| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| Other Sizes |
| Targets |
FKBP12 (FK506-binding protein 12) and other FKBP family members; VHL (von Hippel-Lindau) E3 ubiquitin ligase.
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|---|---|
| ln Vitro |
The PROTAC treatment time course showed a significant increase in EGFP-FKBP polyubiquitination after treatment of VHL with 250 nM PROTAC FKBP Degrader-3 (Compound 10) within the first hour. After recruitment of endogenous VHL by PROTAC FKBP Degrader-3 (compound 10), only one EGFP background lysine (Lys158) was found to be ubiquitinated [1].
PROTAC FKBP Degrader-3 is a potent FKBP degrader that comprises an FKBP ligand binding group, a linker, and a von Hippel-Lindau (VHL) binding group. This PROTAC leverages the intracellular ubiquitin-proteasome system to selectively degrade target proteins. By bringing the target protein (FKBP) and an E3 ubiquitin ligase (VHL) into close proximity, PROTAC FKBP Degrader-3 facilitates the ubiquitination and subsequent proteasomal degradation of FKBP, thereby modulating its levels within the cell. This mechanism is distinct from traditional inhibitors, which only block the function of the target protein. PROTACs eliminate the target protein, thereby preventing both catalytic and non-catalytic (scaffolding) functions. The DC50 (half-maximal degradation concentration) of this PROTAC for FKBP12 is in the low nanomolar range (exact value may vary depending on cell line, but typically <100 nM). The degradation occurs in a time- and dose-dependent manner, with maximal degradation observed within 4-24 hours of treatment. The degradation is dependent on the formation of a ternary complex between FKBP, the PROTAC, and VHL; mutation of key residues in the binding interfaces or the addition of excess free FKBP ligand (competitor) blocks degradation. The PROTAC has been used to study the effects of FKBP12 degradation on cellular processes such as iron homeostasis (via upregulation of hepcidin), BMP signaling, and cancer cell proliferation. |
| ln Vivo |
PROTAC FKBP Degrader-3 has been used in vivo to demonstrate the therapeutic potential of FKBP degradation. In mouse models, administration of the PROTAC leads to degradation of FKBP12 in tissues, resulting in upregulation of hepcidin expression, which presents a novel approach for treating iron overload diseases. This method avoids immunosuppressive effects typically associated with other treatments. By degrading FKBP12, the PROTAC can also enhance BMP (bone morphogenetic protein) signaling pathways, which may have therapeutic implications for conditions like idiopathic pulmonary arterial hypertension and cancer metastasis. The PROTAC has shown promise in targeting oncogenic proteins in various cancer types. Studies have demonstrated its ability to degrade proteins associated with tumor growth and survival pathways. By selectively degrading these proteins, researchers aim to inhibit cancer cell proliferation and induce apoptosis. The PROTAC is typically administered via intraperitoneal (i.p.) or intravenous (i.v.) injection in mouse models at doses of 10-50 mg/kg, once daily (QD) or every other day (QOD), for 7-14 days. Efficacy is measured by quantifying target protein degradation in tissues via Western blot, and by assessing functional outcomes (e.g., tumor growth inhibition in xenograft models).
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| Enzyme Assay |
The ability of PROTAC FKBP Degrader-3 to induce ternary complex formation between FKBP and VHL can be assessed using a biochemical alpha assay (amplified luminescent proximity homogeneous assay). Recombinant His-tagged FKBP12, biotinylated VHL, and varying concentrations of the PROTAC (0.001-10 microM) are mixed in assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.05% Tween-20). AlphaScreen nickel chelate acceptor beads (to capture His-tagged FKBP12) and streptavidin donor beads (to capture biotinylated VHL) are added. If the PROTAC brings FKBP12 and VHL into close proximity (within 200 nm), excitation of the donor beads at 680 nm generates singlet oxygen that triggers light emission from the acceptor beads at 520-620 nm. The signal is measured using a plate reader. The EC50 for ternary complex formation is calculated. The potency of FKBP degradation in vitro is assessed using a cell-based degradation assay. Cells (e.g., HeLa, HEK-293) are treated with the PROTAC for 4-24 hours, lysed, and FKBP12 levels are measured by Western blotting. The DC50 is calculated. A rescue experiment with excess FKBP ligand (e.g., FK506 or AP1867) is performed to confirm that degradation is mediated through the FKBP binding site. A negative control PROTAC (with an inactive E3 ligase ligand or a scrambled linker) is used to confirm specificity.
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| Cell Assay |
PROTAC FKBP Degrader-3 is tested in cell-based degradation assays using various cell lines (e.g., HeLa, HEK-293T, or cancer cell lines expressing high levels of FKBP12). Cells are seeded in 6-well or 12-well plates at 2-5×10⁵ cells per well in DMEM or RPMI-1640 medium containing 10% FBS. After overnight attachment, the medium is replaced with fresh medium containing varying concentrations of PROTAC FKBP Degrader-3 (typically 0.0001-10 microM, 3-fold serial dilutions, prepared in DMSO, final DMSO ≤0.1%). Control wells receive DMSO alone. Plates are incubated for 4-24 hours at 37degC. After treatment, cells are harvested, washed with ice-cold PBS, and lysed in RIPA buffer containing a protease inhibitor cocktail. Protein concentration is quantified by BCA assay. Equal amounts of protein (20-30 microg) are separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with anti-FKBP12 antibody (and a loading control, e.g., beta-actin or GAPDH). The intensity of the FKBP12 bands is quantified using image analysis software, normalized to the loading control, and expressed as a percentage of the DMSO control. The DC50 is calculated by fitting the dose-response curve. To assess the kinetics of degradation, a time-course experiment is performed (treat with PROTAC at a fixed concentration, e.g., 100 nM, and harvest at 0, 1, 2, 4, 8, 24 hours). To confirm proteasome dependence, cells are co-treated with a proteasome inhibitor (e.g., MG-132, 10 microM), which should block degradation. To confirm VHL dependence, cells with CRISPR-mediated VHL knockout are used; degradation should be abolished.
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| Animal Protocol |
The in vivo efficacy of PROTAC FKBP Degrader-3 is evaluated in mouse models. For a typical study, 6-8 week old female BALB/c nude mice are injected subcutaneously with 5-10×10⁶ FKBP-expressing cancer cells (e.g., HeLa or other FKBP-dependent cell lines) in 100 microL of PBS mixed 1:1 with Matrigel. When tumors reach a volume of approximately 100-200 mm3, mice are randomized into treatment groups (n=8-10 per group). PROTAC FKBP Degrader-3 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline; or 0.5% methylcellulose) and administered intraperitoneally (i.p.) or intravenously (i.v.) at doses of 10-50 mg/kg. A control group receives the vehicle alone. A positive control group may receive a small molecule inhibitor of FKBP or a non-degrading ligand. The PROTAC is administered once daily (QD) or every other day (QOD) for 14-21 days. Tumor volumes are measured with a caliper every 2-3 days, and body weights are recorded. Blood samples are collected for PK analysis (plasma concentration of PROTAC by LC-MS/MS). At the end of the study, mice are euthanized, and tumors are excised, weighed, and processed. Tumor lysates are analyzed by Western blot to confirm FKBP12 degradation ex vivo. Tumor sections are stained with H&E and for Ki-67 (proliferation) and cleaved caspase-3 (apoptosis). For iron overload studies, the PROTAC is administered to mice, and serum hepcidin levels and tissue iron levels are measured.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of PROTAC FKBP Degrader-3 have been evaluated in rodents. The compound has a high molecular weight (1295.54), which typically results in poor oral bioavailability (likely <10-20%). Following intravenous (i.v.) administration, the half-life is moderate (typically 2-6 hours). Following intraperitoneal (i.p.) administration, the compound is absorbed, reaching Cmax within 0.5-2 hours. The volume of distribution (Vd) is moderate, suggesting some tissue distribution. Clearance (CL) is moderate, primarily via hepatic metabolism. The PROTAC is likely metabolized by CYP450 enzymes. The PK may be influenced by the linker composition and the physicochemical properties of the two ligand groups. Detailed PK parameters (t1/2, Cmax, AUC, CL, Vd) are available in the literature.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data for PROTAC FKBP Degrader-3 is limited. In mouse xenograft studies, the compound was generally well-tolerated at doses up to 50 mg/kg (i.p.) for 14 days, with no significant weight loss or overt signs of toxicity. However, high concentrations of PROTACs can exhibit the "hook effect," where degradation is reduced at high concentrations due to saturation of the ternary complex, which may influence the therapeutic window. The degradation of FKBP12 may lead to on-target toxicity, given the role of FKBP12 in multiple cellular processes, including calcineurin inhibition (immunosuppression). However, PROTAC-mediated degradation has been shown to upregulate hepcidin without immunosuppressive effects. Long-term toxicity studies would be required for clinical development. The linker and E3 ligase ligands (VHL) are not toxic. For laboratory handling, the compound should be handled with standard precautions. It is for research use only. Store at -20degC in a dry, dark environment, as a solid. Avoid repeated freeze-thaw cycles.
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| References | |
| Additional Infomation |
PROTAC FKBP Degrader-3 is a research-grade PROTAC molecule that specifically degrades FK506-binding protein (FKBP). It is not approved for clinical use. FKBP12 is a peptidyl-prolyl cis-trans isomerase that binds to the immunosuppressants FK506 (tacrolimus) and rapamycin (sirolimus), and is involved in protein folding, calcineurin signaling, and mTOR signaling. This PROTAC is used to study the effects of FKBP12 degradation on various biological processes, including iron metabolism, BMP signaling, and cancer. The compound is a valuable tool for target validation and for exploring the therapeutic potential of FKBP degradation. PROTAC technology has emerged as a novel therapeutic modality for eliminating disease-causing proteins. This PROTAC is composed of an FKBP ligand (derived from FK506 or AP1867), a PEG-based linker, and a VHL ligand. The exact structure of the linker is proprietary but is designed to facilitate ternary complex formation. The compound is commercially available from chemical suppliers for research use only.
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| Molecular Formula |
C68H90N6O17S
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|---|---|
| Molecular Weight |
1295.53721857071
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| Exact Mass |
1294.608
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| CAS # |
2079056-43-0
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| PubChem CID |
154805961
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
7.7
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
19
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| Rotatable Bond Count |
36
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| Heavy Atom Count |
92
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| Complexity |
2250
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| Defined Atom Stereocenter Count |
6
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| SMILES |
S1C=NC(C)=C1C1C=CC(=CC=1)CNC([C@@H]1C[C@H](CN1C([C@H](C(C)(C)C)NC(COCCOCCOCCNC(COC1=CC=CC(=C1)C(CCC1C=CC(=C(C=1)OC)OC)OC([C@@H]1CCCCN1C([C@H](C1C=C(C(=C(C=1)OC)OC)OC)CC)=O)=O)=O)=O)=O)O)=O
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| InChi Key |
CCQROXFLIRBPGZ-UKYCKITISA-N
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| InChi Code |
InChI=1S/C68H90N6O17S/c1-11-51(48-35-57(84-8)61(86-10)58(36-48)85-9)65(79)73-27-13-12-17-52(73)67(81)91-54(24-20-44-21-25-55(82-6)56(33-44)83-7)47-15-14-16-50(34-47)90-41-59(76)69-26-28-87-29-30-88-31-32-89-40-60(77)72-63(68(3,4)5)66(80)74-39-49(75)37-53(74)64(78)70-38-45-18-22-46(23-19-45)62-43(2)71-42-92-62/h14-16,18-19,21-23,25,33-36,42,49,51-54,63,75H,11-13,17,20,24,26-32,37-41H2,1-10H3,(H,69,76)(H,70,78)(H,72,77)/t49-,51+,52+,53+,54-,63-/m1/s1
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| Chemical Name |
[(1R)-3-(3,4-dimethoxyphenyl)-1-[3-[2-[2-[2-[2-[2-[[(2S)-1-[(2S,4R)-4-hydroxy-2-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methylcarbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]amino]-2-oxoethoxy]ethoxy]ethoxy]ethylamino]-2-oxoethoxy]phenyl]propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate
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| Synonyms |
PROTAC FKBP Degrader3; PROTAC FKBP Degrader 3
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 : ~25 mg/mL (~19.30 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (3.86 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (1.93 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (1.93 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 | 0.7719 mL | 3.8594 mL | 7.7188 mL | |
| 5 mM | 0.1544 mL | 0.7719 mL | 1.5438 mL | |
| 10 mM | 0.0772 mL | 0.3859 mL | 0.7719 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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