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| Targets |
PROTAC FKBP12-binding moiety 1 targets FKBP12, a 12 kDa FK506-binding protein that is a member of the immunophilin family. FKBP12 is a peptidyl-prolyl cis-trans isomerase that catalyzes the interconversion of cis and trans isomers of proline-containing peptides, playing a role in protein folding and the regulation of signaling pathways. FKBP12 is also the cellular target of the immunosuppressive drugs FK506 and rapamycin, which bind to FKBP12 and inhibit the activity of calcineurin and mTOR, respectively. By binding to FKBP12, PROTAC FKBP12-binding moiety 1 recruits FKBP12 to the PROTAC molecule, which also contains an E3 ligase ligand. This brings the target protein (which is fused to or associated with FKBP12) into proximity with the E3 ligase, leading to ubiquitination and proteasomal degradation of the target protein. The compound's weaker affinity for FKBP12 compared to FK506 may be advantageous for the development of PROTACs, as it allows for the reversible and tunable degradation of target proteins. The carboxyl group of the compound serves as a handle for conjugation to linkers and E3 ligase ligands, enabling the synthesis of a wide variety of PROTAC molecules targeting different proteins.
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| ln Vitro |
SLF-amido-C2-COOH (PROTAC FKBP12 binding moiety 1) is a synthetic ligand of FKBP (SLF). SLF contains only half of FK506 and has a much lower affinity for FKBP12 [1].
In vitro studies have demonstrated that PROTAC FKBP12-binding moiety 1 binds to FKBP12, although with lower affinity than FK506. The compound's binding to FKBP12 has been characterized using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence polarization (FP) assays, with Kd values typically in the low micromolar range. When conjugated to a linker and an E3 ligase ligand (e.g., pomalidomide, lenalidomide, VHL ligand), the resulting PROTAC molecule induces the degradation of FKBP12-fused target proteins in a concentration- and time-dependent manner. In cell-based assays, PROTACs containing this moiety have been shown to degrade various target proteins, including kinases, transcription factors, and other disease-relevant proteins, with DC₅₀ values in the nanomolar to low micromolar range. The degradation is mediated by the ubiquitin-proteasome system and is dependent on the presence of both the FKBP12-binding moiety and the E3 ligase ligand. The compound itself does not induce protein degradation in the absence of the E3 ligase ligand, confirming that its role is to recruit FKBP12 to the PROTAC complex. In addition to its use in PROTAC development, the compound has been used as a negative control in studies of FKBP12 binding and as a tool for the study of FKBP12 function in cells. |
| ln Vivo |
In vivo studies on PROTAC FKBP12-binding moiety 1 are limited, as the compound itself is not a therapeutic agent but a building block for PROTACs. However, PROTACs containing this moiety have been evaluated in animal models for their efficacy in degrading target proteins and treating diseases. In mouse xenograft models of cancer, PROTACs targeting kinases or other oncogenic proteins have been shown to induce target protein degradation, inhibit tumor growth, and improve survival. The in vivo efficacy of these PROTACs is dependent on their pharmacokinetic properties, including oral bioavailability, tissue distribution, and half-life. The use of FKBP12-binding moieties in PROTACs offers several advantages, including the ability to target proteins that are difficult to inhibit with small molecules and the potential for improved selectivity and reduced off-target effects. However, the development of PROTACs for clinical use is still in its early stages, and further studies are needed to optimize their properties and evaluate their safety and efficacy in humans.
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| Enzyme Assay |
For in vitro binding assays, PROTAC FKBP12-binding moiety 1 is typically evaluated for its affinity to FKBP12 using fluorescence polarization (FP) or surface plasmon resonance (SPR). For FP assays, a fluorescently labeled FKBP12 ligand (e.g., fluorescein-FK506) is incubated with FKBP12 protein and varying concentrations of the compound (0.01-100 µM) in assay buffer (50 mM HEPES, pH 7.4, containing 150 mM NaCl, 0.1% BSA, and 0.01% Tween-20). After incubation for 30-60 minutes at room temperature, the fluorescence polarization is measured using a fluorescence plate reader. The IC₅₀ for displacement of the fluorescent ligand is determined from dose-response curves, and the Ki is calculated using the Cheng-Prusoff equation. For SPR assays, FKBP12 protein is immobilized on a sensor chip, and varying concentrations of the compound are flowed over the chip. The binding affinity (Kd) and kinetic parameters (ka, kd) are determined from the binding curves. For PROTAC synthesis, the compound is conjugated to a linker and an E3 ligase ligand using standard peptide coupling chemistry. The resulting PROTAC is purified by HPLC and characterized by mass spectrometry.
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| Cell Assay |
For in vitro cell-based assays, PROTACs containing PROTAC FKBP12-binding moiety 1 are evaluated for their ability to degrade target proteins in cells. Cells expressing the target protein (either endogenously or as a fusion protein with FKBP12) are seeded in 6- or 12-well plates and treated with the PROTAC at concentrations of 0.001-10 µM for 4-24 hours. The degradation of the target protein is assessed by Western blotting, with loading controls (e.g., GAPDH, actin) used for normalization. The DC₅₀ (concentration for 50% degradation) and Dmax (maximum degradation) are determined from dose-response curves. The kinetics of degradation are assessed by treating cells with the PROTAC for various times (0, 1, 2, 4, 8, 12, 24 hours). The mechanism of degradation is confirmed by co-treatment with a proteasome inhibitor (e.g., MG132) or a neddylation inhibitor (e.g., MLN4924), which should block degradation. The selectivity of degradation is assessed by measuring the levels of other proteins. Cell viability is assessed using MTT assays to ensure that the observed effects are not due to cytotoxicity. All experiments include appropriate positive and negative controls (e.g., PROTAC without the FKBP12-binding moiety, PROTAC without the E3 ligase ligand), and results are expressed as mean ± standard deviation from at least three independent experiments.
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| Animal Protocol |
For in vivo animal experiments, PROTACs containing PROTAC FKBP12-binding moiety 1 are typically administered orally or intraperitoneally to mice. For tumor xenograft models, immunocompromised mice are subcutaneously inoculated with tumor cells expressing the target protein. When tumors reach a volume of 50-100 mm³, mice are randomized into treatment groups (n=6-8 per group) and administered the PROTAC at doses of 1-50 mg/kg/day for 7-21 days. Tumor volume is measured every 2-3 days, and body weights are recorded daily. At the end of the study, tumors are excised and processed for Western blotting to assess target protein degradation, and for histopathological analysis. Blood samples are collected for pharmacokinetic analysis. For pharmacodynamic studies, tissues (e.g., liver, kidney, tumor) are collected at various time points after PROTAC administration for Western blotting analysis. All animal procedures are conducted in accordance with institutional guidelines for the care and use of laboratory animals.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for PROTAC FKBP12-binding moiety 1 itself are limited, as the compound is a building block rather than a therapeutic agent. The compound has a molecular weight of 624.72 g/mol and a molecular formula of C₃₄H₄₄N₂O₉. When incorporated into PROTACs, the pharmacokinetic properties of the resulting molecule are determined by the overall structure of the PROTAC, including the linker and the E3 ligase ligand. PROTACs generally have poor oral bioavailability due to their large size and polar nature, and they are typically administered parenterally. The compound is stable when stored as a powder at -20°C, protected from light and moisture. For in vivo administration, PROTACs containing this moiety can be formulated in 0.5% carboxymethyl cellulose (CMC), saline, or other suitable vehicles with the addition of solubilizing agents such as DMSO or PEG400.
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| Toxicity/Toxicokinetics |
Toxicological data for PROTAC FKBP12-binding moiety 1 are limited, as the compound is a research building block that is not intended for human therapeutic use. The compound has not been evaluated in comprehensive toxicology studies. As with all research chemicals, appropriate safety precautions should be taken when handling the compound, including the use of personal protective equipment and working in a well-ventilated fume hood. The compound is for research use only and is not intended for human therapeutic use.
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| References | |
| Additional Infomation |
PROTAC FKBP12-binding moiety 1 is a research-use only compound and has not been approved for clinical applications by any regulatory authority. It is also known as SLF-amido-C2-COOH. The compound has a molecular formula of C₃₄H₄₄N₂O₉ and a molecular weight of 624.72 g/mol. It is a synthetic ligand for FKBP12 that is used in the synthesis of PROTACs (Proteolysis Targeting Chimeras). The compound serves as a warhead that binds to FKBP12, enabling the recruitment of FKBP12 to a target protein for ubiquitination and degradation via the proteasome. The compound contains a carboxyl group that can be conjugated to a linker and an E3 ligase ligand to form a complete PROTAC molecule. The compound is a valuable tool for the study of targeted protein degradation and for the development of novel therapeutic strategies. The compound is available from various research chemical suppliers with purities typically ≥95% (HPLC). Storage recommendations include keeping the compound in a tightly sealed container, protected from light and moisture, at -20°C.
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| Molecular Formula |
C34H44N2O9
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|---|---|
| Molecular Weight |
624.7212
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| Exact Mass |
624.304
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| CAS # |
1092369-24-8
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| PubChem CID |
121427834
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
16
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| Heavy Atom Count |
45
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| Complexity |
1030
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O([C@@]([H])(C1C([H])=C([H])C([H])=C(C=1[H])N([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O)C([H])([H])C([H])([H])C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])OC([H])([H])[H])C([C@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C(C(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])=O)=O)=O
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| InChi Key |
FTAKPNRZIJEFIG-IZZNHLLZSA-N
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| InChi Code |
InChI=1S/C34H44N2O9/c1-6-34(2,3)31(40)32(41)36-19-8-7-12-25(36)33(42)45-26(15-13-22-14-16-27(43-4)28(20-22)44-5)23-10-9-11-24(21-23)35-29(37)17-18-30(38)39/h9-11,14,16,20-21,25-26H,6-8,12-13,15,17-19H2,1-5H3,(H,35,37)(H,38,39)/t25-,26+/m0/s1
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| Chemical Name |
4-[3-[(1R)-3-(3,4-dimethoxyphenyl)-1-[(2S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carbonyl]oxypropyl]anilino]-4-oxobutanoic acid
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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 : ~100 mg/mL (~160.07 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.00 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. Solubility in Formulation 2: 2.5 mg/mL (4.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6007 mL | 8.0036 mL | 16.0072 mL | |
| 5 mM | 0.3201 mL | 1.6007 mL | 3.2014 mL | |
| 10 mM | 0.1601 mL | 0.8004 mL | 1.6007 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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