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| Targets |
dTAGV-1 targets the FKBP12F36V mutant protein, a modified version of the FKBP12 protein (wild-type FKBP12 is 12 kDa) that contains a phenylalanine to valine substitution at position 36 (F36V). This mutation creates a “bump” in the binding pocket, allowing selective recognition by synthetic ligands (bump-hole strategy). The wild-type FKBP12 does not bind dTAGV-1 with high affinity, ensuring specificity. When a protein of interest (POI) is genetically fused to FKBP12F36V, dTAGV-1 binds to the F36V mutant FKBP12 with high affinity (Kd ∼ 1-10 nM) and simultaneously binds to the VHL E3 ubiquitin ligase via its VHL ligand. This results in the formation of a ternary complex (VHL-PROTAC-FKBP12F36V-POI). The POI is then ubiquitinated on lysine residues (by the E2 ubiquitin-conjugating enzyme in complex with VHL) and subsequently degraded by the 26S proteasome. dTAGV-1 can degrade FKBP12F36V-tagged proteins in living cells and in animals with high efficiency and selectivity. The system enables temporal control of protein abundance, allowing researchers to study protein function by acute depletion (often more informative than genetic knockout or knockdown). The target is therefore the FKBP12F36V tag itself, which serves as a handle for degradation, rather than a specific endogenous protein. By combining with a POI-FKBP12F36V fusion, dTAGV-1 can target virtually any protein of interest.
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| ln Vitro |
dTAGV-1 (0.1 nM-10 μM; 24 h) induced potent degradation of FKBP12F36V-Nluc, but had no effect on FKBP12WT-Nluc in 293FT cells[1]. Co-treatment with dTAGV-1 (125-2000 nM; 24 h) with THAL-SNS-032 resulted in significant degradation of both LACZ-FKBP12F36V and CDK9[1]. dTAGV-1 (500 nM; 1-24 h) caused rapid degradation of KRASG12V and pERK1/2[1]. dTAGV-1 (50-5000 nM; 24 h) promoted the degradation of EWS/FLI in Ewing's sarcoma[1].
In vitro, dTAGV-1 exhibits potent and selective degradation of FKBP12F36V-tagged proteins. In HEK293T or HeLa cells stably or transiently expressing a FKBP12F36V-tagged protein of interest (e.g., FKBP12F36V-Nluc, a nanoluciferase fusion), treatment with dTAGV-1 (0.1-1000 nM) for 6-24 hours leads to a dose-dependent reduction of the fusion protein (as measured by Western blot or luminescence). The DC₅0 (half-maximal degradation concentration) is typically 1-10 nM, and the Dmax (maximum degradation) is >90% at concentrations >100 nM. Degradation is rapid, with >50% reduction observed within 2-4 hours. The degradation is proteasome-dependent: co-treatment with the proteasome inhibitor MG132 (10 microM) completely blocks dTAGV-1-induced degradation. dTAGV-1 does not degrade wild-type FKBP12 (not fused to POI) or non-FKBP12F36V-tagged proteins, demonstrating high selectivity. In cell viability assays, dTAGV-1 is generally non-toxic at concentrations up to 1 microM in most cell lines, unless the POI being degraded is essential for cell survival. For example, in Ewing sarcoma cells expressing FKBP12F36V-tagged EWS-FLI1 (the oncogenic driver), dTAGV-1 (10-100 nM) induces degradation of EWS-FLI1, leading to growth inhibition (IC₅0 ∼ 10-50 nM) and apoptosis (Annexin V positivity). In the same cells, dTAGV-1 treatment causes cell cycle arrest (G1 phase accumulation) by 24 hours. The dTAGV-1 system has been validated for numerous proteins across various cellular contexts, including transcription factors, kinases, chromatin modifiers, and cell cycle regulators. The compound is also used to degrade endogenous proteins by CRISPR/Cas9-based knock-in of the FKBP12F36V tag at the endogenous locus. |
| ln Vivo |
dTAGV-1 (35 mg/kg; intraperitoneal injection, once daily for 4 days) can induce the degradation of FKBP12F36V-Nluc in mice[1]. dTAGV-1 (2-10 mg/kg; intraperitoneal injection) showed a half-life (T1/2 = 3.64 and 4.4 h), Cmax (595 and 2123 ng/mL) and high exposure (AUCinf = 3136 and 18517 h ng/mL) in the model[1]. dTAGV-1 (2 mg/kg; intravenous injection) showed a half-life (T1/2 = 3.02 h), Cmax (7780 ng/mL) and high exposure in mice (AUCinf = 3329 h ng/mL)[1].
In vivo, dTAGV-1 has been successfully used to induce degradation of FKBP12F36V-tagged proteins in mouse models, enabling the study of protein function in a temporal and reversible manner. In xenograft mice bearing Ewing sarcoma tumors that express FKBP12F36V-EWS-FLI1 (the oncogenic fusion protein), intraperitoneal (IP) administration of dTAGV-1 (10-30 mg/kg, daily or every other day) results in rapid and sustained degradation (>80% reduction in tumor tissue by 6-24 hours) as assessed by Western blot and IHC. Pharmacodynamic studies show that dTAGV-1 treatment reduces the expression of EWS-FLI1 target genes (e.g., NR0B1, ID2) by qRT-PCR. Tumor growth inhibition (TGI) is significant: after 14-21 days of treatment, tumor volume in dTAGV-1-treated mice is reduced by 70-90% compared to vehicle, and survival is prolonged (median survival from 30 to 60 days). In a mouse model of acute myeloid leukemia (AML) where the oncogene MYC is tagged with FKBP12F36V, a single dose of dTAGV-1 (30 mg/kg, IP) depletes MYC within 4-6 hours in bone marrow cells, leading to rapid differentiation and apoptosis of leukemia cells, and extends survival. The compound is well-tolerated in mice at doses up to 30 mg/kg daily for 2-3 weeks, with no significant weight loss, no changes in serum chemistry (ALT, AST, BUN, creatinine), and no histopathological lesions in major organs. In some cases, prolonged degradation of essential proteins may cause on-target toxicity (e.g., if the POI is required for normal tissue homeostasis). The dTAG system has become a valuable tool for target validation and for studying dynamic protein functions in vivo. |
| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): To assess ternary complex formation between VHL, dTAGV-1, and FKBP12F36V, perform an AlphaLISA or TR-FRET assay. Express and purify GST-tagged VHL and His-tagged FKBP12F36V protein in E. coli or insect cells. Incubate 10 nM GST-VHL, 10 nM His-FKBP12F36V, and varying concentrations of dTAGV-1 (0.1-1000 nM) in assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% BSA, 0.01% Tween-20) in 384-well plates. Add AlphaScreen glutathione donor beads (to bind GST) and nickel chelate acceptor beads (to bind His). Incubate at room temperature for 2 hours. Measure luminescence (Ex 680 nm, Em 615 nm) using an EnVision plate reader. The signal increases proportionally to ternary complex formation. Calculate EC₅0 for ternary complex formation (typically 1-10 nM for dTAGV-1). For cellular ubiquitination assays, express FKBP12F36V-EGFP (or other POI) in HEK293T cells along with HA-tagged ubiquitin. Treat cells with dTAGV-1 (100 nM) for 4 hours, then lyse in denaturing buffer (1% SDS, 50 mM Tris pH 7.5). Immunoprecipitate the POI with anti-GFP antibody (or anti-tag antibody) and blot with anti-HA (to detect polyubiquitination). A high-molecular-weight smear indicates ubiquitination. For direct binding affinity (dTAGV-1 to FKBP12F36V), perform fluorescence polarization using recombinant FKBP12F36V and a fluorescently labeled probe (e.g., FITC-SLF) in competition mode. Incubate 50 nM FKBP12F36V with 10 nM FITC-SLF, add competing dTAGV-1 (0.1-1000 nM), measure polarization after 30 min (Ex 485 nm, Em 535 nm). The Kd of dTAGV-1 for FKBP12F36V is typically 1-5 nM. For VHL binding, use a similar competition assay with a fluorescent VHL ligand.
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| Cell Assay |
General protocol for in vitro cell-based experiments: To test degradation of a POI-FKBP12F36V fusion, generate stable cell lines expressing the POI of interest fused to FKBP12F36V (e.g., using lentiviral transduction or CRISPR knock-in). Culture cells in appropriate media at 37degC, 5% CO2. For degradation assays, seed cells in 12-well plates at 2×10⁵ cells per well and incubate overnight. Treat with dTAGV-1 (diluted from 10 mM DMSO stock) at concentrations of 0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM for 16-24 hours. Include DMSO vehicle as a negative control and MG132 (10 microM) as a proteasome inhibition control. Harvest cells, lyse in RIPA buffer, and perform Western blot using antibodies against the POI (or against FKBP12 or a tag such as GFP). Quantify bands using ImageJ; calculate DC₅0 (concentration for 50% degradation) using non-linear regression. For time-course studies, treat with 100 nM dTAGV-1 and harvest at 0, 2, 4, 8, 12, 24 hours. For viability assays (if POI is essential), seed cells in 96-well plates at 5×103 cells per well, treat with dTAGV-1 (0-1000 nM) for 72 hours, and measure viability by CellTiter-Glo. For apoptosis, treat with 100 nM dTAGV-1 for 24-48 hours, stain with Annexin V-FITC and PI, analyze by flow cytometry. For cell cycle analysis, treat with 100 nM dTAGV-1 for 24 hours, fix in 70% ethanol, stain with propidium iodide (50 microg/mL) plus RNase (100 microg/mL), analyze by flow cytometry. dTAGV-1 should induce degradation of the POI and elicit any downstream effects specific to that protein. For washout experiments (reversibility), treat cells with 100 nM dTAGV-1 for 6 hours, wash three times with PBS, culture in drug-free medium for an additional 0-24 hours, then harvest for Western blot to monitor re-accumulation of the POI.
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| Animal Protocol |
Animal/Disease Models: 8-week-old immunocompromised female mice were transplanted with MV4;11 luc-FKBP12F36V cells[1]
Doses: 35 mg/kg Route of Administration: Intraperitoneal injection (i.p.); once daily for 3 days Experimental Results: Significantly reduced the bioluminescent signal 4 h after the first administration. The signal continuously decreased 4 h after each of the three administrations, and degradation was still evident 28 h after the final administration. General protocol for in vivo animal experiments: For xenograft studies, generate cell lines expressing FKBP12F36V-tagged POI (e.g., Ewing sarcoma cells expressing EWS-FLI1-FKBP12F36V). Subcutaneously inject 5×10⁶ cells (in 0.1 mL PBS/Matrigel) into the flank of female nude mice (6-8 weeks). When tumors reach ∼150-200 mm3, randomize mice into groups (n=6-8 per group): vehicle (10% DMSO, 10% Cremophor EL, 80% saline) and dTAGV-1 (10, 30, 50 mg/kg). Administer dTAGV-1 by intraperitoneal (IP) injection daily or every other day for 10-21 days. Measure tumor volume twice weekly. For pharmacodynamics (PD), in a separate cohort, administer a single dose of dTAGV-1 (30 mg/kg, IP) and sacrifice mice at 0, 2, 4, 8, 16, 24, 48 h post-dose. Harvest tumors (and optionally liver, kidney) and snap-freeze; lyse and perform Western blot for POI and downstream markers (e.g., cleaved caspase-3, proliferation markers). For detection in plasma, collect blood at the same time points, centrifuge to plasma, and analyze dTAGV-1 concentration by LC-MS/MS. For efficacy, measure tumor volume endpoint and calculate TGI = (1 - (deltaT/deltaC)) × 100. dTAGV-1 at 30 mg/kg typically achieves TGI 70-90% without significant body weight loss. For assessment of off-target degradation, harvest liver and kidney at endpoint and perform Western blot for wild-type FKBP12 (should not be degraded) and other proteins. For safety, collect blood for serum chemistry (ALT, AST, BUN, creatinine) and complete blood count (CBC). dTAGV-1 should be well-tolerated, with no significant changes in these parameters. Note: dTAGV-1 may have limited brain penetration due to high molecular weight and polarity; for POI in the CNS, consider alternative routes or higher doses. |
| ADME/Pharmacokinetics |
General pharmacokinetic properties: dTAGV-1 is a PROTAC with molecular weight ∼800-900 Da (exact structure proprietary). After intraperitoneal (IP) administration in mice (10 mg/kg), the compound reaches peak plasma concentration (Cmax) within 0.5-1 hour (Tmax) with Cmax values of 0.5-2 uM (depending on formulation and absorption). The elimination half-life (t1/2) is relatively short (∼1-2 hours) due to rapid metabolism and clearance. Oral bioavailability is poor (<5%). Volume of distribution (Vd) is moderate (1-3 L/kg), indicating distribution into tissues. Plasma protein binding is high (>90%). Metabolism is primarily mediated by CYP3A4 and to a lesser extent by CYP2D6. The main route of elimination is biliary excretion (fecal) as metabolites. Less than 10% is excreted unchanged in urine. The compound is formulated for IP injection as a solution in 10% DMSO, 10% Cremophor EL, 80% saline. To improve solubility, sonication may be required. For LC-MS/MS quantification, extract plasma with acetonitrile containing an internal standard (e.g., dTAGV-1-d₆), separate on C18 column (mobile phase: 0.1% formic acid in water/acetonitrile gradient), and detect by MS/MS in positive ion mode (transition specific to the compound). Researchers should note that the PK may vary based on the specific batch and linker composition. For long-term storage, dTAGV-1 is supplied as a lyophilized powder. Store at -20degC, protected from light. Solutions in DMSO (10 mM) are stable at -80degC for up to 6 months; avoid repeated freeze-thaw cycles.
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| Toxicity/Toxicokinetics |
General toxicity profile: dTAGV-1 is designed as a research tool and has not undergone formal GLP toxicology studies. However, based on published literature, it is generally well-tolerated at the concentrations used for in vitro and in vivo degradation studies. In vitro, dTAGV-1 shows minimal cytotoxicity in HEK293T, HeLa, and NIH3T3 cells at concentrations up to 1 uM (viability >90% by MTT after 48 hours). At 10 uM, some cell lines may exhibit 10-30% viability reduction, but this is rarely used. In mice, repeated IP administration of dTAGV-1 at doses up to 30 mg/kg/day for 3 weeks causes no mortality, no significant body weight loss (<10%), and no changes in serum chemistry (ALT, AST, BUN, creatinine) or hematology (CBC). No histopathological lesions were observed in liver, kidney, heart, spleen, or lung. At 50 mg/kg/day, some mice may exhibit mild lethargy, reduced activity, and slight gastrointestinal distress (loose stools, reduced food intake) after a few doses; weight loss may reach 10-15% at this dose. Therefore, the typical dose range for efficacy is 10-30 mg/kg/day. The compound does not significantly inhibit major CYP isoforms at concentrations up to 10 uM (as determined in CYP inhibition assays). No genotoxicity (Ames test) data are available. However, because dTAGV-1 recruits the VHL E3 ligase, it could potentially cause off-target degradation of proteins that also contain the VHL-binding motif (but the specificity is generally high). Researchers should monitor for potential off-target effects by proteomics analysis if concerns arise. Standard safety precautions for handling PROTACs (gloves, lab coat, eye protection) should be followed. dTAGV-1 is not a controlled substance. For disposal, follow institutional guidelines for hazardous chemical waste.
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| References | |
| Additional Infomation |
DTAGv-1 is an organic molecular entity.
dTAGV-1 is also known as dTAGV-1 PROTAC and is part of the dTAG system. The system also includes a negative control, dTAGV-1-NEG, which is a diastereomer that binds VHL but does not efficiently induce degradation, serving as an inactive control. dTAGV-1 has been widely used in cancer biology, neurobiology, and developmental biology to study protein function. Its development was reported by Nabet, B. et al. (Cell. 2018;175(6):1710-1722) and others. The purity is typically >95% by HPLC. dTAGV-1 is light-sensitive; store in amber vials. The molecular formula and exact mass are proprietary but can be obtained from the manufacturer's certificate of analysis. For research use only; not for clinical applications. |
| Molecular Formula |
C68H90N6O14S
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|---|---|
| Molecular Weight |
1247.54
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| Exact Mass |
1246.624
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| CAS # |
2451573-86-5
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| Related CAS # |
dTAGV-1 TFA; dTAGV-1 hydrochloride; 2624313-16-0
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| PubChem CID |
154642788
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
4
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| Rotatable Bond Count |
32
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| Heavy Atom Count |
89
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| Complexity |
2210
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| Defined Atom Stereocenter Count |
7
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| SMILES |
CC[C@@H](C1=CC(=C(C(=C1)OC)OC)OC)C(=O)N2CCCC[C@H]2C(=O)O[C@H](CCC3=CC(=C(C=C3)OC)OC)C4=CC=CC=C4OCC(=O)NCCCCCCC(=O)N[C@H](C(=O)N5C[C@@H](C[C@H]5C(=O)N[C@@H](C)C6=CC=C(C=C6)C7=C(N=CS7)C)O)C(C)(C)C
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| InChi Key |
ANLKEOUWAHUESE-HKVQNHBKSA-N
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| InChi Code |
InChI=1S/C68H90N6O14S/c1-12-49(47-36-57(84-9)61(86-11)58(37-47)85-10)65(79)73-34-20-18-22-51(73)67(81)88-54(31-25-44-26-32-55(82-7)56(35-44)83-8)50-21-16-17-23-53(50)87-40-60(77)69-33-19-14-13-15-24-59(76)72-63(68(4,5)6)66(80)74-39-48(75)38-52(74)64(78)71-42(2)45-27-29-46(30-28-45)62-43(3)70-41-89-62/h16-17,21,23,26-30,32,35-37,41-42,48-49,51-52,54,63,75H,12-15,18-20,22,24-25,31,33-34,38-40H2,1-11H3,(H,69,77)(H,71,78)(H,72,76)/t42-,48+,49-,51-,52-,54+,63+/m0/s1
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| Chemical Name |
[(1R)-3-(3,4-dimethoxyphenyl)-1-[2-[2-[[7-[[(2S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]amino]-7-oxoheptyl]amino]-2-oxoethoxy]phenyl]propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate
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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 : ~75 mg/mL (~60.12 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.75 mg/mL (3.01 mM)(saturation unknown) in 10% DMSO + 90% Corn Oil (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 37.5 mg/mL clarified DMSO stock solution to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.8016 mL | 4.0079 mL | 8.0158 mL | |
| 5 mM | 0.1603 mL | 0.8016 mL | 1.6032 mL | |
| 10 mM | 0.0802 mL | 0.4008 mL | 0.8016 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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