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Purity: ≥98%
dCBP-1 is a novel, potent and selective heterobifunctional and PROTAC degrader of p300/CBP with potential anticancer activity. It is highly potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression.
Targets |
p300/CBP; E3 ligase Cereblon
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ln Vitro |
In MM1S cells, dCBP-1 (10–1000 nM; 6 hours) nearly entirely degrades p300/CBP. In additional multiple myeloma cell lines tested, such as MM1R, KMS-12-BM, and KMS34 cells, dCBP-1 can also transduce nearly complete p300/CBP degradation [1]. When dCBP-1 was applied to the human haploid cell line HAP1 for six hours, CBP and p300 were almost completely lost at doses ranging from 10 to 1000 nM. After one hour of treatment, p300/CBP nearly completely degraded, according to time course analysis using 250 nM dCBP-1 [1].
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Enzyme Assay |
dCBP-1 ablates enhancer lysine acetylation and chromatin accessibility[1]
p300/CBP acetyltransferase activity dynamically modulates many lysine residues across thousands of chromatin-associated proteins. Lysine 27 on histone H3 is a substrate that has been shown to be inhibited by both KAT inhibitors and bromodomain inhibitors, and H3K27ac is thought to be required for p300/CBP-mediated enhancer activity itself (Raisner et al., 2018). We performed chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27ac following 6 h treatment of MM1S cells with A-485, GNE-781, the combination of both inhibitors, or dCBP-1. The inhibitors alone or in combination had only a modest effect on H3K27ac levels at enriched sites mapped in control cells, while dCBP-1 caused a near-complete loss of this modification at these sites (Figure 5A). These regions of acetylation loss are highly bound by both p300 and CBP, validating them as direct sites of p300/CBP action. Loss of H3K27ac was also apparent at the p300/CBP-bound oncogenic IGH enhancer that drives high MYC expression (Figure 5B). We also performed chromatin accessibility measurements by ATAC-seq, which revealed significant loss of accessible chromatin at the p300/CBP regulatory site of the IGH enhancer and the MYC promoter only in dCBP-1-treated cells. This suggests that complete ablation of enhancer activity with attenuation of transcription factor association with DNA regulatory elements can be accomplished only by complete removal of p300/CBP from chromatin, achieved most readily by chemical-induced degradation. |
Cell Assay |
Western Blot Analysis[1]
Cell Types: Multiple myeloma cell line MM1S Tested Concentrations: 10 nM, 100 nM, 250 nM, 500 nM, 1000 nM Incubation Duration: 6 hrs (hours) Experimental Results: demonstrated rapid degradation, p300/CBP after 2 hrs (hours) Almost completely lost. Cell viability assays: Cells were plated into 384-well tissue-culture treated plates with 2,000 cells per well in 70 μL of appropriate media. The last column of each plate was seeded with media containing no cells. Plates were centrifuged at 350 g to remove bubbles and placed in an incubator overnight. One day after seeding, cells were treated with 35 nL of dCBP-1, pomalidomide, A-485, or GNE-781 resuspended in DMSO with 384-pin disposable replicators. Plates were centrifuged at 350 g again to ensure proper mixing of the compounds in each well and placed in an incubator. Four days after treatment 7 μL of alamarBlue (Invitrogen) was added per well and incubated for 24 hours. After incubation, individual well fluorescence was read on an Envision multi-well plate reader. Dose-response curves were generated by normalizing signal to DMSO-treated wells. AUC values were calculated using GraphPad PRISM software. For growth-over-time assays, MM1S cells were plated at a density of 3 x 105 cells/mL in 24-well low-attachment tissue culture plates. One day following seeding, cells were treated in triplicate with 100 nM of each compound and cultures were counted manually by hemocytometer over the course of five days of treatment.[1] |
References | |
Additional Infomation |
The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.[1]
In the studies of dCBP-1 activity in multiple myeloma, researchers do observe augmented effects on gene expression programs, antiproliferation, and chromatin structures when compared with bromodomain and KAT domain inhibitor treatment either alone or in combination. This includes a complete loss of oncogenic enhancer chromatin accessibility and H3K27 acetylation that cannot be achieved with equivalent doses of free inhibitors. While CRBN-based heterobifunctional degraders of BET proteins have similar antiproliferative and anti-MYC effects in cellular models of multiple myeloma, the functional domains outside their bromodomains are largely divergent, and so it is likely that the acute effects of p300/CBP loss on chromatin composition and enhancer structures are distinct (Lim et al., 2019). The addition of dCBP-1 to the toolbox of chromatin regulator-targeting degraders (along with degraders of BET factors, BRD9, TRIM24, SMARCA2/4, CDK9, EED/EZH2, PCAF/GCN5, HDAC1/2/3) will thus enable precise investigation of their unique functions (Bassi et al., 2018; Farnaby et al., 2019; Gechijian et al., 2018; Hsu et al., 2019; Olson et al., 2018; Potjewyd et al., 2019; Remillard et al., 2017; Smalley et al., 2020; Winter et al., 2015). Further studies will also assess the tolerability of p300/CBP degradation in animal models, which will enable additional evaluation of the therapeutic potential of dCBP-1 and other pharmacologically optimized analogues.[1] The chromatin regulators CBP and p300 play important roles in maintaining enhancer-driven gene transcription in normal and malignant cells. Their enzymatic activity through ε-N-lysine acetylation establishes these posttranslational marks on histones, transcription factors, and an array of other chromatin regulators. In addition to their acetyltransferase activity, p300/CBP also possess a number of other functional domains that mediate protein:protein interactions and scaffolding functions at enhancers. Several high-quality selective chemical inhibitors of p300/CBP function have been developed, and efforts to develop them as cancer therapeutics are underway. However, inhibition of single domains alone cannot completely ablate p300/CBP activity in cells. We have discovered a highly potent chemical degrader of both p300 and CBP, dCBP-1, that potently ablates enhancer-mediated transcription. We envision that dCBP-1 will be a useful tool for studying the acute effects of p300/CBP loss and may lead to new therapeutic strategies in cancers such as multiple myeloma that are often driven by oncogenic enhancer activity.[1] |
Molecular Formula |
C51H63F2N11O10
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Molecular Weight |
1028.1104
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Exact Mass |
1027.472
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Elemental Analysis |
C, 59.58; H, 6.18; F, 3.70; N, 14.99; O, 15.56
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CAS # |
2484739-25-3
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PubChem CID |
154690309
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Appearance |
Light yellow to yellow solid powder
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LogP |
1.6
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Hydrogen Bond Donor Count |
3
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Hydrogen Bond Acceptor Count |
16
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Rotatable Bond Count |
21
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Heavy Atom Count |
74
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Complexity |
1960
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Defined Atom Stereocenter Count |
0
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InChi Key |
ILVRLRGBSSFKIE-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C51H63F2N11O10/c1-54-51(70)61-17-11-41-40(31-61)47(62-14-3-4-32-26-37(33-29-56-59(2)30-33)38(46(52)53)28-43(32)62)58-64(41)35-9-15-60(16-10-35)45(66)12-18-71-20-22-73-24-25-74-23-21-72-19-13-55-34-5-6-36-39(27-34)50(69)63(49(36)68)42-7-8-44(65)57-48(42)67/h5-6,26-30,35,42,46,55H,3-4,7-25,31H2,1-2H3,(H,54,70)(H,57,65,67)
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Chemical Name |
3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-[1-[3-[2-[2-[2-[2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperidin-4-yl]-N-methyl-6,7-dihydro-4H-pyrazolo[4,3-c]pyridine-5-carboxamide
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Synonyms |
dCBP 1; dCBP-1; CID 154690309; 3-[7-(Difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-[1-[3-[2-[2-[2-[2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperidin-4-yl]-N-methyl-6,7-dihydro-4H-pyrazolo[4,3-c]pyridine-5-carboxamide; SCHEMBL24269061; dCBP1
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HS Tariff Code |
2934.99.9001
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Storage |
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. |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
DMSO : ~50 mg/mL (~48.63 mM)
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Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (4.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear 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 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. Solubility in Formulation 2: ≥ 2.86 mg/mL (2.78 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 28.6 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.  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 0.9727 mL | 4.8633 mL | 9.7266 mL | |
5 mM | 0.1945 mL | 0.9727 mL | 1.9453 mL | |
10 mM | 0.0973 mL | 0.4863 mL | 0.9727 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.