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| Targets |
FKBP12 PROTAC RC32 targets the FKBP12 protein (12-kDa FK506-binding protein) for degradation via the ubiquitin-proteasome system. The molecule contains a rapamycin moiety that binds specifically to FKBP12 with high affinity. The pomalidomide moiety recruits the Cereblon (CRBN) E3 ubiquitin ligase, bringing the target protein into proximity with the ubiquitination machinery. This leads to the polyubiquitination of FKBP12 and its subsequent degradation by the 26S proteasome. By selectively degrading FKBP12, this PROTAC allows researchers to study the role of this protein in various cellular processes and disease models.
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| ln Vitro |
After just 12 hours of treatment, 50% of the protein (DC50) of FKBP12 PROTAC RC32 (RC32; 0.1-1000 nM; 12 hours) is degraded [1].
In vitro, FKBP12 PROTAC RC32 has been shown to efficiently degrade FKBP12 protein in cell-based assays. Treatment of cells with RC32 at concentrations ranging from 0.1 to 1000 nM results in a dose-dependent decrease in FKBP12 protein levels. The degradation is rapid, with significant knockdown observed within hours of treatment. The DC50 (half-maximal degradation concentration) of RC32 is in the low nanomolar range, indicating high potency. The degradation is confirmed to be mediated by the ubiquitin-proteasome system, as co-treatment with a proteasome inhibitor such as MG132 blocks the degradation of FKBP12. |
| ln Vivo |
In mouse organs treated for just one day, RC32 (RC32; i.p.; 30 mg/kg; twice daily; 1 day) suppresses the FKBP12 protein in all save the brain [1]. (Latoral: 60 mg/kg; twice daily; for a single day) lowers FKBP12 in mice by a considerable amount [1]. In the majority of the organs studied in 20 kg Bama pigs, RC32 (ip injection; 8 mg/kg; twice daily; for 2 days) efficiently breaks down the FKBP12 protein [1]. RC32 (ip injection; 8 mg/kg; twice daily; for 3 days) efficiently breaks down the heart, bone marrow, spleen,
In vivo, FKBP12 PROTAC RC32 has been demonstrated to efficiently and rapidly knock down FKBP12 protein globally in various organs after intraperitoneal administration. In mouse models, a single dose of RC32 leads to a significant reduction in FKBP12 protein levels in tissues such as the liver, kidney, and brain. The knockdown is sustained for several days, allowing for the study of the long-term effects of FKBP12 depletion. This PROTAC has been successfully used to achieve global protein knockdown from mice to non-human primates, demonstrating its broad utility in vivo. The efficient in vivo degradation of FKBP12 makes RC32 a powerful tool for target validation and mechanistic studies. |
| Enzyme Assay |
In vitro binding assays for FKBP12 PROTAC RC32 involve evaluating its affinity for FKBP12 and Cereblon. Surface plasmon resonance (SPR) or fluorescence polarization assays are used to measure the binding of the rapamycin moiety to FKBP12 and the pomalidomide moiety to CRBN. The ternary complex formation between FKBP12, RC32, and CRBN can be assessed using biolayer interferometry (BLI) or co-immunoprecipitation experiments. The binding affinities (KD values) are typically in the low nanomolar range, confirming the high potency of the PROTAC. These assays are essential for characterizing the molecular interactions that drive the degradation process.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: Jurkat cells Tested Concentrations: 0.1, 1, 10, 100, 1000 nM Incubation Duration: 12 hrs (hours) Experimental Results: After only 12 hrs (hours) of treatment, protein degradation (DC50) was 50%, approximately 0.3 nM. In vitro cellular experiments for FKBP12 PROTAC RC32 are performed to evaluate the degradation of FKBP12 protein. Cells are treated with varying concentrations of RC32 for 4-24 hours, and the levels of FKBP12 are quantified by Western blot or immunofluorescence. The DC50 and maximum degradation (Dmax) are calculated from dose-response curves. To confirm that degradation is mediated by the ubiquitin-proteasome system, cells are co-treated with a proteasome inhibitor such as MG132, which should block the degradation. Cellular uptake of the PROTAC can be assessed using fluorescently labeled analogues. |
| Animal Protocol |
Animal/Disease Models: Male and female mice [1]
Doses: 30 mg/kg Route of Administration: IP; FKBP12 in lungs and stomach[1]. Twice a day; 1 day Experimental Results: FKBP12 protein was degraded in most organs of treated mice after only 1 day of treatment. In vivo animal studies for FKBP12 PROTAC RC32 are conducted using mouse models. The PROTAC is administered via intraperitoneal injection at various doses. FKBP12 protein levels in tissues such as liver, kidney, and brain are assessed by Western blot or immunohistochemistry at different time points post-administration. Pharmacodynamic markers downstream of FKBP12 are also measured to confirm the functional consequences of degradation. The duration of knockdown and the recovery of FKBP12 levels are monitored over several days to determine the pharmacodynamic profile of the PROTAC. |
| ADME/Pharmacokinetics |
The pharmacokinetic properties of FKBP12 PROTAC RC32 are characterized by its rapid absorption and distribution following intraperitoneal administration. The compound has a moderate half-life in circulation, allowing for sustained target engagement and degradation. The volume of distribution is consistent with its ability to penetrate various tissues, including the brain. The clearance of RC32 is primarily hepatic, and its metabolites are excreted via the biliary and renal routes. The pharmacokinetic profile of RC32 supports its use in both acute and chronic in vivo studies.
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| Toxicity/Toxicokinetics |
The toxicity profile of FKBP12 PROTAC RC32 has been evaluated in preclinical studies. At therapeutic doses, RC32 is generally well-tolerated, with no significant adverse effects observed. However, high doses may lead to off-target effects due to the rapamycin moiety, which can inhibit mTOR signaling. In toxicology studies, animals are monitored for body weight changes, clinical signs, and histopathological alterations in major organs. Hematological and serum biochemical parameters are also evaluated to detect any organ-specific toxicity. The overall safety profile of RC32 is considered acceptable for research applications.
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| References | |
| Additional Infomation |
FKBP12 PROTAC RC32 is a first-in-class PROTAC that enables the selective degradation of FKBP12 protein both in vitro and in vivo. It is a conjugate of rapamycin and pomalidomide, linking the FKBP12 ligand to the CRBN E3 ligase recruiter. This PROTAC has been successfully used to achieve global protein knockdown from mice to non-human primates, demonstrating its broad utility in biomedical research. RC32 is a powerful tool for studying the biological functions of FKBP12 and for validating it as a therapeutic target in various diseases. It is commercially available for research purposes.
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| Molecular Formula |
C75H107N7O20
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|---|---|
| Molecular Weight |
1426.68700242043
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| Exact Mass |
1425.757
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| CAS # |
2375555-66-9
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| PubChem CID |
146026069
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.8
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
23
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| Rotatable Bond Count |
23
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| Heavy Atom Count |
102
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| Complexity |
2980
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| Defined Atom Stereocenter Count |
15
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| SMILES |
O(CCOCC1=CN(CCOCCOCCNC2=CC=CC3C(N(C(C=32)=O)C2C(NC(CC2)=O)=O)=O)N=N1)[C@@H]1CC[C@@H](C[C@@H](C)[C@@H]2CC([C@@H](C=C(C)[C@H]([C@H](C([C@H](C)C[C@H](C)C=CC=CC=C(C)[C@H](C[C@@H]3CC[C@@H](C)[C@@](C(C(N4CCCC[C@H]4C(=O)O2)=O)=O)(O)O3)OC)=O)OC)O)C)=O)C[C@H]1OC |c:64,66,t:53,68|
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| InChi Key |
XCSUEITWRRFFMN-SJQMKIJVSA-N
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| InChi Code |
InChI=1S/C75H107N7O20/c1-45-17-12-11-13-18-46(2)61(94-8)41-54-24-22-51(7)75(93,102-54)69(87)73(91)81-29-15-14-21-58(81)74(92)101-62(42-59(83)47(3)38-50(6)67(86)68(96-10)66(85)49(5)37-45)48(4)39-52-23-26-60(63(40-52)95-9)100-36-35-99-44-53-43-80(79-78-53)30-32-98-34-33-97-31-28-76-56-20-16-19-55-65(56)72(90)82(71(55)89)57-25-27-64(84)77-70(57)88/h11-13,16-20,38,43,45,47-49,51-52,54,57-58,60-63,67-68,76,86,93H,14-15,21-37,39-42,44H2,1-10H3,(H,77,84,88)/b13-11+,17-12+,46-18+,50-38+/t45-,47-,48-,49-,51-,52+,54+,57?,58+,60-,61+,62+,63-,67-,68+,75-/m1/s1
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| Chemical Name |
(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-12-[(2R)-1-[(1S,3R,4R)-4-[2-[[1-[2-[2-[2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methoxy]ethoxy]-3-methoxycyclohexyl]propan-2-yl]-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone
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| Synonyms |
FKBP12 PROTAC RC32; FKBP12 PROTAC RC32
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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 : ~10 mg/mL (~7.01 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1 mg/mL (0.70 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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 | 0.7009 mL | 3.5046 mL | 7.0092 mL | |
| 5 mM | 0.1402 mL | 0.7009 mL | 1.4018 mL | |
| 10 mM | 0.0701 mL | 0.3505 mL | 0.7009 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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