H24N2OS

328.47

328.1609345

3095569-90-4

92288648

Off-white to light yellow ointment

3.8

1

4

5

23

378

1

C[C@@H](C1=CC2=C(C=C1)SC3=CC=CC=C3N2CCCN(C)C)O

ZIJWCRNUEBJMSQ-AWEZNQCLSA-N

InChI=1S/C19H24N2OS/c1-14(22)15-9-10-19-17(13-15)21(12-6-11-20(2)3)16-7-4-5-8-18(16)23-19/h4-5,7-10,13-14,22H,6,11-12H2,1-3H3/t14-/m0/s1

(1S)-1-[10-[3-(dimethylamino)propyl]phenothiazin-2-yl]ethanol

(S)-ACE-OH; (S)-ACE OH;

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)

DMSO: 100 mg/mL (304.4 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.)