VHL; FKBP12F36V fusion protein

在 293FT 细胞中，dTAGV-1（0.1 nM-10 μM；24 h）TFA 会导致 FKBP12F36V-Nluc 强烈降解，但对 FKBP12WT-Nluc 没有影响[1]。 THAL-SNS-032 与 dTAGV-1（125-2000 nM；24 h）TFA 共同处理会导致 CDK9 和 LACZ-FKBP12F36V 显着降解[1]。 dTAGV-1（500 nM；1–24 小时）TFA 导致 pERK1/2 和 KRASG12V 快速降解[1]。在尤文肉瘤中，dTAGV-1（50-5000 nM；24 小时）TFA 促进 EWS/FLI 降解[1]。

dTAGV-1（35 mg/kg；腹膜内注射，每天一次，持续 4 天） 在小鼠中，TFA 会导致 FKBP12F36V-Nluc 降解[1]。小鼠中 TFA 半衰期（T1/2=3.64 和 4.4 h）、Cmax（595 和 2123 ng/mL）以及高暴露量（AUCinf = 3136 和 18517 h·ng/mL）均受 dTAGV-1 影响（ 2-10 毫克/千克；腹膜内注射) TFA[1]。小鼠的半衰期 (T1/2=3.02 h)、Cmax (7780 ng/mL) 和大暴露量 (AUCinf = 3329 h·ng/mL) 均受到 dTAGV-1 (2 mg/kg; iv) TFA 的影响[1]。

FKBP12WT和FKBP12F36V双荧光素酶测定[1]
采用293FT FKBP12WT-Nluc和FKBP12F36V-Nluc细胞进行双荧光素酶检测6。简而言之，将细胞以每孔2000个细胞的速度，在20µL合适的培养基中，在384孔的白色培养板中，粘附过夜，并使用Janus Workstation引脚工具添加100 nL化合物，在37℃下放置24 h。为了评估Fluc信号，将板置于室温，加入20µL Dual-Glo Reagent 10 min，在Envision 2104平板读取仪上测量发光。随后，加入20µL Dual-Glo Stop & Glo Reagent，静置10 min，再次测量发光，捕捉Nluc信号。使用GraphPad PRISM v8分析和绘制Nluc/Fluc信号的dmso归一化比率。

细胞活力分析[1]
使用CellTiter-Glo 检测2d贴壁或超低贴壁3d球体的细胞活力。在Envision 2104平板阅读器和Fluostar Omega阅读器上测量发光，并使用GraphPad PRISM v8分析数据。使用CellTiter-Glo进行协同评估，并对参考文献34中描述的方案进行以下修改。简而言之，将EWS502细胞以每孔1000个细胞的速度，在50µL合适的培养基中，在384孔的白色培养板中粘附过夜，并使用Janus Workstation引脚工具添加100 nL化合物，放置72小时。加入10µL CellTiter-glo测定细胞活力，室温孵育15分钟。在Envision 2104平板阅读器上测量发光，使用GraphPad PRISM v8对数据进行分析。

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 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]

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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

308

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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)

配方 1 中的溶解度: ≥ 3.75 mg/mL (2.75 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 37.5 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 3.75 mg/mL (2.75 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 37.5 mg/mL 的澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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