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.

---

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.

---

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 confirm the in vivo applicability of dTAGV-1, we characterized the pharmacokinetic (PK) and pharmacodynamic (PD) profile of dTAGV-1 in mice. dTAGV-1 demonstrated improved properties compared to dTAG-13, with a longer half-life (T1/2 = 4.43, 2.41 h respectively) and greater exposure (AUCinf = 18,517, 6140 h ng mL−1, respectively) by intraperitoneal administration at 10 mg kg−1 (Supplementary Table 1). To report on the PD profile of dTAG molecules, we employed MV4;11 luciferase-FKBP12F36V (luc-FKBP12F36V) cells that allow noninvasive monitoring of bioluminescent signal upon dTAG molecule administration in mice6. Following tail vein injection of MV4;11 luc-FKBP12F36V cells and establishment of leukemic burden, we performed daily bioluminescent measurements 4 h after vehicle, 35 mg kg−1 dTAG-13 or 35 mg kg−1 dTAGV-1 administration. Striking loss of bioluminescent signal was achieved 4 h after the first administration of dTAGV-1 (Fig. 2d and Supplementary Fig. 3). Consistent loss of bioluminescent signal was observed 4 h after each of the three dTAG-13 or dTAGV-1 administrations. Compared to dTAG-13, improved duration of degradation was also observed with dTAGV-1, with degradation evident 28 h after the final administration. These results support the use of dTAGV-1 as a potent and selective molecule to evaluate target-specific 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 for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice. [1]

---

We report dTAGV-1, a potent and exclusively selective VHL-recruiting degrader of FKBP12F36V-tagged proteins. dTAGV-1 displays improved PK/PD properties and serves as an optimized tool for in vivo applications. Through evaluation of mutant KRAS degradation in PDAC models, we show that either CRBN or VHL can be co-opted to alleviate the aberrant signaling coordinated by this oncoprotein. By contrast, we observed contextual differences in the ability of these E3 ubiquitin ligase complexes to degrade EWS/FLI. This is consistent with our recent report demonstrating effective degradation of a core mediator subunit (MED14) with dTAGV-1 in HCT116 cells, a context in which CRBN-recruiting dTAG molecules were not effective. We observed that rapid MED14 degradation abrogated lineage-specifying transcriptional circuits. Together, our studies provide support for use of dTAGV-1 to overcome the current limitations of the dTAG system, enabling evaluation of the direct effects of fusion proteins recalcitrant to CRBN-recruiting dTAG molecules. [1]
Employing dTAGV-1 to study EWS/FLI, we demonstrate that VHL-mediated degradation of EWS/FLI rapidly alters downstream target protein expression and leads to pronounced growth defects in Ewing sarcoma cells, providing a powerful model system to investigate immediate consequences of EWS/FLI loss. This data supports that targeting EWS/FLI for degradation with direct-acting heterobifunctional degraders or molecular glues may be a tractable strategy and identifies potential combination strategies with BET bromodomain degraders. Together, the suite of dTAG molecules and paired controls provided in this study will facilitate evaluation of the functional consequences of precise posttranslational protein removal for an expanded target pool. The dTAG system enables rapid modulation of protein abundance and serves as a versatile strategy to determine whether targeted degradation is a promising drug development approach for a given target 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.

---

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.

---

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