p300/CBP; E3 ligase Cereblon
dCBP-1 targets human CREB-binding protein (CBP) (DC50 = 1.8 nM, Dmax = 95% in MOLM-13 cells; Ki = 2.3 nM, SPR binding assay) [1]
dCBP-1 targets human E1A-binding protein p300 (p300) (DC50 = 2.5 nM, Dmax = 92% in MOLM-13 cells; Ki = 3.1 nM, SPR binding assay) [1]

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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1. Potently degrades CBP and p300 in a concentration-dependent manner in MLL-rearranged leukemia cell lines: MOLM-13 (CBP DC50 = 1.8 nM, p300 DC50 = 2.5 nM), MV4;11 (CBP DC50 = 2.2 nM, p300 DC50 = 3.0 nM), with Dmax > 90% for both proteins at 10 nM [1]
2. Inhibits CBP/p300 acetyltransferase activity: Reduces acetylation of histone H3K27 (H3K27ac) by 80% and acetylation of MYC (K323ac) by 75% at 10 nM in MOLM-13 cells (western blot) [1]
3. Downregulates MLL-CBP/p300 target gene expression: Reduces HOXA9 (72%), MEIS1 (68%), and MYC (65%) mRNA levels at 10 nM in MV4;11 cells (qPCR) [1]
4. Exhibits antiproliferative activity against MLL-rearranged leukemia cells: IC50 = 3.5 nM (MOLM-13), IC50 = 4.2 nM (MV4;11); no significant effect on non-MLL-rearranged cells (IC50 > 1000 nM for K562, Raji) [1]
5. Induces apoptosis in MOLM-13 cells: 10 nM concentration increases Annexin V-positive cells by 45% after 48 hours; activates caspase-3/7 (2.8-fold increase) [1]
6. Degradation is proteasome-dependent: Pretreatment with bortezomib (1 μM) completely blocks CBP/p300 degradation by dCBP-1 (10 nM) [1]
7. High selectivity: No degradation of other lysine acetyltransferases (p300/CBP-associated factor, PCAF; general control nonderepressible 5, GCN5) at concentrations up to 100 nM [1]

1. In MOLM-13 xenograft nude mice: Oral administration of dCBP-1 (10, 30 mg/kg/day) for 21 days dose-dependently inhibits tumor growth (TGI = 65% at 10 mg/kg, TGI = 88% at 30 mg/kg). Tumor tissues show reduced CBP (78%) and p300 (72%) protein levels, decreased H3K27ac (65%) and MYC K323ac (60%) (western blot) [1]
2. Reduces target gene expression in xenograft tumors: qPCR of tumor tissues from 30 mg/kg group shows decreased HOXA9 (68%), MEIS1 (63%), and MYC (59%) mRNA levels compared to vehicle [1]
3. No significant body weight loss (<5%) or organ toxicity: Histopathological examination of liver, kidney, heart, and spleen shows no abnormal lesions; blood chemistry (ALT, AST, BUN, Scr) and hematology indices are within normal ranges [1]

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.

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1. CBP/p300 acetyltransferase activity assay: Recombinant CBP/p300 catalytic domain is incubated with histone H3 substrate, acetyl-CoA, and serial concentrations of dCBP-1 (0.1 nM-10 μM). After 37°C incubation for 60 minutes, acetylated H3 (H3K27ac) is detected by ELISA. Inhibition rates are calculated relative to vehicle control [1]
2. SPR binding assay: CBP or p300 bromodomain is immobilized on a CM5 sensor chip. dCBP-1 is injected at 0.5 nM-1 μM (flow rate 30 μL/min). Sensorgrams are fitted with a 1:1 binding model to determine Ki values, after subtracting reference channel signals [1]
3. Ubiquitination assay: MOLM-13 cell lysates are incubated with dCBP-1 (10 nM), E1, E2, VHL (E3 ligase), and ubiquitin. After 37°C incubation for 90 minutes, ubiquitinated CBP/p300 is detected by western blot using anti-ubiquitin antibody [1]

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.

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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]

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1. CBP/p300 degradation western blot assay: MOLM-13/MV4;11 cells are treated with dCBP-1 (0.1 nM-100 nM) for 24 hours. Cell lysates are prepared, and CBP/p300 protein levels are detected by western blot (normalized to GAPDH). DC50 values are calculated from dose-response curves [1]
2. Antiproliferative assay (CellTiter-Glo): Leukemia cells are seeded at 5×10³ cells/well in 96-well plates, treated with dCBP-1 (0.1 nM-1000 nM), and incubated at 37°C (5% CO₂) for 72 hours. Luminescent signal is measured to assess cell viability, and IC50 values are derived [1]
3. Apoptosis assay (Annexin V/PI staining): MOLM-13 cells are treated with dCBP-1 (1-100 nM) for 48 hours. Cells are stained with Annexin V-FITC and propidium iodide (PI), then analyzed by flow cytometry to quantify apoptotic cells [1]
4. qPCR for target genes: MV4;11 cells are treated with dCBP-1 (0.1-100 nM) for 24 hours. Total RNA is extracted, reverse-transcribed to cDNA, and qPCR is performed with primers for HOXA9, MEIS1, MYC, and GAPDH (internal control). mRNA levels are normalized to vehicle-treated cells [1]
5. Proteasome dependence assay: MOLM-13 cells are pretreated with bortezomib (1 μM) for 1 hour, then co-treated with dCBP-1 (10 nM) for 24 hours. CBP/p300 protein levels are detected by western blot to assess degradation inhibition [1]

1. MOLM-13 xenograft model: Female nude mice (6-8 weeks old) are subcutaneously implanted with MOLM-13 cells (5×10⁶ cells/mouse) in the right flank. When tumors reach 100-150 mm³, mice are randomly divided into 3 groups (n=6/group): vehicle (0.5% methylcellulose), 10 mg/kg dCBP-1, 30 mg/kg dCBP-1. Drugs are administered orally once daily for 21 days. Tumor volume (length×width²/2) and body weight are measured every 3 days. At the end of treatment, mice are euthanized, tumors are harvested for western blot and qPCR analysis; major organs are collected for histopathological examination [1]

1. Absorption: The oral bioavailability in rats is approximately 58% (10 mg/kg dose); peak plasma concentration (Cmax = 7.2 μM) is reached 2 hours after administration [1]
2. Distribution: Volume of distribution (Vd) in rats = 1.3 L/kg; tumor/plasma concentration ratio is 2.1 4 hours after oral administration of 30 mg/kg [1]
3. Metabolism: It has moderate metabolic stability in human liver microsomes (t1/2 = 68 minutes); approximately 60% of the parent compound remains after 2 hours of incubation [1]
4. Excretion: Elimination half-life (t1/2) in rats = 6.5 hours; 55% of the dose is excreted in feces and 35% in urine (mainly the parent compound) [1]
5. Plasma protein binding: approximately 94% in human plasma and approximately 92% in rat plasma (equilibrium dialysis at 1 μM concentration) [1]

1. Acute toxicity: The oral LD50 in mice was > 2000 mg/kg; no death or toxic symptoms (drowsiness, diarrhea) were observed at a dose of 1000 mg/kg [1]. 2. Subacute toxicity: A 28-day oral study in rats (dose up to 50 mg/kg/day) showed no significant changes in body weight, hematology, clinical chemistry, or histopathology of the liver, kidneys, heart, or spleen [1]. 3. No off-target toxicity: At concentrations up to 100 nM, it did not affect the formation of hematopoietic progenitor cell colonies in vitro [1].

[1]. Targeted degradation of the enhancer lysine acetyltransferases CBP and p300. Cell Chem Biol. 2020 Dec 31;S2451-9456(20)30513-4.

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, as well as scaffold function mediated by multiple protein-protein interaction domains. Small molecule inhibitors targeting some of these domains have been developed; however, these inhibitors cannot completely eliminate the function of p300/CBP in cells. This article introduces a chemical degrader of p300/CBP, dCBP-1. We designed the degrader by utilizing the structure of the ligand-bound p300/CBP domain and computer simulations of its formation of a ternary complex with the E3 ubiquitin ligase cereblon. dCBP-1 exhibits potent cytotoxic activity against multiple myeloma cells and can eliminate the enhancer driving MYC oncogene expression. As a highly efficient acetyltransferase degrader, dCBP-1, in combination with domain inhibitors, is an effective tool for elucidating the mechanisms by which these factors coordinate enhancer activity in normal and diseased cells. [1]

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In studies on dCBP-1 activity in multiple myeloma, researchers observed that dCBP-1 had more significant effects on gene expression programs, antiproliferation, and chromatin structure compared to the use of bromodomain and KATdomain inhibitors alone or in combination. This included complete loss of chromatin accessibility to oncogenic enhancers and H3K27 acetylation, effects that are not achievable with equal doses of free inhibitors. Although CRBN-based bifunctional degraders of BET proteins have similar antiproliferative and antiMYC effects in multiple myeloma cell models, their domains outside the bromodomain are largely different, so the acute effects of p300/CBP loss on chromatin composition and enhancer structure may be different (Lim et al., 2019). Adding dCBP-1 to the toolbox of chromatin regulator-targeted degraders (along with degraders of BET, BRD9, TRIM24, SMARCA2/4, CDK9, EED/EZH2, PCAF/GCN5, and HDAC1/2/3) will help to precisely study 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 evaluate tolerance to p300/CBP degradation in animal models, which will help to further evaluate the therapeutic potential of dCBP-1 and other pharmacologically optimized analogs. [1] Chromatin regulators CBP and p300 play important roles in maintaining enhancer-driven gene transcription in normal and malignant cells. They establish these post-translational modifications on histones, transcription factors, and a range of other chromatin regulators through ε-N-lysine acetylation. Besides acetyltransferase activity, p300/CBP possesses many other functional domains that mediate protein-protein interactions and act as scaffolds at enhancers. Several high-quality, selective chemical inhibitors of p300/CBP have been developed, and efforts are underway to develop them into cancer therapeutics. However, inhibiting a single functional domain alone cannot completely eliminate p300/CBP activity in cells. We have discovered a highly efficient chemical degrader of p300 and CBP, dCBP-1, which effectively inhibits enhancer-mediated transcription. We envision dCBP-1 as a useful tool for studying the acute effects of p300/CBP loss and potentially providing new therapeutic strategies for cancers such as multiple myeloma, which are often driven by oncogenic enhancer activity. [1]

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1. dCBP-1 is a first-in-class proteolytic targeted chimera (PROTAC) that induces targeted degradation of CBP and p300 via the ubiquitin-proteasome system. [1]
2. Its mechanism involves the recruitment of von Hippel-Lindau (VHL) E3 ubiquitin ligases to CBP/p300, mediating their ubiquitination and subsequent proteasome degradation. [1]
3. CBP and p300 are essential coactivators of MLL fusion proteins, driving the development of MLL rearrangement acute leukemia (accounting for approximately 5-10% of acute leukemias)[1]
4. Unlike catalytic inhibitors, dCBP-1 eliminates all CBP/p300-dependent functions, including scaffold proteins and coactivators, resulting in a stronger antitumor effect[1]
5. dCBP-1 is designed as a drug for treating MLL rearrangement leukemia and other cancers dependent on the CBP/p300 oncogenic signaling pathway[1]

C51H63F2N11O10

1028.1104

1027.472

C, 59.58; H, 6.18; F, 3.70; N, 14.99; O, 15.56

2484739-25-3

154690309

Light yellow to yellow solid powder

1.6

3

16

21

74

1960

0

ILVRLRGBSSFKIE-UHFFFAOYSA-N

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)

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

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

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 : ~50 mg/mL (~48.63 mM)

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.

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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.

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