VHL; FKBP12F36V fusion protein

FKBP12F36V-Nluc is not degraded on 293FT FKBP12F36V-Nluc or FKBP12WT-Nluc cells by dTAGV-1-NEG (1-10 µM; 24 hours) [1]. PATU-8902 FKBP12F36V-KRASG12V and KRAS−/− cells do not exhibit KRASG12V protein degradation in response to dTAGV-1-NEG (500 nM; 24 hours) [1].

Western Blot Analysis[1]
Cell Types: PATU-8902 FKBP12F36V-KRASG12V, KRAS−/− Cell
Tested Concentrations: 500 nM
Incubation Duration: 1-24 hrs (hours)
Experimental Results: KRASG12V protein degradation was not induced.

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Cell Viability Assay[1]
Cell Types: EWS502 FKBP12F36V-GFP cells
Tested Concentrations: 1 μM
Incubation Duration: 0, 2, 4, 6, 8 days
Experimental Results: demonstrated relative growth of cells same as the control group, higher than dTAGV-1 treated group.

[1]. Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat Commun. 2020 Sep 18;11(1):4687.

Chemical biology strategies, such as degradation tag (dTAG) systems, can directly disrupt protein homeostasis, offering a time advantage over gene approaches and greater selectivity than small molecule inhibitors. We describe dTAGV-1, a VHL-specific dTAG molecule that rapidly degrades FKBP12F36V-tagged proteins. dTAGV-1 overcomes the limitations of previously reported CRBN-recruited dTAG molecules for degrading recalcitrant oncogenes, supports co-degrader studies, and facilitates the study of protein function in cells and mice. [1]

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We report dTAGV-1, a highly efficient and VHL-specific FKBP12F36V-tagged protein degrader. dTAGV-1 has superior pharmacokinetic/pharmacodynamic properties, making it an optimal tool for in vivo application. Through evaluation of mutant KRAS degradation in a PDAC model, we found that either CRBN or VHL can be synergistically utilized to alleviate aberrant signaling coordinated by this oncoprotein. Conversely, we observed contextual differences in the ability of these E3 ubiquitin ligase complexes to degrade EWS/FLI. This is consistent with our recent report that dTAGV-1 effectively degrades the core-mediated subunit (MED14) in HCT116 cells, while dTAG molecules recruited by CRBN are ineffective in this cell. We observed that rapid degradation of MED14 blocks lineage-specific transcriptional circuits. In conclusion, our study supports the use of dTAGV-1 to overcome the current limitations of the dTAG system, thereby enabling the assessment of the direct effects of fusion proteins that are difficult to degrade by dTAG molecules recruited by CRBN. [1]
We used dTAGV-1 to study EWS/FLI, and the results showed that VHL-mediated EWS/FLI degradation rapidly alters the expression of downstream target proteins and leads to significant growth defects in Ewing sarcoma cells, thus providing a powerful model system for studying the direct consequences of EWS/FLI deficiency. The data support the possibility that targeting the degradation of EWS/FLI with direct-acting heterobifunctional degraders or molecular gels may be a viable strategy, and reveal a potential strategy for combined use with BET bromine domain degraders. The dTAG molecules and their paired controls provided in this study will help assess the functional consequences of precise post-translational protein removal for a wider range of targets. The dTAG system can rapidly modulate protein abundance and can serve as a multifunctional strategy for determining whether targeted degradation is an effective method for drug development against specific targets in vitro and in vivo. [1]

C68H90N6O14S

1247.53901815414

1246.623

C, 65.47; H, 7.27; N, 6.74; O, 17.95; S, 2.57

2451573-87-6

2624313-15-9 (dTAGV-1 TFA); 2624313-16-0 (dTAGV-1 hydrochloride); 2451573-87-6 (dTAGV-1-NEG)

154828676

White to off-white solid powder

10.1

4

16

32

89

2210

7

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

ANLKEOUWAHUESE-SVUPEXKHSA-N

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

[(1R)-3-(3,4-dimethoxyphenyl)-1-[2-[2-[[7-[[(2S)-1-[(2R,4S)-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

dTAGV-1-NEG; 2451573-87-6; [(1R)-3-(3,4-Dimethoxyphenyl)-1-[2-[2-[[7-[[(2S)-1-[(2R,4S)-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; SCHEMBL25887266;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)