FKBP12; DCAF16 (E3 ligase)

Treatment with KB02-SLF (2 µM; 4-72 hours; HEK293T cells) significantly reduces nuclear FKBP12 and this effect lasts for the full 4-72 hours[1]. The selective loss of nuclear-localized FKBP12 in KB02-SLF-treated cells is confirmed by cell imaging experiments. Over a concentration range of approximately 0.5–5µM, KB02-SLF facilitates the loss of FKBP12_NLS, although at higher concentrations, it exhibits varying declines in activity. DCAF16 mediates the FKBP12_NLS degradation brought on by KB02-SLF[1].

Identification of FLAG-FKBP12_NLS interacting proteins[1]
HEK293T light and heavy SILAC cells stably expressing FLAG-FKBP12_NLS were treated with DMSO and KB02-SLF (2 or 10 µM) for 2 h, respectively, in the presence of 10 µM MG132. Heavy and light cells were collected and lysed in 1% NP-40 lysis buffer with complete protease inhibitor cocktail. FLAG immunoprecipitation (20 µL slurry per sample) was performed with 2 mg of total protein lysates to enrich FLAG-FKBP12_NLS from light and heavy cell lysates. After washing the FLAG resin four times with IP washing buffer, FLAG resin from light and heavy samples were combined and washed once with PBS. FLAG-FKBP12_NLS and its associating proteins were eluted by heating at 65 ºC for 10 min with 8 M urea in PBS, then reduced with 12.5 mM DTT at 65 ºC for 15 min and alkylated with 25 mM iodoacetamide at 37 ºC for 30 min. The protein solution was diluted with PBS to 2 M urea and digested with 2 µg trypsin at 37 ºC for 6 h. Tryptic peptides were acidified with 5% formic acid and loaded onto a silica capillary column (250 µm) packed with 3 cm of C18 resin. Peptides were analyzed on LTQ-Orbitrap Elite mass spectrometer coupled with Thermo UltiMate 3000 UHPLC system. Peptides were separated on a capillary column packed with 3 cm of strong cation exchange (SCX) resin, 10 cm of C18 resin and a 5 µm tip. A five-step MudPIT method and CIMAGE software were used to analyze the peptides as previously described51. To generate the plot in Fig. 3d and Supplementary Fig. 5c, the following quality filters were applied: (1) proteins must have at least two quantified peptides; (2) proteins must be absence in control SILAC (light and heavy amino acid-labeled HEK293T cells stably expressing pCDH empty vector were treated for 2 h with DMSO or 10 µM KB02-SLF, respectively in the presence of 10 µM MG132). (3) proteins must be quantified in at least two replicates; (4) the coefficient of variation of heavy/light ratio from three replicates is < 0.5.

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isoTOP-ABPP[1]
HEK293T cells were treated with DMSO or 10 µM KB02-SLFfor 2 h. Cells were collected and subjected to isoTOP-ABPP sample preparation using the same protocol as previously described.

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Far-western blot[1]
HEK293T cells were transfected with HA-DCAF16 plasmid by PEI transfection reagent for 24 h and treated with DMSO or KB02-SLF (5 µM) for 2 h. Cells were collected and lysed in 1% NP-40 lysis buffer with complete protease inhibitor cocktail. HA immunoprecipitation (20 µL slurry anti-HA agarose, 4 °C, 2 h) was performed with 2 mg of total protein lysates to purify HA-DCAF16. After washing the HA resin four times with IP washing buffer, HA affinity gel was heated at 95 °C for 10 min in 2X Laemmli sample buffer to elute HA-DCAF16 protein. Proteins were resolved by 14% Novex Tris-Glycine mini gels and transferred to PVDF membrane. The membrane was blocked with 5% BSA in TBST buffer at room temperature for 1 h. The recombinant His-tagged FKBP12 protein was diluted with fresh 5% BSA in TBST buffer (final concentration: 2 µg/mL) and incubated with membrane at 4 °C overnight (14 h). Membrane was washed three times with TBST buffer and incubated with anti-His-HRP antibody (1:1000 dilution in 5% BSA in TBST) at room temperature for 4 h. Membrane was washed three times with TBST buffer. The chemiluminescence signal in membrane was recorded after developing in ECL plus western blotting detection reagent using CL-XPosure film. The same protein samples were resolved by another 14% Novex Tris-Glycine mini gel and analyzed by anti-HA Western blot in parallel.

Western Blot Analysis[1]
Cell Types: HEK293T cells
Tested Concentrations: 2 µM
Incubation Duration: 4 hrs (hours), 8 hrs (hours), 24 hrs (hours), 48 hrs (hours), 72 hrs (hours)
Experimental Results: Promoted a substantial reduction in nuclear FKBP12 that was sustained across a 4-72 h time frame.

[1]. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat Chem Biol. 2019 Jul;15(7):737-746.

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.[1]

C50H65CLN4O12

949.523713827133

948.428

C, 63.25; H, 6.90; Cl, 3.73; N, 5.90; O, 20.22

2384184-40-9

KB02-COOH;2375196-30-6

137347727

White to yellow solid powder

6.1

2

12

26

67

1600

2

ClCC(N1C2C=CC(=CC=2CCC1)OCC(NCCOCCOCCC(NC1=CC=CC(=C1)[C@@H](CCC1C=CC(=C(C=1)OC)OC)OC([C@@H]1CCCCN1C(C(C(C)(C)CC)=O)=O)=O)=O)=O)=O

OKJBKEQXKMMRPL-WVILEFPPSA-N

InChI=1S/C50H65ClN4O12/c1-6-50(2,3)47(59)48(60)55-23-8-7-14-40(55)49(61)67-41(19-15-34-16-20-42(62-4)43(29-34)63-5)36-11-9-13-37(30-36)53-44(56)21-25-64-27-28-65-26-22-52-45(57)33-66-38-17-18-39-35(31-38)12-10-24-54(39)46(58)32-51/h9,11,13,16-18,20,29-31,40-41H,6-8,10,12,14-15,19,21-28,32-33H2,1-5H3,(H,52,57)(H,53,56)/t40-,41+/m0/s1

[(1R)-1-[3-[3-[2-[2-[[2-[[1-(2-chloroacetyl)-3,4-dihydro-2H-quinolin-6-yl]oxy]acetyl]amino]ethoxy]ethoxy]propanoylamino]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate

KB02-SLF; 2384184-40-9; [(1R)-1-[3-[3-[2-[2-[[2-[[1-(2-chloroacetyl)-3,4-dihydro-2H-quinolin-6-yl]oxy]acetyl]amino]ethoxy]ethoxy]propanoylamino]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate; SCHEMBL22111181;

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 : 100 mg/mL (105.32 mM)

Solubility in Formulation 1: 5 mg/mL (5.27 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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: 5 mg/mL (5.27 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 5 mg/mL (5.27 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 50.0 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.)