BRD4 Research Articles

Bromodomain-containing protein 4 activates androgen receptor transcription and promotes ovarian fibrosis in PCOS

Polycystic ovary syndrome (PCOS) is an endocrine disorder and the main cause of anovulatory infertility, in which persistent activation of androgen receptor (AR) due to aberrant acetylation modifications of transcription is a potential trigger; however, the precise mechanisms of AR activation are poorly understood. In this study, AR activation in dehydroepiandrosterone- and letrozole-induced rat PCOS ovaries coincided with a marked increase of a chromatin acetylation "reader" BRD4. Further bioinformatic analysis showed that the AR promoter contained highly conserved binding motifs of BRD4 and HIF-1α. BRD4 and HIF-1α inducibly bound to the histone 3/4 acetylation-modified AR promoter, while administration of a BRD4-selective inhibitor JQ1 reduced the binding and AR transcription and improved the adverse expression of the core fibrotic mediators in PCOS ovaries and DHT-treated granulosa cells. Our data indicate that BRD4 upregulation and the resultant AR transcriptional activation constitute an important regulatory pathway that promotes ovarian fibrosis in PCOS.

Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 inHSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

Discovery of a potent, orally available tricyclic derivative as a novel BRD4 inhibitor for melanoma

The epigenetic regulation of the protein bromodomain-containing protein 4 (BRD4) has emerged as a compelling target for cancer treatment. In this study, we outline the discovery of a novel BRD4 inhibitor for melanoma therapy. Our initial finding was that benzimidazole derivative 1, sourced from our library, was a powerful BRD4 inhibitor. However, it exhibited a poor pharmacokinetic (PK) profile. To address this, we conducted a scaffold-hopping procedure with derivative 1, which resulted in the creation of benzimidazolinone derivative 5. This new derivative displayed an improved PK profile. To further enhance the BRD4 inhibitory activity, we attempted to introduce hydrogen bond acceptors. This indeed improved the activity, but at the cost of decreased membrane permeability. Our search for a potent inhibitor with desirable permeability led to the development of tricyclic 18. This compound demonstrated powerful inhibitory activity and a favorable PK profile. More significantly, tricyclic 18 showed antitumor efficacy in a mouse melanoma xenograft model, suggesting that it holds potential as a therapeutic agent for melanoma treatment.

BRD4 as a Therapeutic Target in Pulmonary Diseases.

Bromodomain and extra-terminal domain (BET) proteins are epigenetic modulators that regulate gene transcription through interacting with acetylated lysine residues of histone proteins. BET proteins have multiple roles in regulating key cellular functions such as cell proliferation, differentiation, inflammation, oxidative and redox balance, and immune responses. As a result, BET proteins have been found to be actively involved in a broad range of human lung diseases including acute lung inflammation, asthma, pulmonary arterial hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). Due to the identification of specific small molecular inhibitors of BET proteins, targeting BET in these lung diseases has become an area of increasing interest. Emerging evidence has demonstrated the beneficial effects of BET inhibitors in preclinical models of various human lung diseases. This is, in general, largely related to the ability of BET proteins to bind to promoters of genes that are critical for inflammation, differentiation, and beyond. By modulating these critical genes, BET proteins are integrated into the pathogenesis of disease progression. The intrinsic histone acetyltransferase activity of bromodomain-containing protein 4 (BRD4) is of particular interest, seems to act independently of its bromodomain binding activity, and has implication in some contexts. In this review, we provide a brief overview of the research on BET proteins with a focus on BRD4 in several major human lung diseases, the underlying molecular mechanisms, as well as findings of targeting BET proteins using pharmaceutical inhibitors in different lung diseases preclinically.

Chemical Epigenetic Regulation of Adeno-Associated Virus Delivered Transgenes.

Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

BRD7 suppresses tumor chemosensitivity to CHK1 inhibitors by inhibiting USP1-mediated deubiquitination of CHK1

Checkpoint kinase 1 (CHK1), a key effector in the cellular response to DNA lesions, is a crucial component of all cell cycle checkpoints. Recent reports have revealed that CHK1 is highly expressed in numerous cancer types in the clinical settings. However, the mechanisms underlying the regulation of CHK1 expression in tumor cells remain unclear. Here, we report that CHK1 is negatively regulated by the bromodomain-containing protein 7 (BRD7). Specifically, BRD7 silencing increased CHK1 (but not CHK2) expression at both mRNA and protein levels, in a p53-independent manner in multiple tumor cell lines. Furthermore, BRD7 silencing stabilized CHK1 via reducing its ubiquitination. Mechanistically, BRD7 knockdown not only increased the levels of USP1, a deubiquitinase for CHK1, but also promoted the interaction between CHK1 and USP1, subsequently enhancing the de-ubiquitination of CHK1. USP1 knockdown abrogated BRD7 silencing-induced CHK1 induction. Biologically, the increased expression of CHK1 in tumor cells caused by BRD7 silencing significantly increased cell sensitivity to CHK1 inhibitors by enhancing tumor cell apoptosis, and this effect was reversed by the simultaneous knockdown of CHK1 or USP1. Taken together, our findings suggest that BRD7 is a potential genetic or drug target that may help to improve the efficacy of chemotherapeutic drugs targeting CHK1 in combinatorial therapy.

Transcriptional factor BRD4 promotes the stemness of esophageal cancer by activating the nuclear PD-L1/RelB axis.

Esophageal cancer (EC) is a prevalent malignancy associated with therapeutic resistance and poor prognosis. This study investigates the role of programmed death-ligand 1 (PD-L1) in esophageal cancer stem cell (ECSC) formation. ECSCs were enriched and characterized using various assays. We found that both PD-L1 and bromodomain-containing protein 4 (BRD4) were upregulated in ECSCs, promoting their stemness. Inhibiting BRD4 suppressed ECSC markers expression and sphere formation. Furthermore, BRD4 inhibitors downregulated membrane and nuclear PD-L1 levels, with knockdown of PD-L1 inhibiting ECSC formation. PD-L1 degraders also affected PD-L1 and its downstream effector RelB expression. Moreover, inhibiting RelB influenced sphere formation through interleukin-6 expression. This study reveals the critical role of the BRD4/nuclear PD-L1/RelB axis in ECSC formation, highlighting nuclear PD-L1 as a potential immunotherapeutic target for refractory EC.

Inhibition of Brd4 alleviates osteoarthritis pain via suppression of neuroinflammation and activation of Nrf2-mediated antioxidant signalling.

Osteoarthritis (OA) pain remains a major clinical problem. It is urgent to identify novel therapeutic approaches for OA pain states. Bromodomain and extra-terminal (BET) protein inhibitors have robust anti-inflammatory effects in several pain models. However, the underlying mechanisms of these inhibitors in OA pain have not been determined. We, therefore, investigated the effects and the underlying mechanism(s) of BET inhibition on pain-related behaviours in a rat model of OA. The OA model was established by intra-articular injection of monosodium iodoacetate (MIA) in rat knees. Pain behaviours were assessed in rats by hindlimb weight-bearing asymmetry, mechanical allodynia and thermal hyperalgesia. Possible mechanisms underlying BET inhibition were explored in the MIA-induced OA pain model in the spinal cord and dorsal root ganglia (DRG). Inhibiting bromodomain-containing protein 4 (Brd4) with either JQ1 or MS417, or using AAV2/9-shRNA-Brd4-EGFP-mediated knockdown of Brd4 genes, significantly attenuated MIA-induced pain behaviours. Brd4 inhibition suppressed NF-κB and NF-κB-mediated inflammatory cytokines in both the spinal cord and DRG in rats with MIA-induced OA pain. Brd4 inhibition also attenuated the oxidative stress and promoted nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidant genes in both the spinal cord and DRG in our odel of MIA-induced OA pain. In conclusion, Brd4 inhibition alleviated MIA-induced OA pain in rats, via suppression of neuroinflammation and activation of Nrf2-mediated antioxidant signalling. Although our model does not perfectly represent how OA develops in humans, inhibition of Brd4 may provide novel insights into possible treatments for OA pain.

BRD4 Inhibition Enhances the Antitumor Effects of Radiation Therapy in a Murine Breast Cancer Model.

Bromodomain-containing protein 4 (BRD4) is an intracellular protein that regulates expression of various cellular functions. This study investigated whether BRD4 inhibition can alter the immunomodulatory and antitumor effects of radiation therapy (RT). A murine breast cancer cell line was implanted into BALB/c mice. The dual-tumor model was used to evaluate the abscopal effects of RT. A total of 24 Gy was delivered and BRD4 inhibitor was injected intravenously. Tumor size was measured, and in vivo imaging was performed to evaluate tumor growth. Flow cytometry and immunohistochemistry were performed to examine immunologic changes upon treatment. The combination of BRD4 inhibitor and RT significantly suppressed tumor growth compared to RT alone. BRD4 inhibitor reduced the size of the unirradiated tumor, indicating that it may induce systemic immune responses. The expression of HIF-1α and PD-L1 in the tumor was significantly downregulated by the BRD4 inhibitor. The proportion of M1 tumor-associated macrophages (TAMs) increased, and the proportion of M2 TAMs decreased upon BRD4 inhibition. BRD4 inhibitor expanded CD4+ and CD8+ T cell populations in the tumor microenvironment. Additionally, splenic monocytic myeloid derived suppressor cells, which were increased by RT, were reduced upon the addition of BRD4 inhibitor. Therefore, the addition of BRD4 inhibitor significantly enhanced the systemic antitumor responses of local RT.

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents.

A Functional Bayesian Model for Hydrogen-Deuterium Exchange Mass Spectrometry.

Proteins often undergo structural perturbations upon binding to other proteins or ligands or when they are subjected to environmental changes. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to explore conformational changes in proteins by examining differences in the rate of deuterium incorporation in different contexts. To determine deuterium incorporation rates, HDX-MS measurements are typically made over a time course. Recently introduced methods show that incorporating the temporal dimension into the statistical analysis improves power and interpretation. However, these approaches have technical assumptions that hinder their flexibility. Here, we propose a more flexible methodology by reframing these methods in a Bayesian framework. Our proposed framework has improved algorithmic stability, allows us to perform uncertainty quantification, and can calculate statistical quantities that are inaccessible to other approaches. We demonstrate the general applicability of the method by showing it can perform rigorous model selection on a spike-in HDX-MS experiment, improved interpretation in an epitope mapping experiment, and increased sensitivity in a small molecule case-study. Bayesian analysis of an HDX experiment with an antibody dimer bound to an E3 ubiquitin ligase identifies at least two interaction interfaces where previous methods obtained confounding results due to the complexities of conformational changes on binding. Our findings are consistent with the cocrystal structure of these proteins, demonstrating a bayesian approach can identify important binding epitopes from HDX data. We also generate HDX-MS data of the bromodomain-containing protein BRD4 in complex with GSK1210151A to demonstrate the increased sensitivity of adopting a Bayesian approach.

Targeted delivery of PROTAC-based prodrug activated by bond-cleavage bioorthogonal chemistry for microneedle-assisted cancer therapy

Proteolysis-targeting chimera (PROTAC) is emerging as a new strategy to degrade target proteins in a precise way by taking advantage of the cellular ubiquitin-proteasome system. However, the potential cytotoxicity of PROTAC should be avoided to mitigate the off-target degradation of proteins in healthy tissues or cells. To address this issue, we herein present a strategy to cage a PROTAC with 4-(vinyloxy) benzyl carbonate (MZ1-O), which can be eliminated through a 3,6-dimethyl-1,2,4,5-tetrazine (Tz)-mediated inverse electron-demand Diels-Alder (iEDDA) reaction to generate a BRD4 (bromodomain-containing protein 4) degrader, MZ1. We further propose a dissolvable microneedle-assisted strategy for site-specific activation of MZ1-O that is delivered by a targeted delivery vector through systemic route in vivo, and demonstrate such a bioorthogonal strategy is efficient and precise for tumor treatment. Our study suggests that the bioorthogonal activation of PROTAC-based prodrug offers a highly specific and precise approach for cancer therapy.

Rational Design and Development of Selective BRD7 Bromodomain Inhibitors and Their Activity in Prostate Cancer.

Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) is implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity toward BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.

NcBAF enhances PXR-mediated transcriptional activation in the human and mouse liver

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug-drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

Spotlight on Plant Bromodomain Proteins.

Bromodomain-containing proteins (BRD-proteins) are the "readers" of histone lysine acetylation, translating chromatin state into gene expression. They act alone or as components of larger complexes and exhibit diverse functions to regulate gene expression; they participate in chromatin remodeling complexes, mediate histone modifications, serve as scaffolds to recruit transcriptional regulators or act themselves as transcriptional co-activators or repressors. Human BRD-proteins have been extensively studied and have gained interest as potential drug targets for various diseases, whereas in plants, this group of proteins is still not well investigated. In this review, we aimed to concentrate scientific knowledge on these chromatin "readers" with a focus on Arabidopsis. We organized plant BRD-proteins into groups based on their functions and domain architecture and summarized the published work regarding their interactions, activity and diverse functions. Overall, it seems that plant BRD-proteins are indispensable components and fine-tuners of the complex network plants have built to regulate development, flowering, hormone signaling and response to various biotic or abiotic stresses. This work will facilitate the understanding of their roles in plants and highlight BRD-proteins with yet undiscovered functions.

Bellidifolin ameliorates isoprenaline-induced cardiac hypertrophy by the Nox4/ROS signalling pathway through inhibiting BRD4

To date, there is no effective therapy for pathological cardiac hypertrophy, which can ultimately lead to heart failure. Bellidifolin (BEL) is an active xanthone component of Gentianella acuta (G. acuta) with a protective function for the heart. However, the role and mechanism of BEL action in cardiac hypertrophy remain unknown. In this study, the mouse model of cardiac hypertrophy was established by isoprenaline (ISO) induction with or without BEL treatment. The results showed that BEL alleviated cardiac dysfunction and pathological changes induced by ISO in the mice. The expression of cardiac hypertrophy marker genes, including ANP, BNP, and β-MHC, were inhibited by BEL both in mice and in H9C2 cells. Furthermore, BEL repressed the epigenetic regulator bromodomain-containing protein 4 (BRD4) to reduce the ISO-induced acetylation of H3K122 and phosphorylation of RNA Pol II. The Nox4/ROS/ADAM17 signalling pathway was also inhibited by BEL in a BRD4 dependent manner. Thus, BEL alleviated cardiac hypertrophy and cardiac dysfunction via the BRD4/Nox4/ROS axes during ISO-induced cardiac hypertrophy. These findings clarify the function and molecular mechanism of BEL action in the therapeutic intervention of cardiac hypertrophy.

Hydrogen Peroxide-Inducible PROTACs for Targeted Protein Degradation in Cancer Cells.

Proteolysis-targeting chimeras (PROTACs) provide a powerful technique to degrade targeted proteins utilizing the cellular ubiquitin-proteasome system. The major concern is the host toxicity resulting from their poor selectivity. Inducible PROTACs responding to exogenous stimulus, such as light, improve their specificity, but it is difficult for photo-activation in deep tissues. Herein, we develop H2 O2 -inducible PROTAC precursors 2/5, which can be activated by endogenous H2 O2 in cancer cells to release the active PROTACs 1/4 to effectively degrade targeted proteins. This results in the intended cytotoxicity towards cancer cells while targeted protein in normal cells remains almost unaffected. The higher Bromodomain-containing protein 4 (BRD4) degradation activity and cytotoxicity of 2 towards cancer cells is mainly due to the higher endogenous concentration of H2 O2 in cancer cells (A549 and H1299), characterized by H2 O2 -responsive fluorescence probe 3. Western blot assays and cytotoxicity experiments demonstrate that 2 degrades BRD4 more effectively and is more cytotoxic in H2 O2 -rich cancer cells than in H2 O2 -deficient normal cells. This method is also extended to estrogen receptor (ER)-PROTAC precursor 5, showing H2 O2 -dependent ER degradation ability. Thus, we establish a novel strategy to induce targeted protein degradation in a H2 O2 -dependent way, which has the potential to improve the selectivity of PROTACs.

Pyronaridine as a Bromodomain-Containing Protein 4-N-Terminal Bromodomain (BRD4-BD1) Inhibitor: In Silico Database Mining, Molecular Docking, and Molecular Dynamics Simulation.

BRD4 (bromodomain-containing protein 4) is an epigenetic reader that realizes histone proteins and promotes the transcription of genes linked to cancer progression and non-cancer diseases such as acute heart failure and severe inflammation. The highly conserved N-terminal bromodomain (BD1) recognizes acylated lysine residues to organize the expression of genes. As such, BD1 is essential for disrupting BRD4 interactions and is a promising target for cancer treatment. To identify new BD1 inhibitors, a SuperDRUG2 database that contains more than 4600 pharmaceutical compounds was screened using in silico techniques. The efficiency of the AutoDock Vina1.1.2 software to anticipate inhibitor-BRD4-BD1 binding poses was first evaluated based on the co-crystallized R6S ligand in complex with BRD4-BD1. From database screening, the most promising BRD4-BD1 inhibitors were subsequently submitted to molecular dynamics (MD) simulations integrated with an MM-GBSA approach. MM-GBSA computations indicated promising BD1 binding with a benzonaphthyridine derivative, pyronaridine (SD003509), with an energy prediction (ΔGbinding) of -42.7 kcal/mol in comparison with -41.5 kcal/mol for a positive control inhibitor (R6S). Pharmacokinetic properties predicted oral bioavailability for both ligands, while post-dynamic analyses of the BRD4-BD1 binding pocket demonstrated greater stability for pyronaridine. These results confirm that in silico studies can provide insight into novel protein-ligand regulators, specifically that pyronaridine is a potential cancer drug candidate.

Self-Assembled Nano-PROTAC Enables Near-Infrared Photodynamic Proteolysis for Cancer Therapy.

Confining the protein degradation activity of proteolysis-targeting chimera (PROTAC) to cancer lesions ensures precision treatment. However, it still remains challenging to precisely control PROTAC function in tumor regions in vivo. We herein describe a near-infrared (NIR) photoactivatable nano-PROTAC (NAP) for remote-controllable proteolysis in tumor-bearing mice. NAP is formed by molecular self-assembly from an amphiphilic conjugate of PROTAC linked with an NIR photosensitizer through a singlet oxygen (1O2)-cleavable linker. The activity of PROTAC is initially silenced but can be remotely switched on upon NIR photoirradiation to generate 1O2 by the photosensitizer. We demonstrated that NAP enabled tumor-specific degradation of bromodomain-containing protein 4 (BRD4) in an NIR light-instructed manner. This in combination with photodynamic therapy (PDT) elicited an effective suppression of tumor growth. This work thus presents a novel approach for spatiotemporal control over targeted protein degradation by PROTAC.

Orchestrated copper-based nanoreactor for remodeling tumor microenvironment to amplify cuproptosis-mediated anti-tumor immunity in colorectal cancer

Owing to the deficiency of infiltrating immune cells and the accumulation of immunosuppressive cells in tumor microenvironment (TME), immunotherapy remains enormous challenges in colorectal cancer (CRC). Here, CRC was found to be sensitive to cuproptosis which could further evoke immune responses and mediate immune resistance. Thus, a cuproptosis-mediated immunotherapy nanoreactor (CCJD-FA) was constructed by encapsulating copper-based shell-coated CaO2 and bromodomain-containing protein 4 inhibitor JQ-1 with DSPE-PEG-FA. Within tumor cells, CCJD-FA was disassembled under GSH environment, and the exposed CaO2 could generate H2O2 under the acidic condition, which reacted with the released Cu2+ via Fenton-like reaction to produce O2 for relieving hypoxia and Cu+ for inducing cuproptosis. Furthermore, CCJD-FA could also inhibit intracellular glycolysis and ATP generation, then blocking the Cu+ efflux protein ATP7B. Meanwhile, the IFN-γ-induced PD-L1 expression could be exactly reduced by the BRD4 suppression and oxygen supply. These effects sensitized cancer cells to cuproptosis, further evoking systemic immune responses and reshaping immunosuppressive TME to inhibit tumor growth.