VHL; FKBP12F36V fusion protein

In 293FT cells, dTAGV-1 (0.1 nM-10 μM; 24 h) TFA causes strong degradation of FKBP12F36V-Nluc while having no effect on FKBP12WT-Nluc[1]. The co-treatment of THAL-SNS-032 with dTAGV-1 (125-2000 nM; 24 h) TFA causes a significant degradation of both CDK9 and LACZ-FKBP12F36V[1]. dTAGV-1 (500 nM; 1–24 h) TFA causes pERK1/2 and KRASG12V to degrade quickly[1]. In Ewing sarcoma, dTAGV-1 (50-5000 nM; 24 h) TFA promotes EWS/FLI degradation[1].

dTAGV-1 (35 mg/kg; intraperitoneally once day for 4 days) In mice, TFA causes FKBP12F36V-Nluc to degrade[1]. TFA half-lives (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 mice are all affected by dTAGV-1 (2-10 mg/kg; ip) TFA[1]. Mice's half-life (T1/2=3.02 h), Cmax (7780 ng/mL), and great exposure (AUCinf = 3329 h·ng/mL) are all affected by dTAGV-1 (2 mg/kg; iv) TFA[1].

FKBP12WT and FKBP12F36V dual luciferase assay [1]
Dual luciferase assays were performed using 293FT FKBP12WT-Nluc and FKBP12F36V-Nluc cells6. In brief, cells were plated at 2000 cells per well in 20 µL of appropriate media in 384-well white culture plates, allowed to adhere overnight, and 100 nL of compounds were added using a Janus Workstation pin tool for 24 h at 37 °C. To evaluate Fluc signal, plates were brought to room temperature, 20 µL of Dual-Glo Reagent was added for 10 min and luminescence was measured on an Envision 2104 plate reader. Subsequently, 20 µL of Dual-Glo Stop & Glo Reagent was added for 10 min and luminescence was again measured to capture Nluc signal. DMSO-normalized ratios of Nluc/Fluc signal was analyzed and plotted using GraphPad PRISM v8.

Analysis of cell viability [1]
Cell viability was assayed in 2D-adherent or ultra-low adherent 3D-spheroids using CellTiter-Glo. Luminescence was measured on an Envision 2104 plate reader and Fluostar Omega Reader and data was analyzed using GraphPad PRISM v8. Synergy assessments were performed using CellTiter-Glo with the following modifications to the protocol described in the ref. 34. In brief, EWS502 cells were plated at 1000 cells per well in 50 µL of appropriate media in 384-well white culture plates allowed to adhere overnight, and 100 nL of compounds were added using a Janus Workstation pin tool for 72 h. Cell viability was measured by addition of 10 µL of CellTiter-glo, followed by incubation for 15 minutes at room temperature. Luminescence was measured on an Envision 2104 plate reader and data was analyzed using GraphPad PRISM v8.

Animal/Disease Models: 8weeks old immunocompromised female mice were transplanted with MV4;11 luc-FKBP12F36V cells[1]
Doses: 35 mg/kg
Route of Administration: Ip one time/day for 4 days
Experimental Results: Observed striking loss of bioluminescent signal 4 h after the first and three administrations. Degradation evident 28 h after the final administration.

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Animal studies: compound formulation [1]
For IP injections, dTAG-13 and dTAGV-1 were formulated by dissolving into DMSO and then diluting with 20% solutol: 0.9% sterile saline (w:v) with the final formulation containing 5% DMSO. Maximal solubility of 35 mg kg−1 and 40 mg kg−1 were observed for dTAG-13 and dTAGV-1, respectively. Formulations were stable at room temperature for 7 days. For IV injections, dTAG-13 and dTAGV-1 were formulated by dissolving into DMSO and then diluting with 5% solutol: 0.9% sterile saline (w:v) with the final formulation containing 5% DMSO.

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Animal studies: pharmacokinetic (PK) evaluation [1]
PK was assessed in 8-week-old C57BL/6J male mice with blood collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, and 8 h (2 mg kg−1 dTAG-13 intravenous (IV) tail vein, 10 mg kg−1 dTAG-13 intraperitoneal (IP), and 2 mg kg−1 dTAGV-1 IV tail vein administrations) and 0.25, 0.5, 1, 2, 4, 6, 8, 24 and 48 h (2 mg kg−1 dTAGV-1 IP and 10 mg kg−1 dTAGV-1 IP administrations). Plasma was generated by centrifugation and plasma concentrations were determined by LC-MS/MS following the mass transition 49600à340 AMU. PK parameters were calculated using Phoenix WinNonlin to determine peak plasma concentration (Cmax), oral bioavailability (%F), exposure (AUC), half-life (t1/2), clearance (CL), and volume of distribution (Vd).

To validate the in vivo suitability of dTAGV-1, we characterized its pharmacokinetic (PK) and pharmacodynamic (PD) properties in mice. Compared to dTAG-13, dTAGV-1 exhibited superior properties, including a longer half-life (T1/2 of 4.43 h and 2.41 h, respectively) and higher drug exposure after intraperitoneal injection of a 10 mg kg−1 dose (AUCinf of 18,517 h ng mL−1 and 6,140 h ng mL−1, respectively) (Supplementary Table 1). To report the pharmacodynamic characterization of the dTAG molecule, we used MV4;11 luciferase-FKBP12F36V (luc-FKBP12F36V) cells, a cell line capable of non-invasively monitoring bioluminescent signals following dTAG molecule administration in mice.⁶ After tail vein injection of MV4;11 luc-FKBP12F36V cells and establishment of leukemia burden, we performed daily bioluminescence measurements 4 hours after administration of the vector, 35 mg kg−1 dTAG-13, or 35 mg kg−1 dTAGV-1, respectively. The bioluminescence signal was significantly reduced 4 hours after the first administration of dTAGV-1 (Fig. 2d and Supplementary Fig. 3). A sustained reduction in bioluminescence signal was observed 4 hours after three administrations of dTAG-13 or dTAGV-1. The degradation of dTAGV-1 lasted longer than that of dTAG-13, with degradation still observed 28 hours after the last administration. These results support the use of dTAGV-1 as a highly efficient and selective molecule for evaluating targeted degradation in vitro and in vivo. [1]

[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]

C70H91F3N6O16S

1361.56

1360.616

2624313-15-9

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

154828675

White to light brown solid powder

5

21

32

96

2290

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.C(=O)(C(F)(F)F)O

KSEWNBIDXKMTNT-LNVAYBNASA-N

InChI=1S/C68H90N6O14S.C2HF3O2/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;3-2(4,5)1(6)7/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);(H,6,7)/t42-,48+,49-,51-,52-,54+,63+;/m0./s1

[(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;2,2,2-trifluoroacetic acid

dTAGV-1 TFA; 2624313-15-9; dTAGV-1 (TFA);

2934.99.9001

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 and light.

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

DMSO :~37.5 mg/mL (~27.54 mM)

Solubility in Formulation 1: ≥ 3.75 mg/mL (2.75 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 37.5 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.

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Solubility in Formulation 2: ≥ 3.75 mg/mL (2.75 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 clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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