Discovery of a peptide proteolysis-targeting chimera (PROTAC) drug of p300 for prostate cancer therapy

Summary

Background

The E1A-associated protein p300 (p300) has emerged as a promising target for cancer therapy due to its crucial role in promoting oncogenic signaling pathways in various cancers, including prostate cancer. This need is particularly significant in prostate cancer. While androgen deprivation therapy (ADT) has demonstrated promising efficacy in prostate cancer, its long-term use can eventually lead to the development of castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC). Notably, p300 has been identified as an important co-activator of the androgen receptor (AR), highlighting its significance in prostate cancer progression. Moreover, recent studies have revealed the involvement of p300 in AR-independent oncogenes associated with NEPC. Therefore, the blockade of p300 may emerge as an effective therapeutic strategy to address the challenges posed by both CRPC and NEPC.

Methods

We employed AI-assisted design to develop a peptide-based PROTAC (proteolysis-targeting chimera) drug that targets p300, effectively degrading p300 in vitro and in vivo utilizing nano-selenium as a peptide drug delivery system.

Findings

Our p300-targeting peptide PROTAC drug demonstrated effective p300 degradation and cancer cell-killing capabilities in both CRPC, AR-negative, and NEPC cells. This study demonstrated the efficacy of a p300-targeting drug in NEPC cells. In both AR-positive and AR-negative mouse models, the p300 PROTAC drug showed potent p300 degradation and tumor suppression.

Interpretation

The design of peptide PROTAC drug targeting p300 is feasible and represents an efficient therapeutic strategy for CRPC, AR-negative prostate cancer, and NEPC.

Funding

The funding details can be found in the Acknowledgements section.

Research in context.

Evidence before this study

Prostate cancer stands as the second leading cause of mortality and the foremost cause of morbidity in males. Androgen deprivation therapy (ADT) serves as the prevailing standard treatment for prostate cancer. The activation of the androgen receptor (AR) signaling pathway by AR coactivators represents a primary contributor to castration resistance. Of these coactivators, the E1A-associated protein p300 (p300) has garnered significant attention due to its pivotal regulatory role in diverse cancer-related genes. Given its involvement in prostate cancer and its potential as a therapeutic target, p300 has become an area of profound interest. Peptide PROTAC drugs present notable advantages in drug design, including heightened specificity, enhanced efficacy, and reduced toxicity. These advantages emanate from their capacity to leverage larger interacting surfaces, thereby surmounting the limitations associated with the shallow binding pockets typically encountered in small molecular drugs.

Added value of this study

Our research introduces a peptide PROTAC that successfully targets the cysteine-histidine-rich 1 (CH1) domain of p300, resulting in the degradation of p300. In contrast to previous p300 PROTAC drugs, our approach utilizes E3 ubiquitin-protein ligase Mdm2 (MDM2) as an E3 enzyme, which not only facilitates p300 degradation but also promotes the release of tumor suppressor p53 (p53), thereby augmenting the therapeutic effect in cancer treatment. We demonstrate the efficacy of a p300-targeting PROTAC drug in diverse cell types in vitro, including AR-positive, castration-resistant prostate cancer (CRPC), and neuroendocrine prostate cancer (NEPC) cells. Moreover, our p300 peptide PROTAC drug exhibits notable efficacy in both AR-positive (CWR22Rv1) and AR-negative (PC-3) xenograft models. Besides, the drug's safety profile was assessed. These results indicate our p300 peptide PROTAC presents a promising avenue for prostate cancer therapy, holding considerable value.

Implications of all the available evidence

We have developed a peptide PROTAC that targets the CH1 domain to efficiently degrade p300, offering a promising solution for drug development targeting p300. Our drug demonstrates substantial efficacy in treating prostate cancer, including potent effects against neuroendocrine prostate cancer (NEPC) in vitro, irrespective of its dependence on the androgen receptor (AR) signaling pathway. This breakthrough holds great potential for improving the treatment of prostate cancer by targeting p300.

Introduction

Despite significant advancements in cancer treatment, it remains one of the most formidable challenges to human health.^1,2 To address this, scientists are exploring new targets and treatment strategies to develop effective drugs for cancer therapy.3, 4, 5 The E1A-associated protein p300 (p300) has garnered increasing interest among researchers due to its pivotal role in gene transcription and its close association with tumor progression.6, 7, 8, 9, 10 Extensive research endeavors have consistently revealed the heightened expression of p300 in tumor cells, also in drug-resistant populations.¹¹ Notably, p300 plays a pivotal role in the transcriptional activation of oncogenes, exerting substantial influence on various critical aspects of tumor biology including survival, proliferation, metastasis, immune evasion, drug resistance and so on.12, 13, 14, 15, 16 The p300 protein is comprised of several domains, including the acetyltransferase (HAT) domain¹⁷ and the bromodomain (BRD),¹⁸ which are crucial for its acetyltransferase activity. The HAT domain transfers acetyl groups from Ac-CoA to histone lysine residues,¹⁹ while the BRD recognizes and binds acetyl-lysine residues to facilitate transcriptional complex recruitment, ultimately enhancing the activity of transcription factors and downstream signaling pathways.²⁰ Given p300's vital role in regulating onco-signaling pathways, researchers have been actively developing small molecule inhibitors and degraders targeting p300/CBP (CREB-binding protein).^7,21, 22, 23, 24 Currently, the small-molecule inhibitor of p300/CBP, Inobrodib (CCS1477), is in clinical phase I/IIa trials for patients with advanced drug-resistant prostate cancer.^25,26 And FT-7051, another inhibitor of p300/CBP, is ongoing a multi-center, phase I, open-label clinical trial in patients with metastatic castration-resistant prostate cancer.²⁷ Combined with the existing studies, p300 has been emerging as a potential therapeutic target for cancer.

In the context of prostate cancer, p300 assumes particular significance, as numerous studies have consistently demonstrated its heightened expression within prostate cancer cells.^23,28,29 The current primary treatment approach for prostate cancer remains androgen deprivation therapy (ADT).³⁰ Unfortunately, the long-term use of castration therapy in prostate cancer treatment inevitably leads to the development of drug resistance, resulting in the progression to castration-resistant prostate cancer (CRPC).^31,32 Even more concerning is the potential induction of neuroendocrine prostate cancer (NEPC), an aggressive and refractory form of prostate cancer characterized by high mortality rates.33, 34, 35, 36 The overexpression and hyperactivation of p300 have been identified as significant contributors to the development and progression of castration-resistant prostate cancer (CRPC).^11,37,38 As a well-established AR coactivator, p300 primarily initiates downstream transcription of the AR by participating in the formation of the AR activation complex.39, 40, 41 In addition to its involvement in the androgen receptor (AR) pathway, p300 is implicated in various other critical signaling pathways related to DNA damage repair, WNT-β catenin, and other tumor-related pathways.42, 43, 44, 45 Notably, several key oncogenes and pathways regulated by p300, including EZH2, MYCN, and FOXA2, have been implicated in the development of neuroendocrine prostate cancer (NEPC).46, 47, 48, 49 These findings suggest that inhibitors or degraders targeting p300 could be effective treatment options for NEPC, thereby highlighting the therapeutic potential of targeting p300 in this aggressive form of prostate cancer.

Proteolysis Targeting Chimera (PROTAC) has emerged as a promising technology for protein degradation in drug development. This innovative approach facilitates the targeted degradation of specific proteins of interest (POIs), presenting new opportunities for therapeutic interventions.^50,51 To date, several small molecular PROTAC drugs named JQAD1 and dCBP-1, and CBPD-268 have been reported for targeting p300,^21,24,52 all of which primarily target the bromodomain or HAT domain, the structure homologous to both p300 and CBP. Consequently, these drugs may induce degradation of both p300 and CBP to a similar extent. Also, these PROTACs leverage the E3 ligase cereblon (CRBN) to promote the degradation of p300. The capacity to degrade p300 may be comparable in both cancerous and normal cellular contexts.

Given the considerable homology in the structures of p300 and CBP, both proteins hold significant importance in transcriptional processes within normal cellular functions. The development of cancer-specific and selectively targeted p300-degrading drugs remains an area yet to be adequately addressed. The peptide PROTACs are crafted by leveraging the interaction surface, thus circumventing the constraints encountered by small molecule PROTACs, which necessitate shallow binding pockets.^53,54 In contrast to previously reported p300 PROTAC drugs, our design focuses on a peptide antagonist sequence specifically targeting the cysteine-histidine-rich 1 (CH1) domain of p300, distinct from the HAT and BRD domains. To achieve the selective degradation of p300 within cancer cells, we have employed MDM2, an E3 ligase known for its high expression in prostate cancer cells, as the basis for our PROTAC drug design.55, 56, 57, 58 Previous studies have demonstrated the efficacy of selecting E3 ligases that are overexpressed in cancer cells for achieving targeted protein degradation.⁵⁵ Additionally, our design involves partially binding MDM2 at the same interface utilized by p53. Consequently, the peptide PROTAC drug developed could inhibit the MDM2/p53 interaction and release p53. Through the integration of these three design aspects, we have successfully developed a comprehensive p300-targeted PROTAC design. This innovative approach, encompassing the specific targeting of the CH1 domain, the utilization of MDM2 as an E3 ligase for p53 release, and the use of nano-selenium as a drug delivery vehicle, represents a significant advancement in p300-targeted therapy.

To evaluate the efficacy of our PROTAC drug, we assessed its capacity to degrade p300 and induce cell death in AR-positive prostate cancer cells, such as C4-2 and CWR22Rv1 cells. Furthermore, we investigated the potential of our p300-targeted PROTAC drug in AR-negative prostate cancer and NEPC cells, validating our hypothesis regarding its therapeutic applicability. Additionally, RNA-Seq analysis of NE 1.3 cells treated with our p300 PROTAC drug unveiled its potential mechanism of action in NEPC, revealing significant induction of apoptosis and modulation of immune-related pathways. Moreover, our PROTAC drug exhibited favorable efficacy and safety in the PC3 and CWR22Rv1 graft tumor models. In conclusion, our study underscores the effectiveness and safety of the p300 PROTAC drug in prostate cancer cells, encompassing advanced ADT-resistant and NEPC cells.

Methods

Cell culture

C4-2(RRID: CVCL_4782), 22Rv1(RRID: CVCL_1045), PC3(RRID: CVCL_0035), and DU145(RRID: CVCL_0105) cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 5% saturated humidity of CO2, 37 °C, and cell culture density was up to 90% confluency. NE 1.3 cells were kindly provided by Dr Jun Qin (Chinese Academy of Sciences). The culture conditions of the NE 1.3 cell line remain unchanged except for the replacement of the serum in the above conditions with 10% androgen-removing FBS. 293T (RRID:CVCL_0063) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. H660 (RRID: CVCL_1576) were purchased from ATCC cultured in RPMI 1640 containing 5% FBS and added 0.005 mg/ml Insulin, 0.01 mg/ml Transferrin, 30 nM Sodium selenite (final conc.), 10 nM Hydrocortisone (final conc.),10 nM beta-estradiol (final conc.), extra 2 mM l-glutamine (for final conc. of 4 mM). RWPE-1 (RRID: CVCL_3791) was purchased from ATCC cultured in Keratinocyte Serum Free Medium (K-SFM) and added 0.05 mg/ml BPE and 5 ng/ml EGF provided with the K-SFM kit. All cell lines were recently validated with STR analysis, and confirmed to be mycoplasma-free by EZ-Detect™ Kit (Ubigene Biosciences, #YK-DP-20, Guangzhou, China).

Plasmids

HA-p300 was purchased from MiaoLing Plasmid and Flag-MDM2 and His-ub was purchased from Addgene. pLKO-shP300 was customized from Xi'an Biokeeper Biotechnology Co., Ltd. All si-RNA used was customized from GenePharma.

Antibodies

Anti-p300 (Cell Signaling Technology Cat# 54062, RRID: AB_2799450) antibody and Anti-p300 (Cell Signaling Technology Cat# 86377, RRID: AB_2800077), Anti-CBP (Cell Signaling Technology Cat# 7389, RRID: AB_2616020), Anti-EZH2 (Cell Signaling Technology Cat# 4905, RRID: AB_2278249), Anti-N-Ca (Cell Signaling Technology Cat# 13116 (also 13116T, 13116S), RRID: AB_2687616), Anti-MMP9 (Cell Signaling Technology Cat# 13667, RRID: AB_2798289), Anti-Vimentin (Cell Signaling Technology Cat# 5741 (also 5741S, 5741P), RRID: AB_10695459), Anti-ZEB1 (Cell Signaling Technology Cat# 3396 (also 3396S, 3396P), RRID: AB_1904164), Anti-puma (Cell Signaling Technology Cat# 4976, RRID: AB_2064551), Anti-HA-Tag (Cell Signaling Technology Cat# 3724 (also 3724S), RRID:AB_1549585) antibodies were purchased from cell signaling technology (CST). Anti-vinculin antibody (Sigma-Aldrich Cat# V4505, RRID: AB_477617) and Anti-HA agarose beads (Sigma-Aldrich Cat# A2095, RRID: AB_257974) were purchased from Sigma-Aldrich. Anti-p53 (Santa Cruz Biotechnology Cat# sc-126, RRID:AB_628082), Anti-KLK3 (Santa Cruz Biotechnology Cat# sc-7316, RRID:AB_2279058), Anti-AR (Santa Cruz Biotechnology Cat# sc-7305, RRID:AB_626671) were purchased from Santa Cruz. Anti-Noxa (Millipore Cat# OP180, RRID: AB_2268468) was purchased from Calbiochem. Anti-MDM2 (Proteintech Cat# 27883-1-AP, RRID:AB_2881003), Anti-p21 (Proteintech Cat# 10355-1-AP, RRID:AB_2077682), Anti-FOXA2 (Proteintech Cat# 22474-1-AP, RRID:AB_2879110) was purchased from proteintech. Anti-FOXA1 (GeneTex Cat# GTX100308, RRID: AB_1240823) was purchased from GeneTex. The secondary anti-mouse antibody (ABclonal Cat# AS003, RRID:AB_2769851), secondary anti-rabbit antibody (ABclonal Cat# AS014, RRID:AB_2769854), Anti-MMP2 (ABclonal Cat# A6247, RRID:AB_2766854), Anti-C-MYC (ABclonal Cat# A1309, RRID:AB_2759938), Anti-N-MYC(ABclonal Cat# A22174), Anti-K48-linkage Specific Polyubiquitin (ABclonal Cat# A18163, RRID:AB_2861948), Anti-K63-linkage Specific Polyubiquitin (ABclonal Cat# A18164, RRID:AB_2861949), Anti-GAPDH (ABclonal Cat# AC033, RRID:AB_2769570) and Anti-β-Actin Rabbit mAb (High Dilution) (ABclonal Cat# AC026, RRID:AB_2768234) were purchased from ABclonal. All antibodies were used at the concentration recommended by the instructions in 3% bovine serum albumin (BSA) in Tris-HCl and tween 20 (TBST) buffer for Western blots.

Reagents and drugs

Insulin (human) (TargetMol Cat# T8221), Hydrocortisone (TargetMol Cat# T1614), β-Estradiol (TargetMol Cat# T1048), dCBP-1 (TargetMol Cat# T9370), JQAD1 (TargetMol Cat# T41180) and I-CBP112 (TargetMol Cat# T3969) was purchased from TargetMol. Sodium selenite (Sigma-Aldrich Cat# S5261), L-glutamine (Sigma-Aldrich Cat# G7513) and MG-132 (Sigma-Aldrich Cat# 474791) were purchased from Sigma-Aldrich. Transferrins (MedChemExpress Cat# HY-P3267) and Cycloheximide (MedChemExpress Cat# HY-12320) were purchased from MedChemExpress.

All reagents and drugs were dissolved and stored in the recommended solvents according to the manufacturer's instructions, and then diluted to the required concentrations for subsequent cell culture or drug treatment experiments.

Peptide drug design

Based on published interaction interfaces between p300 and CITED2 complex structures, virtual screening was performed using Rosetta software and an online server to screen out the peptide that strongly interacted with p300. In brief, 10 interface sites were defined at which mutations to all 20 naturally occurring amino acids (except proline) will be allowed in the simulations. The overall interface binding score and fold stability score of each protein chain of the native protein–protein complex were first calculated by Rosetta. Finally, the frequency of each amino acid being included in the standby at that site was counted, and the amino acid with the highest frequency at each interface site was selected as the specific selected amino acid at that site for generating the final peptide sequence.

Fluorescence polarization assay

The p300 CH1 domain and MDM2 N-terminal domain proteins were expressed by E. coli. Additionally, the peptide p300 PROTAC drug was labeled with rhodamine to detect fluorescence. To explore the interaction between the peptide p300 PROTAC drug and p300/MDM2, rhodamine-labeled peptide p300 PROTAC drug was loaded into a 384-well plate at a concentration of 10 nM per well. Subsequently, we systematically decreased the concentration of p300/MDM2 from 10 μM to 10 nM via equal dilution steps. Following a 30-min incubation period, fluorescence polarization values were measured using a Tecan M1000 microplate reader. Affinity values were calculated using GraphPad software.

Stability serum resistance

The p300 PROTAC peptide and nano-conjugated p300 PROTAC drug (40 μl of 1 mg/ml stock solution in dH2O) were combined with 20% FBS (60 μl) and incubated at 37 °C in a standard laboratory incubator. At each designated time point, a 40 μl aliquot was withdrawn, and an equal volume of acetonitrile (ACN) was added for subsequent LC-MS analysis.

CCK-8 cell viability assay

For cell viability assays, cells were seeded into 96-well plates at an appropriate density and multiple wells were set. After the cells adhered to the plates, different drugs, and different concentrations were given. After 48 h of drug treatment, CCK8 reagent was added, and after 2 h of reaction, the absorbance at 450 nm excitation wavelength was measured using a microplate reader.

Colony formation assay

The cells were cultured in a 6-well plate and treated with different concentrations of p300 PROTAC drug for 20 days after the cells were attached to the plates for 48 h. After the treatment, the cell-cultured plates were removed from the incubator and washed with PBS 3 times, then fixed with 1 ml 4% paraformaldehyde for 30 min, then stained with 2 ml 0.1% crystal violet for 30 min, and finally cleaned with PBS 3 times to remove the residual dye. Then, under the developer, the picture was taken and the amount of clone formation was counted by image j.

Wound healing assay

The migration ability of PC3 and DU145 prostate cancer cells with a density of 100% was determined by the wound healing assay. A 200 μl pipette was used to mark the distance, and then PBS was used to flush the floating cells. The p300 PROTAC drug was dissolved in a serum-free medium and added into 6-well plates with 2 ml per well. Photographs were taken under the microscope after 24 h and 48 h to observe the healing of the wound.

Transwell invasion assays

For transwell invasion assays, a 45 μl mixture of Matrigel (Corning Cat#354234)/FBS-free RPMI-1640 at 1:8 ratio was plated onto the upper chamber. After 4 h for the mixture to set, prostate cancer cells in 250 μl serum-free RPMI-1640 absence or presence of p300 peptide PROTAC were added into the upper chamber and 750 μl medium containing 10% FBS was added in the lower chamber. After incubated for 36 h, the chamber was washed by PBS and fixed with 4% formalin at room temperature for 15 min. Subsequently, the chamber was stained with 0.1% crystal violet for 20 min at room temperature. An inverted light microscope was used to count the number of cells which had invaded in 3 randomly selected fields.

Apoptosis detection by flow cytometry

The ability of the p300 PROTAC drug to promote apoptosis in prostate cancer cells was evaluated by flow cytometry using an apoptosis detection kit (Biolegend Cat# 640932).

Cells were seeded at a density of 3 × 10⁵ cells/ml in 6-well plates and treated with the p300 PROTAC drug, while a none-treated control, nano vehicle control, and peptide control. After 24 h incubation, cells were collected and strictly followed the procedures required by the apoptosis detection kit. After that, the apoptotic cells were analyzed by FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) based on the principle of fluorescence-activated cell sorting.

Stable transfectants, and transfection

All transfection steps were strictly conducted following the recommended protocols and transfection reagents (PEI or Lipofectamine 2000) as specified in the instruction manuals.

For lentivirus infection, the packaging vectors (pMD2.G and psPAX2) and PLKO-shRNA were first co-transfected into 293T cells, and the medium containing the virus was collected 48 h later. After filtration with a Millex®-HV Filter Unit (Sterile), polybrene was added at the concentration of 4 μg/ml, and the virus-containing medium was used for transfection of the target cells. After transfection, cells were cultured with fresh medium and selected with 1.5 μg/ml puromycin for about 1 week. When no obvious cell death was observed, the stable cell lines were considered successfully established.

Immunoblotting analysis

When the sample was to be collected, the medium of the cells was discarded and washed three times with PBS. After removal of residual PBS, radioimmunoprecipitation assay (RIPA) buffer (Beijing Dingguo Changsheng Biotechnology Co, Ltd Cat# WB-0072) containing protease inhibitors (MedChemExpress Cat# HY-K0010) and phosphatase inhibitors (MedChemExpress Cat# HY-K0021, Cat# HY-K0022) was added and lysed for 5 min on ice. Subsequently, the lysate was scraped with the cell scraper and transferred to a 1.5 ml micro centrifuge tube, centrifugated at 13,000×g, 4 °C for 15 min. The precipitation was discarded and the supernatant was transferred to a new tube. 5 μl of the supernatant was used for BCA protein quantification (Thermo Fisher Cat# 23225), and the rest was added 5× DualColor Protein loading Buffer (Fudebio-tech Cat# FD002), then boiled at 100 °C for 5 min to obtain the protein samples. Based on BCA quantification, equal amounts of protein samples were subjected to SDS-PAGE on a 6%, 8% or 10% Tris-glycine gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Cat# IPVH00010). After completion of the transfer, the membranes were blocked with 5% milk for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. The next day, after antibody withdrawal, the membrane was washed three times with TBST for 5 min each time, then incubated with secondary antibody at room temperature for 1 h. After completion of incubation, the membrane was washed three more times with TBST and subsequently detected and visualized by the ECL detection system (BIO-RAD ChemiDoc XRS+).

All Western blot analysis in this study, except for special labeling, the treatment of drugs was 24 h, and samples that plasmid transfected were collected after 48 h. The concentration of CHX used was 50 μg/ml, and the duration of CHX treatment is shown in the figure. MG-132 was used at the concentration of 10 μM for 24 h or 20 μM for 6 h before the sample collection.

Co-immunoprecipitation

After being transfected with the specific plasmid, the cells were lysed with IP buffer containing protease inhibitors and phosphatase inhibitors on ice for 15 min. And then it's centrifuged at 13,000×g, 4 °C, for 15 min. The supernatant was then incubated with anti-HA agarose beads for 12 h at 4 °C in the absence or presence of p300 peptide PROTAC. Subsequently, cell lysates were washed with IP buffer and the proteins were extracted from the beads by adding protein loading Buffer and boiling for 5 min. The protein samples then subjected to Western blot analysis.

Intracellular ubiquitination assays

The His-ub and other corresponding plasmids were co-transfected into 293T cells. After 48 h transfection, 1 μM of the p300 PROTAC drug was added to further treat for 24 h. Meanwhile, 20 μM MG-132 was added 6 h before collection. The collected cell samples were placed on ice and lysed in IP buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; and 0.1% NP-40) containing protease inhibitors and phosphatase inhibitors for 10 min, followed by sonication for 15 s. Subsequently, the lysates were centrifuged at 13,000×g, 4 °C, for 15 min. The supernatant was collected, anti-HA agarose beads were added and incubated overnight with rotation. The next day, the beads were washed three times with IP buffer. Protein loading Buffer was then added to the protein bounded beads according to the volume. After boiled for 5 min, the prepared protein samples will be used for subsequent immunoblotting analysis.

The ubiquitin degradation pathway, which p300 peptide PROTAC drug induced, was detected by using anti-K48-linkage Specific Polyubiquitin and anti-K63-linkage Specific Polyubiquitin antibodies.

Confocal fluorescence microscopy and immunofluorescence staining

The cells were seeded on glass confocal plates and treated accordingly. Upon sample collection, cells were washed twice with PBS, followed by fixation with 4% paraformaldehyde for 15 min. Subsequently, cells were permeabilized with 0.1% Triton X-100 and incubated with primary antibodies overnight at 4 °C. The next day, cells were incubated with fluorescent secondary antibodies at room temperature for 1 h, mounted with glycerol, and stained with DAPI. Observations and photographs were taken under a confocal microscope.

RNA-seq and data analysis

Three independent replicates of the experiment were performed for each group. After 24 h of treatment with 800 nM PROTAC for C4-2 or 1 μM for NE1.3, cells in the experimental and control groups were lysed with Trizol and followed by immediate liquid nitrogen flash freezing for subsequent storage and delivery. Library preparation and sequencing analysis were performed by Genenergy (Shanghai).

Analysis of differentially expressed genes (DEGs) in bulk sequencing. Preprocessing of the expression profile from bulk sequencing included background correction, gene symbol transformation and normalization using RStudio programming. Significant DEGs in these datasets were found using the limma package (version 3.48.3, Bioconductor). A significant change is defined by a q value <0.05 and | log2FC | ≥1.8.

Gene ontology (GO), gene set enrichment analysis (GSEA). Candidate genes resulting from bulk sequencing analysis analysis were used to conduct GO analyses, using the clusterprofiler package (v.4.8.1, Bioconductor). Gene sets were evaluated according to the hallmark and KEGG gene sets in the MSigDB database (gsea-msigdb.org/gsea/msigdb/index.jsp). The results met the requirements, with an adjusted p-value of <0.05. Furthermore, the GSEA was utilized to identify the enriched pathways from the previous analyses of bulk sequencing.

Total RNA extraction and quantitative real-time polymerase chain reaction (qRT‒PCR) analysis

Total RNA was isolated using an RNAfast200 kit (Fastagen Cat# 220010). cDNA was synthesized using PrimeScript RT Master Mix (TAKARA Cat# RR036A), and qRT-PCR was performed with TB Green® Premix Ex Taq™ II (TAKARA Cat# RR820A) and a CFX96 detection system (Bio-Rad). The primer sequences for target genes were listed in Supplementary Data (Table 1).

Immunohistochemical analysis

Tumor tissue specimens from mice were stained using the standard immunohistochemistry (IHC) protocol and images were captured using a Leica SCN400 microscope. Each image was scored according to the following criteria: according to the intensity of cell staining, the score was 4 grades, 0 for no positive staining (negative), 1 for light yellow (weakly positive), 2 for brown (positive), and 3 for tan (strongly positive). Meanwhile, according to the percentage of positive cells, the score was graded on a scale of 4 levels, with ≤25% scored as 1, 25%–50% scored as 2, 50%–75% scored as 3, and >75% scored as 4. The final score was calculated by multiplying these two scores.

Experimental therapy of xenograft mouse models

For AR-negative cell line PC-3 xenograft studies, the 25 4-week-old BALB/c nude mice (male) were randomly separated into 5 groups (5 mice/group), and then 8 × 10⁶ PC-3 cells were subcutaneously injected into the right shoulder. We attempted to start drug treatment (i.e., day 1) after the tumors became palpable (around ∼ 200 mm³) in 3 weeks. Each group of mice received an intraperitoneal (i.p.) injection every other day for 18 days in the five treatment groups. The tumor length and width were measured by the equation: tumor volume (V) = length × width²/2. All mice were euthanized at the end of the experiment. Mice heart, liver, spleen, kidney, and tumor tissue sections were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining, and immunostaining of Ki-67 and Tunel.

For AR-positive cell line 22Rv1 xenograft studies, the operation is basically the same as the above steps. Briefly, the 35 4-week-old BALB/c nude mice (male) were randomly separated into 7 groups (5 mice/group), and then 6 × 10⁶ 22Rv1 cells 1:1 mixed with Matrigel (Corning Cat# 354234) were subcutaneously injected into the right shoulder. After tumor formation, the drugs were administered via intraperitoneal (i.p.) injection every other day for 21 days in each group. After measuring and recording tumor volume growth, the mice were euthanized. Tissues were then subjected to immunohistochemical staining using corresponding antibodies or H&E staining.

Biodistribution and toxicity analyses

Tumor-bearing mice were injected intraperitoneally with p300 PROTAC drug labeled with rhodamine B at different time points, resulting in drug exposure durations of 0, 12, 24, 48, and 72 h respectively. Subsequently, the mice were euthanized, and the hearts, livers, spleens, lungs, and kidneys were dissected out. Imaging was immediately performed using a small animal in vivo fluorescence imaging system to assess the biodistribution of the drug based on the fluorescence intensity.

20 normal 4-week-old BALB/c mice without bearing tumors were randomly and equally divided into 4 groups. Each group received the drug at a dose of 3 times of therapeutic index every other day for 21 days. Then, blood samples were collected from the orbital venous plexus for toxicity testing of biochemistry such as liver and renal function. After euthanasia, each organ of the mice was dissected out and fixed in 4% paraformaldehyde for HE staining to observe if there were any drug-induced toxicity.

Ethical approval

All experimental procedures involving animals were conducted by institutional guidelines and were approved by the Laboratory Animal Center and Biomedical Ethics Committee of Xi'an Jiaotong University. The approval number is NO:2022-1302.

Statistics

All data are expressed as the mean ± SD, and experimental group data were normalized to the non-treated group. Statistics were generated using GraphPad Prism software. p-values of 0.05 or less were considered statistically significant for single comparisons.

Role of the funding source

The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Results

P300 peptide PROTAC drug design and nano delivery system construction and evaluation

PROTAC drugs function by mediating targeting protein interaction with an E3 ligase, peptide sequence targeting p300 and E3 ligase-MDM2 was designed, respectively. A 15-amino acid leading peptide sequence was derived from the p300/CITED2 protein complex structure (PDB: 1p4q) (Figs. 1a and S1). A hot-spot amino acid screening with Rosetta was performed to achieve a higher-affinity p300 targeting peptide sequence. As shown in Fig. 1b and c, we achieved a p300 targeting peptide sequence: PWIWDGDNKDDNSTD by Rosetta. To design the p300 targeting peptide PROTAC drug, the MDM2 targeting peptide sequence was used as our previous publication and GGGSGGG was used as a flexible linker sequence.^59,60 Taken together, the p300 peptide PROTAC drug sequence is PWIWDGDNKDDNSTDGGGSGGGTSFEQFWAWLWP. MDM2 is not only a broad E3 enzyme in cells but also functions as an oncoprotein by degrading p53. Our p300 targeting PROTAC drug design uses homologous sequences targeting MDM2/p53, thus releasing p53 while utilizing the E3 enzymatic activity of MDM2. The p53 protein has also been reported to be an important therapeutic target for cancer therapy including prostate cancer, even for neuroendocrine prostate cancer.⁶¹ Compared with the p300 PROTAC drug that has been reported, our p300 targeting PROTAC drug design not only degrades p300 but also releases p53. To realize the subsequent coupling of p300 peptide PROTAC drugs with nano vehicle systems, cysteine was added to the N terminal of the peptide sequence to utilize the sulfhydryl of cysteine. The final p300 peptide PROTAC drug sequence is CPWIWDGDNKDDNSTDGGGSGGGTSFEQFWAWLWP.

Fig. 1.

P300 PROTAC drug design and nano delivery system construction and evaluation. (a) The protein complex structure of p300 with a 15 amino acids peptide derived from CITED2 (PDB: 1p4q). (b) Amino acids of p300 targeting peptide which is ranked individually for each sequence position by computationally predicted frequency. The original peptide residues are shown in red. (c) Sequence logos of predicted sequence mutations for p300 targeting peptide. (d) The binding affinities between p300 PROTAC and p300/MDM2 were detected by fluorescence polarization, respectively. (e) Immunoblotting analysis of whole cell lysis and anti-HA agarose immunoprecipitate from 293T cells transfected with the indicated plasmids - MDM2 and p300, with or without p300 PROTAC drug treatment. (f) Hydrodynamic diameter analysis of p300 PROTAC drug by Malvern laser particle size analyzer. (g) Transmission electron microscope (TEM) analysis of p300 PROTAC drug. (h) The serum resistance of the p300 PROTAC drug was tested in PBS containing 10% fetal bovine serum. The half-life of the p300 PROTAC drug is 10.19 h. (i) Confocal analysis of C4-2 cells incubated with 500 nM p300 PROTAC drug with rhodamine (red). The cell nucleus was stained with DAPI (blue). All images were acquired with the same excitation wavelength and detector gain settings (scale bar: 50 μm).

Fluorescence polarization assays are widely used to detect the binding affinity between drugs and their protein targets. In this study, the p300 PROTAC drug was labeled with rhodamine to detect its binding affinity to p300/MDM2 using fluorescence polarization assays. The principle of this assay is based on the fact that the fluorescence polarization value increases with the increase in molecular weight of the bound complex. The results of the fluorescence polarization assay showed that the p300 PROTAC drug has a binding affinity of 78.3 nM and 101.4 nM to p300 and MDM2, respectively (Fig. 1d). The binding ability of our drugs to MDM2 was also demonstrated in the isothermal titration calorimetry (ITC) experiment (Fig. S2). To investigate the binding ability between p300 and MDM2 in the presence of the p300 PROTAC drug, immunoprecipitation experiments were performed in 293T cells transfected with HA-p300 or Flag-MDM2 plasmids with or without the p300 PROTAC drug. The results showed that p300 is weakly bound to MDM2 under natural conditions, but the binding ability was significantly enhanced in the presence of the p300 PROTAC drug, indicating the rationality of the p300 PROTAC drug design (Fig. 1e).

The use of a nano-selenium system as a delivery vehicle for the p300 PROTAC drug has overcome the poor stability and cell membrane penetrating ability of peptide drugs.

As shown in Fig. 1f and g, after conjugating with the nano-selenium system the p300 PROTAC drug maintains a uniform nanoparticle size of around ∼178 nm. The stability detection proved that the p300 PROTAC drug coupled with nano-selenium has high stability which could function effectively in vivo (Fig. 1h). To detect the cell-penetrating ability of the p300 PROTAC drug, confocal microscopy was used to detect the internalization of the p300 PROTAC drug. The positive surface charge of nano-selenium could help the p300 peptide PROTAC drug enter cells (Fig. S3). As shown in Figs. 1i, S4 and S5, confocal microscopy and flow cytometer analysis have confirmed the internalization of the p300 PROTAC drug in a time-dependent manner in prostate cancer cells.

The p300 degradation ability detection of the p300 PROTAC drug

In order to evaluate the efficacy of the p300 PROTAC drug in degrading p300, we conducted immunoblot analysis of p300 in AR-positive and AR-negative prostate cancer cells following treatment with the p300 PROTAC drug. Our results, depicted in Fig. 2a, demonstrate that the p300 PROTAC drug effectively degraded p300 in both AR-positive prostate cancer cells (C4-2 and CWR22Rv1) and AR-negative prostate cancer cells (DU145 and PC-3). Consistent with these findings, the p300 PROTAC drug exhibited strong inhibition of proliferation in both AR-positive and AR-negative prostate cancer cells, while the free peptide control and nano vehicle showed no toxicity, as shown in Fig. 2b–e. Immunofluorescence staining of p300 in C4-2 cells after treatment with the p300 PROTAC drug confirmed its ability to degrade p300, as illustrated in Fig. 2f. Additionally, the proteasome inhibitor MG132 was able to prevent the degradation of p300 by the p300 PROTAC drug, indicating that its function is mediated by the proteasome pathway, as depicted in Fig. S6. To further evaluate the impact of the p300 PROTAC drug on the half-life of p300, we performed Western blot analysis on cells treated with cycloheximide (CHX). Our results, shown in Fig. 2g and h, demonstrate that the p300 PROTAC drug induced the degradation of p300 in a time-dependent manner, effectively accelerating its degradation.

Fig. 2.

The p300 degradation ability detection of the p300 PROTAC drug. (a) Immunoblotting analysis of p300 in both AR-positive and AR-negative prostate cancer cells treated with the indicated concentrations of p300 PROTAC drug for 24 h. (b–e) Cell viability assay of AR-positive and AR-negative prostate cancer cells after 48 h of treatment with indicated concentrations of the p300 PROTAC drug, nano vehicle (Se nanoparticles without loading p300 peptide PROTAC), and free peptide control. The nano vehicle and free p300 PROTAC peptide groups showed no toxicity in the prostate cancer cells, while the p300 PROTAC drug showed dose-dependent growth inhibition in the prostate cancer cells. (f) Immunofluorescence staining of p300 (green) in C4-2 cells after p300 PROTAC drug treatment at different time points. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. (g and h) Immunoblotting assay was applied to detect p300 protein degradation after treatment with CHX, with or without 500 nM p300 PROTAC for the different time points (0, 2, 4, 8, 12, 24 h). The signal of p300 protein intensity in PC-3 and C4-2 cells normalized to vinculin were quantified separately.

To evaluate the advantages of the PROTAC drug we designed, we investigated the efficacy and specificity of dCBP-1 and JQAD1 in prostate cancer cells. Surprisingly, JQAD1 exhibited poor performance in prostate cancer cell lines. Although dCBP-1 demonstrated effective p300 degradation, it also resulted in a reduction of CBP. However, the potency of our peptide PROTAC drug in reducing CBP was notably weaker compared to its effect on p300 (Fig. S7).

To further address the specificity of p300 PROTAC drug in prostate cancer cells, we conducted an overlap analysis of the differentially expressed genes (DEGs) affected by p300 knockdown and PROTAC drug treatment in C4-2 and NE1.3 cell lines (Fig. S8). Our analysis revealed a significant overlap, with nearly 50% DEGs caused by p300 knockdown observed overlap in C4-2 cells and 70% in NE1.3 cells. This finding underscores the specificity of the p300 PROTAC drug treatment and provides additional evidence supporting its therapeutic potential.

The p300 PROTAC drug relies on ubiquitination mediated by MDM2 and showed a potent ability to cure prostate cancer in vitro

To elucidate the mechanism and specificity of the p300 PROTAC drug, our initial investigation aimed to determine whether this drug could enhance the binding of MDM2 to p300. As depicted in Figs. 1e and 3a, our findings demonstrate that the p300 PROTAC drug effectively augments the interaction between MDM2 and p300. Subsequent concurrent knockdown of MDM2 inhibited the degradation of p300 induced by p300 PROTAC drug (Fig. 3b). Building upon these observations, we further determine the ubiquitin degradation pathway of the p300 PROTAC (Fig. 3c). To analyze drug specificity of p300 PROTAC drug, we initially used shRNA to knockdown p300 in prostate cancer cells and subsequently treated them with the p300 PROTAC drug. Our findings indicate that in the shRNA control group, the p300 PROTAC drug exhibited a notable killing effect. However, upon p300 knockdown, the efficacy of the p300 PROTAC drug was diminished, demonstrating its dependence on the p300 target and thereby confirming its specificity (Fig. 3d).

Fig. 3.

The p300 PROTAC drug relies on ubiquitination mediated by MDM2 and effectively inhibits tumor-progression related proteins. (a) The p300 PROTAC drug enhances the binding ability of MDM2 to p300 in C4-2 cells. (b) After knockdown MDM2, the ability of p300 PROTAC drug to induce p300 degradation is inhibited. (c) The p300 PROTAC drug takes effect through the K48-linked ubiquitination but not K63-linked ubiquitination. (d) After knockdown of p300, the killing ability of the p300 PROTAC drug against prostate cancer cells is inhibited. (e) The p300 PROTAC drug can induce the downregulation of key tumor-related proteins in both AR-positive and AR-negative cell lines.

Furthermore, to explore downstream alterations induced by the p300 PROTAC drug, we assessed the expression levels of known p300-associated proteins in AR-positive and AR-negative cells. Our results indicate that the p300 PROTAC drug effectively diminishes the levels of these critical oncogenic proteins (Fig. 3e). Moreover, in AR-positive cell lines, p300 can influence AR-related signaling pathways. Thus, we investigated the impact of p300 PROTAC treatment on the downstream AR signaling pathway. Our findings revealed a significant inhibition of the AR signaling pathway following p300 PROTAC treatment (Figs. S9 and S10). To further investigate whether the reduction of these proteins is indeed due to the p300 inhibition or affected by apoptosis induced by the PROTAC, we knockdown p300 in C4-2 and NE1.3 cells to assess whether the protein levels would parallel those observed with p300 PROTAC drug treatment (Fig. S11). Besides, we examined the raw data from RNA-seq analysis to find the changes of these genes on mRNA levels (Fig. S12). The results demonstrated that p300 knockdown led to reduced levels of proteins including c-Myc, AR, and FOXA1, among others.

As described above, the p300 PROTAC drug we designed can not only degrade p300 but also activate p53 as the MDM2 targeting sequence is the MDM2/p53 homologous binding sequence. Immunoblotting analysis of p53 and downstream proteins after p300 PROTAC drug treatment was performed in prostate cancer cells carrying wild-type p53. As shown in Fig. S13a and b, the p300 PROTAC drug effectively increased the protein level of p53 and its downstream proteins in C4-2 and CWR22Rv1 cells. Moreover, although some have proposed that p300 is a p53 coactivator,⁶² as shown in Fig. S13c, the effect of p300 PROTAC on increasing p53 level was not affected by p300 protein. Besides, RNA-seq results also demonstrated the activation of p53 signaling pathway after drug treatment in C4-2 (Fig. S10).

To further evaluate the effects of the drug on prostate cancer cells, clone formation assays, apoptosis detection, and wound healing assays were performed to evaluate the efficacy of the p300 PROTAC drug. Clonogenic assays are widely used in the field of cancer research as the formation of clones is interpreted as a trait of cancer cells with tumor-initiating capabilities. As shown in Fig. S13d–g, the p300 PROTAC drug effectively inhibited colony formation in prostate cancer cells indicating that the p300 PROTAC drug functions in the initiation of cancer cell proliferation. As shown in Fig. S13h and i, the p300 PROTAC drug under high concentrations effectively induced apoptosis in both AR-positive and AR-negative cell lines. The wound healing and transwell invasion assays proved that the p300 PROTAC drug can effectively inhibit the migration and invasion of prostate cancer cells (Figs. S13j and k and S14).

To explain the mechanism for the anti-tumor effect of p300 PROTAC, in AR-negative cell lines DU145 and PC-3, where p53 is mutant or absent, we have found the p300 PROTAC drug can exert its tumor suppression effect just by degrading the p300 protein. Furthermore, in AR-positive cell lines such as C4-2 and CWR22Rv1, which carrying wild type p53, in addition to the degradation of the p300 protein, the anti-tumor effect of the p300 PROTAC may be also partly due to the release of p53. To find out, we constructed p53 knockdown cell lines and conducted efficacy testing (Fig. S15). Our results indicate that at low concentrations, the drug exhibits reduced efficacy in killing cells compared to p53 wild-type cells. However, at high concentrations, due to the complete degradation of p300, the p300 PROTAC drug can still achieve complete inhibition of prostate cancer cell proliferation. These findings highlight the mechanisms of action of our designed drug and provide insights into its efficacy in prostate cancer cells.

The p300 PROTAC drug achieves anti-neuroendocrine prostate cancer (NEPC) potency

As p300 regulates MYCN, EZH2, and FOXA2, etc. onco-pathways have been reported to involve in the process of NEPC, we proposed that p300 PROTAC drug is capable of NEPC therapeutic effect. We evaluated the efficacy of the p300 PROTAC drug in NEPC cells to demonstrate our proposal. Consistent with the results of the p300 PROTAC drug on other prostate cells, the p300 PROTAC drug can enter the NE 1.3 cells efficiently (Fig. S16).

Moreover, we evaluated the cytotoxic effects of p300 PROTAC on NEPC cells using two distinct NEPC cell lines, NE1.3 and H660. As illustrated in Fig. 4a and e, p300 PROTAC exhibited robust cytotoxicity in both NE1.3 and H660 cell lines. Furthermore, the treatment led to the degradation of p300 and related crucial cancer-promoting proteins (Fig. 4b and f). Remarkably, significant downregulation of NEPC-related markers was observed after p300 PROTAC treatment (Fig. 4c and g). Additionally, p300 PROTAC effectively induced apoptosis in NE1.3 and H660 cells (Fig. 4d and h). These findings collectively underscore the therapeutic potential of p300 PROTAC in NEPC therapy.

Fig. 4.

The p300 PROTAC drug achieves anti-neuroendocrine prostate cancer (NEPC) potency. (a) Cell viability assay of NE 1.3 cells after 48 h of treatment with indicated concentrations of the p300 PROTAC drug, nano vehicle (Se nanoparticles without loading p300 peptide PROTAC), and free peptide control. (b) Changes in p300 and NEPC-related proteins were detected after treatment with p300 PROTAC drug treatment in NE1.3 cells. (c) Changes in neuroendocrine markers were detected after treatment with 1 μM p300 PROTAC drug for 24 h in NE1.3 cells. (d) Representative apoptosis assay and statistical analysis of NE 1.3 cells after treatment with nano vehicle, peptide control, and p300 PROTAC drug (The data are presented as mean ± sd (n = 3), statistical analysis was used t-test, ns represents p > 0.05, ∗∗∗represents p < 0.001). (e) Cell viability assay of H660 cells after 48 h of treatment with indicated concentrations of the p300 PROTAC drug, nano vehicle (Se nanoparticles without loading p300 peptide PROTAC), and free peptide control. (f) Changes in p300 and NEPC-related proteins were detected after treatment with p300 PROTAC drug treatment in H660 cells. (g) Changes in neuroendocrine markers were detected after treatment with 1 μM p300 PROTAC drug for 24 h in H660 cells. (h) Representative apoptosis assay and statistical analysis of H660 cells after treatment with nano vehicle, peptide control, and p300 PROTAC drug (The data are presented as mean ± sd (n = 3), statistical analysis was used t-test, ns represents p > 0.05, ∗∗∗represents p < 0.001, ∗∗∗∗represents p < 0.0001).

To explore the mechanism of action of p300-targeted therapy in NEPC, RNA-seq analysis was performed after p300 PROTAC drug treatment. As shown in Figs. S17a and c, S18 and S19, the degradation of p300 induced a large number of cancer-related genes and immune-related gene changes. Particularly, the p53 pathway was significantly enriched. Besides, in NE1.3 cells, we observed a concentration-dependent increase in p53 protein concentration which is similar to in C4-2 and CWR22Rv1 cells (Fig. S20). Consequently, our findings suggest that in NEPC cells with wild-type p53, the p300 PROTAC drug we developed has the potential to enhance p53 protein levels. Also, gene set enrichment analysis (GSEA) after p300 degradation shows significant alterations in DNA repair and ferroptosis pathways (Fig. S17b and d).

The p300 PROTAC drug inhibits prostate tumor growth in vivo

To assess the therapeutic potential of the p300 PROTAC drug for advanced prostate cancer in vivo, we established two xenograft mouse models of advanced prostate cancer using CWR22Rv1 and PC-3 cells.

In the CWR22Rv1 model, the dosage of p300 PROTAC drug was 5 mg/kg. And we used I-CBP112, dCBP-1, and JQAD1 as controls at the dose of 5 mg/kg. All drug treatments were administered via intraperitoneal injection every other day. As shown in Fig. 5a–c, neither the nano vehicle nor the peptide control had any effect on CWR22Rv1 tumor growth, whereas the p300 PROTAC drug demonstrated significant efficacy in vivo. The p300 PROTAC drug showed better efficacy compared with I-CBP112, dCBP-1, and JQAD1. Also, the p300 PROTAC drug effectively degraded the p300, AR and Ki-67, and effectively induced the increase of tumor apoptosis (Fig. 5d–h).

Fig. 5.

The p300 PROTAC drug inhibits prostate tumor growth in the CWR22Rv1 model. (a) Tumor growth curves of CWR22Rv1 xenografts in nude mice treated as indicated (n = 5 per group) (The data are presented as the mean ± SD values (n = 5). Statistical analysis was performed using the t-test; ns represents p > 0.05, ∗ represents p < 0.05, and ∗∗∗ represents p < 0.001). (b) Photos of CWR22Rv1 tumors excised at the end of the experiment after different drug treatments. (c) Average weight of tumors excised from each group of mice at the end of drug treatment (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ns represents p > 0.05, ∗ represents p < 0.05, ∗∗ represents p < 0.01). (d) IHC analysis of p300 in CWR22Rv1 tumors from each treatment group (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗∗∗ represents p < 0.0001). (e) IHC analysis of AR in CWR22Rv1 tumors from each treatment group (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗∗∗ represents p < 0.0001). (f) IHC analysis of Ki-67 in CWR22Rv1 tumors from each treatment group (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗∗∗ represents p < 0.0001). (g) IHC analysis of TUNEL in CWR22Rv1 tumors from each treatment group (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗∗∗ represents p < 0.0001). (h) Representative photo of IHC analysis of p300, AR, Ki67, and TUNEL.

In the PC-3 model, we randomly divided 25 tumor-bearing mice into five groups and treated them every 2 days for 18 days with either PBS, the peptide control, the nano vehicle, p300 PROTAC drug, or I-CBP112 at a dose of 5 mg/kg. As depicted in Fig. 6a–c, neither the nano vehicle nor the peptide control had any effect on PC-3 tumor growth, whereas the p300 PROTAC drug demonstrated significant efficacy in vivo. Remarkably, the p300 PROTAC showed better efficacy compared to small molecular p300/CBP inhibitor I-CBP112 at the same dose. To evaluate the in vivo effect of p300 PROTAC, we performed histopathological analysis of tumors using hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and anti-Ki-67 immunohistochemistry. As shown in Fig. 6d–h, p300 PROTAC drug treatment significantly induced a higher level of apoptotic tumor cells in tumors compared with I-CBP112, indicating stronger potency. Pharmacodynamic tests at the animal level verified that our designed p300 PROTAC drug exhibits greater efficacy than small molecule p300 inhibitor in vivo.

Fig. 6.

The p300 PROTAC drug inhibits prostate tumor growth in the PC-3 model. (a) Tumor growth curves of PC-3 xenografts in nude mice treated as indicated (n = 5 per group) (The data are presented as the mean ± SD values (n = 5). Statistical analysis was performed using the t-test; ∗ represents p < 0.05, and ∗∗∗∗ represents p < 0.0001). (b) Photos of PC-3 tumors excised at the end of the experiment after different drug treatments. (c) The average weight of tumors excised from each group of mice at the end of drug treatment (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001). (d) IHC analysis of Ki-67 in PC-3 tumors from each treatment group (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗ represents p < 0.01). (e) IHC analysis of TUNEL in PC-3 tumors from each treatment group. Tunel positive area percent were determined with ImageJ (The data are presented as the mean ± SD (n = 5). Statistical analysis was performed using the t-test; ∗∗ represents p < 0.01). (f–h) Histopathological analysis of the excised tumors using Ki-67 assay (f), TUNEL assay (g), and H&E staining (h) (scale bar: 10 μm).

The biodistribution and toxicity analysis of the p300 PROTAC drug

Biodistribution and toxicity analysis of the p300 PROTAC drug were performed to assess the druggability potential of the p300 PROTAC drug. To facilitate fluorescence detection, the p300 PROTAC drug was labeled with rhodamine. As shown in Fig. 7a, the p300 PROTAC drug accumulated within the tumor obviously as an EPR effect (enhanced permeability and retention effect) of the nanosystem. It should be noted that the duration time of the p300 PROTAC drug in the tumor site is longer than that in the liver indicating a stronger effect in tumor cells and less toxicity in liver cells. The accumulation of the p300 PROTAC drug in the tumor site and liver was also confirmed by in vivo experiments in mice (Fig. S21).

Fig. 7.

The biodistribution and toxicity analysis of the p300 PROTAC drug. (a) The biodistribution analysis of p300 PROTAC at different time points in PC-3 xenograft models. (b and c) Evaluation of renal and hepatic toxicity of p300 PROTAC at 15 mg/kg in mice (The data are presented as the mean ± SD values (n = 5). Statistical analysis was performed using the t-test; ns represents p > 0.05). (d) Histopathological analysis of the excised organs after p300 PROTAC treatment at 15 mg/kg by H&E staining (scale bar: 10 μm).

To evaluate the toxicity of the p300 PROTAC drug more accurately, we treated the BALB/c mice with three times the concentration of the drug efficacy experiment (15 mg/kg) for 3 weeks. The changes in kidney and liver function indexes and the damage of major organs were analyzed. The results showed that at a concentration of 15 mg/kg, the p300 PROTAC drug had no significant effect on renal and liver function and no toxicity to major organs (Fig. 7b–d). The toxicity evaluation proved that the p300 PROTAC drug has great safety and clinical application potential.

Discussion

Although AR-targeting therapies have achieved a great curative effect and clinical benefits for patients with prostate cancer, they will eventually develop drug resistance, and some of them will develop NEPC which is fatal.⁶³ Treatment for advanced ADT-resistance prostate cancer and NEPC remains a challenge.⁶⁴ Exploring targeted therapies other than AR is one of the main directions for the development of treatment for advanced CRPC and AR-negative prostate cancer including NEPC. In this study, we proposed p300 as a drug target for advanced prostate cancer therapy including NEPC. With the continuous in-depth research on the mechanism of p300, in addition to being an AR coactivator, the carcinogenic function of p300 independent of AR in prostate cancer has been widely reported. Particularly, the regulation of TMPRSS2 in prostate cancer by p300 independent of AR has been identified.^65,66 Several NEPC-related genes have also been reported to be regulated by p300. By combining existing clues, we propose that p300 targeting therapy can treat not only AR pathway-dependent prostate cancer but also AR pathway-independent prostate cancer including NEPC.

In recent years, several small molecular inhibitors and degraders with clinical potential have been developed against p300.^7,21, 22, 23, 24 So far, only Inobrodib (CCS1477) and FT-7051 have entered clinical phase I for patients with prostate cancer and no p300-targeting drug has finally entered clinical use. PROTAC drug design has emerged as a new drug design strategy providing another solution for p300 targeting.²¹ PROTAC drug has the advantage of completely inducing protein degradation at the protein level rather than just inhibiting protein function.

Compared with the reported p300 targeting small molecular PROTAC drug, we used MDM2 as an E3 ligase rather than CRBN. By interacting with the N-terminal domain of MDM2, our designed p300 PROTAC drug can release the tumor suppressor protein p53 whose loss and aberration are critical in the progression of CPRC as well as NEPC.⁶¹ And unlike those based on p300's HAT and BRD domains, according to its CH1 domain we have chosen a protein–protein interface targeting p300 to further improve the specificity and safety of the p300 PROTAC drug design. Our results showed that the p300 PROTAC drug had excellent p300 degradation ability and cancer cell-killing ability in AR-positive prostate cancer cells. Furthermore, as p300 is known to regulate genes involved in various pathways, including EZH2,⁴⁶ MYCN,^24,67 FOXA2,^33,48,49 angiogenesis,⁶⁸ DNA damage-repair,⁶⁹ and WNT-β catenin pathways,⁷⁰ which are all implicated in neuroendocrine prostate cancer (NEPC), we investigated the efficacy of p300-targeted drugs in AR-negative prostate cancer and NEPC cells.

The significant killing effect of our p300 PROTAC drug was validated in prostate cancer whether it is dependent on the AR signaling pathway or not, including NEPC. Our designed p300 PROTAC drug could eliminate the p300 protein under 1 μM in different prostate cancer cell lines. In NEPC cell lines, the p300 PROTAC drug demonstrated potent p300 cleavage ability, cancer cell proliferation inhibition ability, and apoptosis-inducing ability. Through RNA-seq analysis of NE 1.3 cells after p300 PROTAC drug treatment, we demonstrated that p300 degradation suppresses cancer-related pathways and upregulates immune-related pathways indicating p300 targeting therapeutic potential in NEPC treatment.

Considering the stability of our PROTAC, in this study, we conjugated our p300 PROTAC with a nano-selenium delivery system to make it function in vitro and in vivo. In the following further research, the search for a safer and more durable delivery system, as well as increasing the specific targeting of tumor tissues without affecting normal tissues, may be the key to enhancing the efficacy of the peptide-based drug and improving clinical transformation.⁷¹

In summary, we developed a p300 PROTAC drug and verified its efficacy and safety. In AR-positive prostate cancer, the p300 PROTAC drug functions mainly by decreasing the transcriptional activity of AR and also interfering other tumorigenic effects of p300. In AR-negative prostate cancer, including NEPC, p300 PROTAC drugs exert their tumor-suppressive effects independently of AR inhibition. Given p300's extensive involvement in transcriptional regulation in cancer, the p300 PROTAC drug induces the inhibition of various important oncogenic proteins, such as EZH2, c-Myc, N-Myc, among others. This broad inhibitory role contributes to suppressing the progression of AR-negative prostate cancer (Fig. 8). Our peptide-based PROTAC drug design has several advantages. Firstly, the peptide antagonist sequence targeting the CH1 domain of p300 with high selectivity and specificity, which provides a new approach to drug development. Secondly, in our design, application of MDM2 instead of CRBN as the E3 ligase allowed p53 to be released along with degradation, would result in a synergistic therapeutic effect. Compared with the existing small molecular drugs, our p300 PROTAC provides an innovative and safer approach to degrade the target p300. Thirdly, we preliminarily verified the feasibility of targeting p300 to treat NEPC and analyzed the possible mechanism. Based on the existing data, we provide a good drug for the treatment of prostate cancer, especially NEPC.

Fig. 8.

Different mechanisms of the function of p300 PROTAC drugs in AR-positive and negative cells. In AR-positive prostate cancer cells, the degradation of p300 can effectively reduce the transcriptional activity of AR, thereby inhibiting prostate cancer cell viability. In AR-negative prostate cancer cells, the degradation of p300 can inhibit other onco-pathways independent of the AR signaling pathway, thereby killing cancer cells.

Contributors

Investigation: D. Z. Z., B. H. M.; Conceptualization and Funding acquisition: B. H. M., L. L.; Methodology and data curation: D. Z. Z., B. H. M., D. H. L., T. Y. Z.; Data verification: D. Z. Z., B. H. M, Y. L. J.; Software: W. W., C. L. Y., Y. B. G.; Visualisation: B. H. M., Y. C. Q.; Writing—original draft: D. Z. Z., B. H. M.; Writing—review & editing: Y. Z. F., Y. G., Y. L. C., J. M., S. X., L. L.; Supervision and project administration: L. L. All authors read and approved the final manuscript.

Data sharing statement

All data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declaration of interests

The authors declare no conflict of interest.