BRD4; DCAF16 (E3 ligase);

Endogenous BRD4 in HEK293T cells is degraded in a concentration-dependent manner after KB02-JQ1 (5–40 μM) treatment for 24 hours[1].
Protein degradation by low fractional DCAF16 engagement.[1]
Researchers next investigated whether covalent modification of DCAF16 could support the degradation of another nuclear protein. For these studies, Researchers selected BRD4 as the nuclear protein, as it has a potent and selective ligand JQ1 that has been successfully coupled to other E3 ligands to promote degradation13, 15, 45-47. We found that a KB02-JQ1 (9) bifunctional compound (Fig. 6a) promoted, in a concentration-dependent manner, the degradation of BRD4 in HEK293T cells (Fig. 6b), and this effect was blocked by MG132 or MLN4924 (Fig. 6c). BRD4 was not degraded in cells treated with either the two separate components (KB02 and JQ1) of KB02-JQ1 or the FKBP12-directed bifunctional compound KB02-SLF (Fig. 6d). Consistent with DCAF16 mediating the degradation of BRD4 by KB02-JQ1, Researchers found that the KB02-JQ1-induced, but not the KB02-SLF-induced HMW form of DCAF16 co-immunoprecipitated with BRD4 (Fig. 6e). KB02-JQ1-induced degradation of BRD4 was also substantially blocked in DCAF16−/− cells (Fig. 6f).

Researchers noted that much higher concentrations of KB02-JQ1 (20–40 µM; Fig. 6b) were required to degrade BRD4 compared to the degradation of FKBP12_NLS by KB02-SLF (0.5–2 µM; Fig. 2e, f). This reduction in potency may reflect a combination of factors, including differential cellular uptake of the two KB02 bifunctional compounds (KB02-JQ1 showed a rightward shift in cytotoxicity (IC50 > 50 µM) compared to KB02-SLF (IC50 = 14 ± 1.1 µM) (Supplementary Fig. 11)) and less efficient DCAF16-mediated degradation of BRD4 compared to FKBP12. To explore the latter possibility, Researchers measured the relative cellular engagement of DCAF16 by KB02-JQ1 and KB02-SLF. Specifically, Researchers used competitive ABPP to quantitatively map the fractional blockade of IA-alkyne-modified cysteines on endogenously expressed DCAF16 in HEK293T cells treated with concentrations of KB02-SLF (2 µM) or KB02-JQ1 (20 µM) that support FKBP12 and BRD4 degradation, respectively. These chemical proteomic data revealed that KB02-SLF and KB02-JQ1 produced ~10% and 40% engagement, respectively, of the DCAF16 peptide (aa 168–184) containing cysteines 173 and 177–179 (Fig. 6g, Supplementary Fig. 12, and Supplementary Dataset 4). Another IA-alkyne-reactive cysteine, C119, showed no evidence of engagement in these chemical proteomic experiments (Fig. 6g, Supplementary Fig. 12, and Supplementary Dataset 4). Researchers measured a similar pattern of engagement by comparing the HMW and LMW forms of recombinantly expressed DCAF16 in HEK293T cells treated with KB02-SLF (5 µM, 13% engagement) and KB02-JQ1 (20 µM, 35% engagement) (Supplementary Fig. 13). Taken together, these data indicate that differential amounts of DCAF16 engagement are required to support FKBP12 and BRD4 degradation by their respective electrophilic PROTACs, but, in neither case is a substantial fraction (> 50%) of DCAF16 diverted to a PROTAC-modified state.

Finally, MS-based proteomic analysis of KB02-JQ1-treated HEK293T cells revealed selective degradation of BRD4, but not BRD2 or BRD3 (Fig. 6h and Supplementary Dataset 5). BRD2 appeared to be stabilized by KB02-JQ1, as has been found for JQ1 itself48. One additional protein across the proteome – ACAT1 – displayed substantially decreased abundance in KB02-JQ1-treated cells (Fig. 6h and Supplementary Dataset 5). Control cells treated with KB02-SLF also showed reductions in ACAT1, but not BRD4 (Fig. 6h and Supplementary Dataset 5). Interestingly, ACAT1 harbors a highly reactive cysteine (C126)27, 28, 36 that was fully engaged by KB02-JQ1 or KB02-SLF in HEK293T cells (Supplementary Fig. 12b and Supplementary Dataset 4). These data suggest that covalent modification of C126 by KB02-containing compounds could lead to the degradation of ACAT1, possibly by disrupting homo-oligomeric forms of the enzyme49. While BRD2 was the only protein that showed a > two-fold increase in abundance in KB02-JQ1-treated cells, a handful of additional proteins (nine total) showed more modest elevations (~1.5–1.8-fold) (Supplementary Dataset 5). None of these proteins were altered in KB02-SLF-treated cells (Supplementary Dataset 5). Whether these proteins represent potential endogenous substrates of DCAF16 that are stabilized by the higher cellular engagement of this E3 ligase by KB02-JQ1 versus KB02-SLF in cells, or, alternatively, are stabilized by direct modification of KB02-JQ1 represents an important topic for future investigation.

Proteome-wide identification of KB02-JQ1- or KB02-SLF-induced protein degradation[1]
HEK293T light and heavy SILAC cells were treated with DMSO and KB02-JQ1 (20 µM) or KB02-SLF (2 µM) for 24 h, respectively. Light and heavy cells were collected and lysed in 1% NP-40 lysis buffer with cOmplete protease inhibitor cocktail. Cells were vortexed and sonicated for 5 pulses (40%, 4). The supernatant was collected after centrifugation at 16,000 g for 10 min at 4 °C. Protein concentration was determined by DC assay. 50 µg proteome from light and heavy samples were mixed, followed by methanol/chloroform precipitation. Protein pellets were heated 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. 5 µg peptides were loaded onto a silica capillary column (250 µm) packed with 3 cm of C18 resin. The same MudPIT method and CIMAGE software as described above were used to analyze the peptides. The median SILAC ratios from quantified peptides was used as measures of protein abundance. The following quality filters were further applied to generate the plot in Fig. 6h: (1) proteins must have at least two quantified peptides; (2) proteins must be present in both replicates; (3) for each protein, the standard deviation of measured ratios of peptides must be < 1.0.

Western Blot Analysis[1]
Cell Types: HEK293T cells
Tested Concentrations: 5 μM, 10 μM, 20 μm or 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Concentration-dependent degradation of endogenous BRD4 in HEK293T cells.

[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]

C38H43CL2N7O6S

796.7623

795.237

C, 57.28; H, 5.44; Cl, 8.90; N, 12.31; O, 12.05; S, 4.02

2384184-44-3

KB02-COOH;2375196-30-6

137347731

White to off-white solid powder

4.4

2

10

16

54

1300

1

ClC([H])([H])C(N1C2C([H])=C([H])C(=C([H])C=2C([H])([H])C([H])([H])C1([H])[H])OC([H])([H])C(N([H])C([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC([H])([H])C([H])([H])N([H])C(C([H])([H])[C@@]1([H])C2=NN=C(C([H])([H])[H])N2C2=C(C(C([H])([H])[H])=C(C([H])([H])[H])S2)C(C2C([H])=C([H])C(=C([H])C=2[H])Cl)=N1)=O)=O)=O

XIYYDTXOEVAZGI-PMERELPUSA-N

InChI=1S/C38H43Cl2N7O6S/c1-23-24(2)54-38-35(23)36(26-6-8-28(40)9-7-26)43-30(37-45-44-25(3)47(37)38)20-32(48)41-12-15-51-17-18-52-16-13-42-33(49)22-53-29-10-11-31-27(19-29)5-4-14-46(31)34(50)21-39/h6-11,19,30H,4-5,12-18,20-22H2,1-3H3,(H,41,48)(H,42,49)/t30-/m0/s1

N-[2-[2-[2-[[2-[[1-(2-chloroacetyl)-3,4-dihydro-2H-quinolin-6-yl]oxy]acetyl]amino]ethoxy]ethoxy]ethyl]-2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetamide

KB02-JQ1; 2384184-44-3; N-[2-[2-[2-[[2-[[1-(2-Chloroacetyl)-3,4-dihydro-2H-quinolin-6-yl]oxy]acetyl]amino]ethoxy]ethoxy]ethyl]-2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetamide; SCHEMBL22075929;

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)

DMSO : 200 mg/mL (251.02 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (6.28 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 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.

---

Solubility in Formulation 2: 5 mg/mL (6.28 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.

---

View More

Solubility in Formulation 3: ≥ 5 mg/mL (6.28 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.

---

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