Research Progress of p300/CBP PROTAC Degrader
What are p300 and CBP?
Chrivia et al. discovered a protein that specifically binds to phosphorylated c-AMP reaction element binding protein (CREB), named CREB-binding protein (CBP). p300 is a multidomain macromolecular protein that can interact with adenovirus oncoprotein E1A, and is named p300 because of its molecular weight of 300 KDa. At the amino acid level, the two proteins were highly homologous. Although the two are encoded by different genes, they have very similar amino acid sequences and similar functions, and actually belong to the same protein family. P300/CBP are critical transcriptional coactivators involved in cell cycle progression and cell growth, differentiation and development. Studies have shown that p300/CBP are highly expressed and activated in a variety of different tumors, so p300/CBP and their mechanism of action have attracted more and more attention.
Structure and function of p300/CBP
Structure of p300/CBP
The P300/CBP family is one of the four major histone acetyltransferase (HAT) polygene families in mammals. CBP is composed of 2441 amino acids, p300 is composed of 2414 amino acids, and these two proteins have about 75% sequence similarity and 63% identity.
Organisation of p300/CBP proteins. (A) Comparison of p300 and CBP. (B) The functional domains in p300 are indicated, including the cysteine/histidine-rich domains CH1, CH2 and CH3, the KIX domain, the bromodomain (BRD) and the ADA2 homology region. (Chan, H. M., 2001)
P300/CBP proteins are divided into structured and unstructured regions, of which the unstructured region accounts for more than 50% of the whole, and structured regions include: transcription adapter zinc finger domain 1 (TAZ1), kinase-induced CREB interaction domain (KIX), BRD, plant homology domain (PHD), HAT domain, ZZ type zinc finger domain, and transcription adapter zinc finger domain 2 (TAZ2).
Functions of p300/CBP
Acetylation and deacetylation of histones is a reversible post-translational modification (PTM) that plays a key role in the regulation of gene expression in eukaryotic cells. P300/CBP remodel chromatin by acetylating lysine of histones (H2A, H2B, H3, H4) to alter their structures and affect gene transcription. HAT activity of p300/CBP also acetylates some transcription factors (such as p53), thereby modulating the function of key transcription regulators.
The domains of P300/CBP consist of 110 amino acid residues, and their characteristic structure consists of four α helices (αZ, αA, αB, αC) connected by helical rings, called BRD folds. Proteins with BRD domains are able to read acetyllysine markers associated with epigenetic information transmission and thus play a role in genetic regulation, determining regulation of protein function.
P300/CBP have a wide range of substrates. In order to study the function of P300/CBP, three methods were used to detect protein acetylation after inhibition of p300/CBP. 1. For histone acetyltransferase (HAT), KAT inhibitors (Cmpd-R, A-485) were added; 2. for BRD protein binding functional domain, add BRD inhibitor (CBP112); 3. CBP/p300 gene knockout. In total, the three methods detected more than 20,000 sites of acetylation changes on about 5,300 proteins.
It was also found that CBP/p300 can regulate the acetylation levels of H3K18, H3K27, and H3K36 on H2A, H2B and H3, among which the N-terminal acetylation site of H2B is the most rapidly regulated. CBP/p300 can regulate the expression of downstream genes through acetylation, thus changing the function of proteome.
By transcriptome sequencing, it was found that the KO cells were very consistent with the changes induced by the treatment of the two KAT inhibitors, suggesting that the function of CBP/p300 may be mainly dependent on its acetylation catalytic activity. Further total proteome sequencing showed that KAT inhibitor could completely inhibit downstream gene transcription, while BET inhibitor could only partially inhibit it, indicating that the effect of CBP/p300 on proteome was mainly accomplished through transcriptional events.
p300/CBP and different malignant tumors
p300/CBP are positive regulators of cancer progression and are strongly associated with various human tumor diseases. In colon cancer, human small cell lung cancer, and non-small cell lung cancer, upregulation of p300/CBP is an indicator of poor prognosis. High expression of p300/CBP in breast cancer may promote tumor recurrence and is associated with the aggressive character of breast cancer. In hepatocellular carcinoma, high expression of p300/CBP is associated with enhanced vascular invasion, intrahepatic metastasis, and shortened threshold. In prostate cancer, androgen-induced recruitment of androgen receptor (AR) to chromatin is closely related to the acetylation of H3K27. By blocking the acetylation of H3K27, the function of coactivator on AR is prevented, thereby blocking the expression of key proliferating genes and tumor growth. It shows the potential of p300/CBP inhibitors in the treatment of prostate cancer. Simultaneous mutated p300/CBP is associated with many hematologic malignancies (AML, ALL).
Research progress of p300/CBP PROTAC degraders
p300/CBP are one of the important targets of tumor diseases, and researchers have been working for a long time to design and discover high-quality small molecule inhibitors targeting p300/CBP. However, the currently reported small molecule inhibitors only act on the HAT domain or bromine domain of CBP/p300 alone and may not be able to completely eliminate its carcinogenic function. Therefore, a new strategy is urgently needed to solve this problem. In recent years, protein degradation targeting chimera (PROTAC) technology is a hot research field. The PROTAC molecule recruits both target protein and E3 ligase, which in turn causes the target protein to be ubiquitinated, which is subsequently recognized by the proteasome and triggers degradation. Due to its special mechanism of action, PROTAC molecules can cause the target protein to be degraded and completely lose its carcinogenic activity.
p300/CBP PROTAC for hepatocellular carcinoma
In February 2024, Yingxia Li's group at Fudan University and Xun Huang's group at Lingang Laboratory published a paper entitled "Discovery of Novel PROTAC Degraders of p300/CBP as Potential Therapeutics for Hepatocellular Carcinoma" in the Journal of Medicinal Chemistry. This study reports the novel p300/CBP PROTAC degrader QC-182 and its potential anti-hepatocellular carcinoma activity and mechanism. In this study, CCS1477, the p300/CBP bromine domain inhibitor that has made the fastest progress in clinical trials, was selected as the target protein ligand prototype to build p300/CBP PROTAC degraders. Through the complex crystal structure analysis combined with biochemical experiments, the appropriate junction site was identified and verified. Thus, the PROTAC molecule was constructed and the target protein degradation activity in hepatocellular cancer cells was screened, and the optimal p300/CBP PROTAC depressant QC-182 was finally obtained. The molecule can effectively degrade p300 and CBP proteins in SK-HEP-1 cells through PROTAC mechanism. Sequencing analysis showed that QC-182 significantly down-regulates transcriptome levels regulated by p300/CBP, especially oncogene transcription involved in epithelial-mesenchymal transformation and G2/M phase checkpoint. Compared with the prototype inhibitor CCS1477 and the recently reported depressant dCBP-1, QC-182 has more significant anti-proliferative activity, and can induce apoptosis and G2/M phase arrest. In vivo studies in mice showed that QC-182 could degrade p300 and CBP proteins in tumor tissues of SK-HEP-1 mice. In summary, QC-182 is an effective p300/CBP PROTAC depressant, and further optimization is expected to obtain lead compounds that inhibit the growth of hepatocellular carcinoma and other solid tumors.
Related ligand for target protein at BOC Sciences
Xu Yong's team at Chinese Academy of Sciences, reported a series of novel and highly effective PROTAC inhibitors targeting CBP/p300 for the treatment of acute myeloid leukemia (AML). Through systematic structure-activity relationship studies and a series of biological activity evaluations, the team found the highly active CBP/p300 degrader XYD129. The degrader can effectively bind CBP/p300 and CRBN to form a stable ternary complex. XYD129 inhibited the proliferation of AML cell lines MV4-11 and MOLM-16 with IC50 values of 49 nM and 7.4 nM, respectively, and degraded CBP and p300 proteins through the proteasome pathway. In MOLM-16 mouse xenografted tumor models, XYD129 significantly inhibited tumor growth (TGI = 60%). This study provides a promising drug candidate for the treatment of AML.
XYD129 can effectively degrade CBP and p300 and inhibit tumor growth in vivo.(Wu, T., 2024)
Ligand for CRBN E3 ligase at BOC Sciences
Recent research by Shaomeng Wang's team focuses on the development of a highly effective oral PROTAC molecule CBPD-268 targeting CBP/p300 for the treatment of prostate cancer. CBP/p300 are key co-activators of AR and other transcription factors, and their overexpression is associated with prostate cancer treatment resistance. The experimental design was based on GNE-049 as a CBP/p300 inhibitor and TX-16 as a CRBN ligand. Through multiple rounds of structural optimization, CBPD-268 was finally determined as the optimal molecule. Experimental data showed that CBPD-268 induced deep degradation of CBP/p300 protein in a variety of AR+ prostate cancer cell lines and effectively inhibited cell growth. In mouse and rat models, CBPD-268 showed excellent oral bioavailability and PK properties, with a single oral dose of 0.3-3mg/kg significantly reducing CBP and p300 protein levels in tumor tissue. In a xenograft mouse model of prostate cancer, CBPD-268 achieved tumor regression, exhibited strong antitumor activity, and did not cause significant weight loss or signs of toxicity at multiple doses, showing good tolerance and therapeutic indices. Combined with the experimental results, CBPD-268 has been confirmed as a highly effective, orally effective CBP/p300 depressant with promise as a potential novel therapy for AR-positive prostate cancer and other cancers. The team then replaced the CRBN ligand with GNE-049 as an inhibitor and retained the same ligand site, and found another CBP/p300 PROTAC degrader CBPD-409 as a highly effective, selective and orally effective CBP/ P300 PROTAC for the treatment of advanced prostate cancer.
Using AI-assisted design, Lei Li's team at the First Affiliated Hospital of Xi'an Jiaotong University has successfully developed a peptide-based PROTAC drug that can target and degrade p300 protein. In this study, nano-selenium was used as the delivery system of peptide drugs, which ensured the effective delivery of drugs in vivo and in vitro and the degradation of p300. The peptide PROTAC targeting p300 not only showed significant p300 degradation effect in castration-resistant prostate cancer (CRPC), AR-negative and neuroendocrine prostate cancer (NEPC) cells, but also effectively inhibited the growth of cancer cells. In both AR positive and AR negative mouse models, the drug effectively degraded p300 and inhibited tumor growth. The findings suggest that designing peptide PROTAC drugs that target p300 is an effective therapeutic strategy for CRPC, AR-negative prostate cancer, and NEPC.
With a comprehensive and advanced platform, BOC Sciences provides PROTAC molecular discovery package services to customers around the world to meet new drug discovery goals.
References:
- Chan, H. M., La Thangue, N. B. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. Journal of Cell Science. 2001, 114(13): 2363-2373.
- Chang, Q., et al. Discovery of novel PROTAC degraders of p300/CBP as potential therapeutics for hepatocellular carcinoma. Journal of Medicinal Chemistry. 2024, 67(4): 2466-2486.
- Wu, T., et al. Discovery of a promising CBP/p300 degrader XYD129 for the treatment of acute myeloid leukemia. Journal of Medicinal Chemistry. 2024.
- Chen, Z., et al. Discovery of CBPD-409 as a highly potent, selective, and orally efficacious CBP/p300 PROTAC degrader for the treatment of advanced prostate cancer. Journal of Medicinal Chemistry. 2024, 67(7): 5351-5372.
