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| Targets |
Cereblon
PROTAC-O4I2 targets splicing factor 3B1 (SF3B1), a core component of the U2 small nuclear ribonucleoprotein (snRNP) complex essential for pre-mRNA splicing. The compound simultaneously binds SF3B1 and the E3 ubiquitin ligase cereblon (CRBN) via its thalidomide moiety, forming a ternary complex that induces ubiquitination and subsequent proteasomal degradation of SF3B1. This targeted degradation mechanism is cereblon-dependent, and the compound shows preferential degradation of SF3B1 over other cellular proteins. The parental compound O4I2 functions as an SF3B1 activator, but PROTAC-O4I2 represses splicing activity by degrading the target protein. |
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| ln Vitro |
PROTAC-O4I2 preferentially degrades SF3B1 and prevents the formation of tumors in cells by introducing thalidomide into the ubiquitin E3 ligase cereblon (CRBN) [1]. STAT-O4I2 inhibits and degrades SF3B1 in K562 cells. SF3B1 WT, SF3B1 OE, and SF3B1 K700E cells are non-proliferatively affected by PROTAC-O4I2, with IC50 values of 228, 63, and 90 nM, respectively [1]. With an IC50 value of 0.244 μM in K562 cells, PROTAC-O4I2 causes FLAG-SF3B1 degradation in a concentration-dependent manner [1].
In vitro, PROTAC-O4I2 induces FLAG-SF3B1 degradation in K562 cells with an IC₅₀ of 0.244 μM (244 nM). The compound demonstrates anti-proliferative effects on SF3B1 wild-type, SF3B1-overexpressing, and SF3B1 K700E mutant cells with IC₅₀ values of 228 nM, 63 nM, and 90 nM, respectively. PROTAC-O4I2 induces apoptosis in K562 wild-type cells. The degradation occurs in a concentration-dependent manner. Compared to pladienolide B (IC₅₀ 76 nM), PROTAC-O4I2 shows approximately 3-fold lower potency with an IC₅₀ of 228 nM in certain assays. The compound effectively inhibits tumor cell growth through SF3B1 degradation. |
| ln Vivo |
PROTAC-O4I2 (10 μM) inhibits the maintenance and development of the tumor, hence considerably increasing the survival rate of Drosophila in the intestinal tumor model [1].
In vivo, PROTAC-O4I2 significantly improves survival in a Drosophila intestinal tumor model by interfering with tumor maintenance and proliferation. Fruit flies were fed PROTAC-O4I2 at 10 μM in 5% sucrose solution on round filter paper, maintained at 18°C, and transferred to fresh vials every 2 days. The compound effectively suppressed tumor growth and extended organismal survival in this model system. These findings demonstrate that SF3B1 degradation via PROTAC-O4I2 can impair tumor maintenance in vivo, supporting the therapeutic potential of targeting SF3B1 through protein degradation in cancer models. |
| Enzyme Assay |
In vitro enzyme/receptor binding (non-cellular) assays for PROTAC-O4I2 typically involve biochemical binding studies to confirm ternary complex formation between SF3B1, CRBN, and the PROTAC molecule. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can be employed to measure binding affinities of PROTAC-O4I2 to both SF3B1 and CRBN individually. Ubiquitination assays using recombinant E1/E2/E3 enzymes in cell-free systems assess the efficiency of SF3B1 ubiquitination induced by the compound. These assays are performed in buffer systems containing ATP and ubiquitin, with reaction products analyzed by immunoblotting using anti-ubiquitin and anti-SF3B1 antibodies to confirm target ubiquitination prior to proteasomal degradation.
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| Cell Assay |
Cell Proliferation Assay[1]
Cell Types: K562 control cells (WT), cells overexpressing SF3B1 (OE), and cells expressing SF3B1K700E (K700E) Tested Concentrations: 1 pM, 0.1 nM, 10 nM, 1 μM, 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: In K562 WT cells, the parental compound O4I2 demonstrated a marginal anti-proliferation effect (IC50 >10 μM). In In contrast, PROTAC-O4I2 demonstrated Dramatically higher toxicity with an IC50 of 228 nM, nearly 3-fold less potent than pladienolide B (IC50, 76 nM). Cells overexpressing SF3B1 WT was slightly resistant to pladienolide B (IC50, 134 nM), but more sensitive to PROTAC-O4I2 (an IC50 value of 63 nM). Apoptosis Analysis[1] Cell Types: K562 cell Tested Concentrations: 1 μM Incubation Duration: 48 h Experimental Results: Induced cellular apoptosis s in cells expressing SF3B1WT or SF3B1K700E. In vitro cellular experiments with PROTAC-O4I2 are performed in K562 human chronic myelogenous leukemia cells. Cells are treated with varying concentrations of the compound (typically 0.01-10 μM) for 24-72 hours. FLAG-SF3B1 degradation is assessed by Western blot analysis using anti-FLAG or anti-SF3B1 antibodies, with IC₅₀ determined from concentration-response curves. Cell viability is measured using MTT or CellTiter-Glo assays after 72 hours of treatment. Apoptosis is evaluated by Annexin V-FITC/PI staining and flow cytometry. Cells are cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO₂. |
| Animal Protocol |
Animal/Disease Models: Drosophila melanogaster[1]
Doses: 10 μM Route of Administration: Flies were fed on a round filter paper loaded with PROTAC-O4I2 in a 5% sucrose solution , maintained at 18℃ and flipped into a freshly prepared vial every 2 days. Experimental Results: diminished stem cell activity, blocked the initiation and growth of tumor, and improved the survival of the Drosophila ISC tumor model. In vivo animal studies with PROTAC-O4I2 have been conducted in a Drosophila melanogaster intestinal tumor model. Flies are fed on round filter paper loaded with 10 μM PROTAC-O4I2 dissolved in 5% sucrose solution. The flies are maintained at 18°C and transferred to freshly prepared vials every 2 days. Survival rates are monitored over time to assess the compound's effect on tumor progression and organismal viability. Tumor maintenance and proliferation are evaluated by microscopic examination of intestinal tissues. This model allows for rapid in vivo assessment of anti-tumor efficacy through targeted protein degradation. |
| ADME/Pharmacokinetics |
Comprehensive pharmacokinetic data for PROTAC-O4I2 are not extensively reported in the available literature. As a PROTAC molecule with molecular weight 609.1 g/mol, it is expected to have moderate oral bioavailability and cellular permeability typical of such bifunctional degraders. The compound is soluble in DMSO and can be formulated for in vivo administration using appropriate vehicles. PROTACs generally exhibit favorable tissue distribution due to their modular design, though detailed parameters such as half-life, clearance, and volume of distribution for this specific compound require further investigation from primary research publications.
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| Toxicity/Toxicokinetics |
Toxicological information for PROTAC-O4I2 is primarily derived from cellular assays where the compound induces apoptosis in K562 cells through SF3B1 degradation. The compound demonstrates concentration-dependent cytotoxicity with IC₅₀ values in the nanomolar range across various SF3B1-expressing cell lines. As a cereblon-recruiting PROTAC containing a thalidomide analog, potential off-target effects and developmental toxicity concerns should be considered, though specific toxicology studies are not detailed in the available literature. Standard safety precautions for handling potent investigational compounds apply, including use of personal protective equipment and proper chemical waste disposal.
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| References | |
| Additional Infomation |
Protac-O4I2 is an organic molecular entity.
PROTAC-O4I2 (CAS 2785323-62-6) is a research-grade PROTAC degrader developed for targeted protein degradation studies. The compound has a molecular formula C₂₉H₂₉ClN₆O₅S and molecular weight 609.1 g/mol. It appears as a yellow solid and is soluble in DMSO. The compound should be stored as powder at -20°C for up to 3 years or in solvent at -80°C for 1 year. Purity is typically ≥99%. PROTAC-O4I2 is exclusively for research purposes and not approved for clinical use in humans. Its mechanism involves cereblon-dependent ubiquitination and proteasomal degradation of SF3B1, representing a novel approach to modulating RNA splicing in cancer research. |
| Molecular Formula |
C29H29CLN6O5S
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| Molecular Weight |
609.10
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| Exact Mass |
608.16
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| CAS # |
2785323-62-6
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| PubChem CID |
162641046
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| Appearance |
White to yellow solid powder
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| Density |
1.453±0.06 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
42
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| Complexity |
1030
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=CC(=CC=1)NC1=NC(=CS1)C(NCCCCCCNC1=CC=CC2C(N(C(C=21)=O)C1C(NC(CC1)=O)=O)=O)=O
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| InChi Key |
UWYLVQJEOCYCEW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H29ClN6O5S/c30-17-8-10-18(11-9-17)33-29-34-21(16-42-29)25(38)32-15-4-2-1-3-14-31-20-7-5-6-19-24(20)28(41)36(27(19)40)22-12-13-23(37)35-26(22)39/h5-11,16,22,31H,1-4,12-15H2,(H,32,38)(H,33,34)(H,35,37,39)
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| Chemical Name |
2-(4-chloroanilino)-N-[6-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]hexyl]-1,3-thiazole-4-carboxamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 250 mg/mL (410.44 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6418 mL | 8.2088 mL | 16.4177 mL | |
| 5 mM | 0.3284 mL | 1.6418 mL | 3.2835 mL | |
| 10 mM | 0.1642 mL | 0.8209 mL | 1.6418 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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