Discovery of a potent orally available pyrazolopyridone derivative as a novel selective bromodomain and extra-terminal domain (BET)-first bromodomain (BD1) inhibitor.

Clinical studies have shown that inhibitors of bromodomain and extra-terminal domain (BET) proteins, particularly BRD4, have antitumor activity and efficacy. The BET protein has two domains, BD1 and BD2, and we previously focused on BD1 and reported orally bioavailable BD1-selective inhibitors. In this study, we obtained a BD1 inhibitor, a more potent and highly selective pyrazolopyridone derivative 13a, and confirmed its in vivo efficacy.

Discovery of a Highly Potent and Selective BRD9 PROTAC Degrader Based on E3 Binder Investigation for the Treatment of Hematological Tumors.

BRD9 is a pivotal epigenetic factor involved in cancers and inflammatory diseases. Still, the limited selectivity and poor phenotypic activity of targeted agents make it an atypically undruggable target. PROTAC offers an alternative strategy for overcoming the issue. In this study, we explored diverse E3 ligase ligands for the contribution of BRD9 PROTAC degradation. Through molecular docking, binding affinity analysis, and structure-activity relationship study, we identified a highly potent PROTAC E5, with excellent BRD9 degradation (DC50 = 16 pM) and antiproliferation in MV4-11 cells (IC50 = 0.27 nM) and OCI-LY10 cells (IC50 = 1.04 nM). E5 can selectively degrade BRD9 and induce cell cycle arrest and apoptosis. Moreover, the therapeutic efficacy of E5 was confirmed in xenograft tumor models, accompanied by further RNA-seq analysis. Therefore, these results may pave the way and provide the reference for the discovery and investigation of highly effective PROTAC degraders.

NUAK2 Inhibitors, KHKI-01128 and KHKI-01215, Exhibit Potent Anticancer Activity Against SW480 Colorectal Cancer Cells.

BACKGROUND/AIM: NUAK family kinase 2 (NUAK2) is a promising target for cancer therapeutics due to its reported role in protein phosphorylation, a critical process in cancer cell survival, proliferation, invasion, and senescence. This study aimed to identify novel inhibitors that disrupt NUAK2 activity. We have already identified two KRICT Hippo kinase inhibitor (KHKI) compounds, such as KHKI-01128 and KHKI-01215. Our aim was to evaluate the impact of KHKI-01128 and KHKI-01215 on NUAK2 activity and elucidate its mechanism in colorectal cancer cells. MATERIALS AND METHODS: To evaluate anticancer properties of these inhibitors, four in vitro assays in the SW480 cell line (time-resolved fluorescence resonance energy transfer assay, KINOMEscan kinase profiling, viability, and apoptosis assays) and two pharmacological mechanism analyses (Gene Set Enrichment Analysis and western blotting) were performed. RESULTS: KHKI-01128 and KHKI-01215 exhibited potent inhibitory activity against NUAK2 (half-maximal inhibitory concentration=0.024±0.015 µM and 0.052±0.011 µM, respectively). These inhibitors suppressed cell proliferation, with half-maximal inhibitory concentrations of 1.26±0.17 µM and 3.16±0.30 µM, respectively, and induced apoptosis of SW480 cells. Gene Set Enrichment Analysis revealed negative enrichment scores of -0.84 for KHKI-01128 (false-discovery rate=0.70) and 1.37 for KHKI-01215 (false-discovery rate=0.18), indicating that both effectively suppressed the expression of YES1-associated transcriptional regulator (YAP) target genes. CONCLUSION: These results suggest that KHKI-01128 and KHKI-01215 are potent NUAK2 inhibitors with promising potential for pharmaceutical applications.

BRD4 as a therapeutic target for atrial fibrosis and atrial fibrillation.

OBJECTIVE: This study aimed to elucidate the molecular mechanisms by which BRD4 play a role in atrial fibrillation (AF). METHODS AND RESULTS: We used a discovery-driven approach to detect BRD4 expression in the atria of patients with AF and in various murine models of atrial fibrosis. We used a BRD4 inhibitor (JQ1) and atrial fibroblast (aFB)-specific BRD4-knockout mice to elucidate the role of BRD4 in AF. We further examined the underlying mechanisms using RNA-seq and ChIP-seq analyses in vitro, to identify key downstream targets of BRD4. We found that BRD4 expression is significantly increased in patients with AF, with accompanying atrial fibrosis and aFB differentiation. We showed that JQ1 treatment and shRNA-based molecular silencing of BRD4 blocked ANG-II-induced extracellular matrix production and cell-cycle progression in aFBs. BRD4-related RNA-seq and ChIP-seq analyses in aFBs demonstrated enrichment of a subset of promoters related to the expression of profibrotic and proliferation-related genes. The pharmacological inhibition of BRD4 in vivo or in aFB-specific BRD4-knockout in mice limited ANG-II-induced atrial fibrosis, atrial enlargement, and AF susceptibility. CONCLUSION: Our findings suggest that BRD4 plays a key role in pathological AF, at least partially by activating aFB proliferation and ECM synthesis. This study provides mechanistic insights into the development of BRD4 inhibitors as targeted antiarrhythmic therapies.

Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

A photoactivatable upconverting nanodevice boosts the lysosomal escape of PROTAC degraders for enhanced combination therapy.

PROteolysis TArgeting Chimeras have received increasing attention due to their capability to induce potent degradation of various disease-related proteins. However, the effective and controlled cytosolic delivery of current small-molecule PROTACs remains a challenge, primarily due to their intrinsic shortcomings, including unfavorable solubility, poor cell permeability, and limited spatiotemporal precision. Here, we develop a near-infrared light-controlled PROTAC delivery device (abbreviated as USDPR) that allows the efficient photoactivation of PROTAC function to achieve enhanced protein degradation. The nanodevice is constructed by encapsulating the commercial BRD4-targeting PROTACs (dBET6) in the hollow cavity of mesoporous silica-coated upconversion nanoparticles, followed by coating a Rose Bengal (RB) photosensitizer conjugated poly-L-lysine (PLL-RB). This composition enables NIR light-activatable generation of cytotoxic reactive oxygen species due to the energy transfer from the UCNPs to PLL-RB, which boosts the endo/lysosomal escape and subsequent cytosolic release of dBET6. We demonstrate that USDPR is capable of effectively degrading BRD4 in a NIR light-controlled manner. This in combination with NIR light-triggered photodynamic therapy enables an enhanced antitumor effect both in vitro and in vivo. This work thus presents a versatile strategy for controlled release of PROTACs and codelivery with photosensitizers using an NIR-responsive nanodevice, providing important insight into the design of effective PROTAC-based combination therapy.

Discovery of indole-2-one derivatives as BRD4 (BD1) selective inhibitors.

Bromodomain protein 4 (BRD4) is a member of the BET family, and its overexpression is closely associated with the development of many tumors. Inhibition of BRD4 shows great therapeutic potential in anti-tumor, and pan-BRD4 inhibitors show adverse effects of dose limiting toxicity and thrombocytopenia in clinical trials. To improve clinical effects and reduce side effects, more efforts have focused on seeking selective inhibitors of BD1 or BD2. Herein, a series of indole-2-one derivatives were designed and synthesized through docking-guided optimization to find BRD4-BD1 selective inhibitors, and their BRD4 inhibitory and antiproliferation activities were evaluated. Among them, compound 21r had potent BRD4 inhibitory activity (the IC50 values of 41 nM and 313 nM in BD1 and BD2 domain), excellent anti-proliferation (the IC50 values of 4.64 ± 0.30 µM, 0.78 ± 0.03 µM, 5.57 ± 1.03 µM against HL-60, MV-4-11 and HT-29 cells), and displayed low toxicity against normal cell GES-1 cells. Further studies revealed that 21r inhibited proliferation by decreasing the expression of proto-oncogene c-Myc, blocking cell cycle in G0/G1 phase, and inducing apoptosis in MV-4-11 cells in a dose-dependent manner. All the results showed that compound 21r was a potent BRD4 inhibitor with BD1 selectivity, which had potential in treatment of leukemia.

Semisynthesis and biological evaluation of novel honokiol thioethers against colon cancer cells HCT116 via inhibiting the transcription and expression of YAP protein.

Honokiol, derived from Magnolia officinalis (a traditional Chinese medicine), has been reported to have anticancer activity. Here, a series of novel honokiol thioethers bearing a 1,3,4-oxadiazole moiety were prepared and evaluated for their anticancer activities against three types of digestive system tumor cells. Biological evaluation showed that honokiol derivative 3k exhibited the best antiproliferative activity against HCT116 cells with an IC50 value of 6.1 µmol/L, superior to the reference drug 5-fluorouracil (IC50: 9.63 ± 0.27 µmol/L). The structure-activity relationships (SARs) indicated that the introduction of -(4-NO2)Ph, 3-pyridyl, -(2-F)Ph, -(4-F)Ph, -(3-F)Ph, -(4-Cl)Ph, and -(3-Cl)Ph groups was favorable for enhancing the anticancer activity of the title honokiol thioethers. Further study revealed that honokiol thioether 3k can well inhibit the proliferation of colon cancer cells HCT116, arresting the cells in G1 phase and inducing cell death. Moreover, a preliminary mechanism study indicated that 3k directly inhibits the transcription and expression of YAP protein without activating the Hippo signaling pathway. Thus, honokiol thioether 3k could be deeply developed for the development of honokiol-based anticancer candidates.

A BRD4-Targeting Photothermal Agent for Controlled Protein Degradation.

BRD4 protein plays a pivotal role in cell cycle regulation and differentiation. Disrupting the activity of BRD4 has emerged as a promising strategy for inhibiting the growth and proliferation of cancer cells. Herein, we introduced a BRD4-targeting photothermal agent for controlled protein degradation, aiming to enhance low-temperature photothermal therapy (PTT) for cancer treatment. By incorporating a BRD4 protein inhibitor into a cyanine dye scaffold, the photothermal agent specifically bond to the bromodomain of BRD4. Upon low power density laser irradiation, the agent induced protein degradation, directly destroying the BRD4 structure and inhibiting its transcriptional regulatory function. This strategy not only prolonged the retention time of the photothermal agent in cancer cells but also confined the targeted protein degradation process solely to the tumor tissue, minimizing side effects on normal tissues through the aid of exogenous signals. This work established a simple and feasible platform for future PTT agent design in clinical cancer treatment.

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma.

Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

Novel therapeutic strategies targeting bypass pathways and mitochondrial dysfunction to combat resistance to RET inhibitors in NSCLC.

RET fusion is an oncogenic driver in 1-2 % of patients with non-small cell lung cancer (NSCLC). Although RET-positive tumors have been treated with multikinase inhibitors such as vandetanib or RET-selective inhibitors, ultimately resistance to them develops. Here we established vandetanib resistance (VR) clones from LC-2/ad cells harboring CCDC6-RET fusion and explored the molecular mechanism of the resistance. Each VR clone had a distinct phenotype, implying they had acquired resistance via different mechanisms. Consistently, whole exome-seq and RNA-seq revealed that the VR clones had unique mutational signatures and expression profiles, and shared only a few common remarkable events. AXL and IGF-1R were activated as bypass pathway in different VR clones, and sensitive to a combination of RET and AXL inhibitors or IGF-1R inhibitors, respectively. SMARCA4 loss was also found in a particular VR clone and 55 % of post-TKI lung tumor tissues, being correlated with higher sensitivity to SMARCA4/SMARCA2 dual inhibition and shorter PFS after subsequent treatments. Finally, we detected an increased number of damaged mitochondria in one VR clone, which conferred sensitivity to mitochondrial electron transfer chain inhibitors. Increased mitochondria were also observed in post-TKI biopsy specimens in 13/20 cases of NSCLC, suggesting a potential strategy targeting mitochondria to treat resistant tumors. Our data propose new promising therapeutic options to combat resistance to RET inhibitors in NSCLC.

Leveraging the Fragment Molecular Orbital and MM-GBSA Methods in Virtual Screening for the Discovery of Novel Non-Covalent Inhibitors Targeting the TEAD Lipid Binding Pocket.

The Hippo pathway controls organ size and homeostasis and is linked to numerous diseases, including cancer. The transcriptional enhanced associate domain (TEAD) family of transcription factors acts as a receptor for downstream effectors, namely yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which binds to various transcription factors and is essential for stimulated gene transcription. YAP/TAZ-TEAD facilitates the upregulation of multiple genes involved in evolutionary cell proliferation and survival. TEAD1-4 overexpression has been observed in different cancers in various tissues, making TEAD an attractive target for drug development. The central drug-accessible pocket of TEAD is crucial because it undergoes a post-translational modification called auto-palmitoylation. Crystal structures of the C-terminal TEAD complex with small molecules are available in the Protein Data Bank, aiding structure-based drug design. In this study, we utilized the fragment molecular orbital (FMO) method, molecular dynamics (MD) simulations, shape-based screening, and molecular mechanics-generalized Born surface area (MM-GBSA) calculations for virtual screening, and we identified a novel non-covalent inhibitor-BC-001-with IC50 = 3.7 µM in a reporter assay. Subsequently, we optimized several analogs of BC-001 and found that the optimized compound BC-011 exhibited an IC50 of 72.43 nM. These findings can be used to design effective TEAD modulators with anticancer therapeutic implications.

BET inhibition decreases HMGCS2 and sensitizes resistant pancreatic tumors to gemcitabine.

Efforts to develop targetable molecular bases for drug resistance for pancreatic ductal adenocarcinoma (PDAC) have been equivocally successful. Using RNA-seq and ingenuity pathway analysis we identified that the superpathway of cholesterol biosynthesis is upregulated in gemcitabine resistant (gemR) tumors using a unique PDAC PDX model with resistance to gemcitabine acquired in vivo. Analysis of additional in vitro and in vivo gemR PDAC models showed that HMG-CoA synthase 2 (HMGCS2), an enzyme involved in cholesterol biosynthesis and rate limiting in ketogenesis, is overexpressed in these models. Mechanistic data demonstrate the novel findings that HMGCS2 contributes to gemR and confers metastatic properties in PDAC models, and that HMGCS2 is BRD4 dependent. Further, BET inhibitor JQ1 decreases levels of HMGCS2, sensitizes PDAC cells to gemcitabine, and a combination of gemcitabine and JQ1 induced regressions of gemR tumors in vivo. Our data suggest that decreasing HMGCS2 may reverse gemR, and that HMGCS2 represents a useful therapeutic target for treating gemcitabine resistant PDAC.

Discovery of potent small molecule ubiquitin-specific protease 10 inhibitors with anti-hepatocellular carcinoma activity through regulating YAP expression.

High expression of ubiquitin-specific protease 10 (USP10) promote the proliferation of hepatocellular carcinoma (HCC), thus the development of USP10 inhibitors holds promise as a novel therapeutic approach for HCC treatment. However, the development of selective USP10 inhibitor is still limited. In this study, we developed a novel USP10 inhibitor for investigating the feasibility of targeting USP10 for the treatment of HCC. Due to high USP10 inhibition potency and prominent selectivity, compound D1 bearing quinolin-4(1H)-one scaffold was identified as a lead compound. Subsequent research revealed that D1 significantly inhibits cell proliferation and clone formation in HCC cells. Mechanistic insights indicated that D1 targets the ubiquitin pathway, facilitating the degradation of YAP (Yes-associated protein), thereby triggering the downregulation of p53 and its downstream protein p21. Ultimately, this cascade leads to S-phase arrest in HCC cells, followed by cell apoptosis. Collectively, our findings highlight D1 as a promising starting point for USP10-positive HCC treatment, underscoring its potential as a vital tool for unraveling the functional intricacies of USP10.

Discovery of potent and novel dual NAMPT/BRD4 inhibitors for efficient treatment of hepatocellular carcinoma.

The NAPRT-induced increase in NAD+ levels was proposed as a mechanism contributing to hepatocellular carcinoma (HCC) resistance to NAMPT inhibitors. Thus, concurrently targeting NAMPT and NAPRT could be considered to overcome drug resistance. A BRD4 inhibitor downregulates the expression of NAPRT in HCC, and the combination of NAMPT inhibitors with BRD4 inhibitors simultaneously blocks NAD+ generation via salvage and the PH synthesis pathway. Moreover, the combination of the two agents significantly downregulated the expression of tumor-promoting genes and strongly promoted apoptosis. The present work identified various NAMPT/BRD4 dual inhibitors based on the multitargeted drug rationale. Among them, compound A2, which demonstrated the strongest effect, exhibited potent inhibition of NAMPT and BRD4 (IC50 = 35 and 58 nM, respectively). It significantly suppressed the growth and migration of HCC cells and facilitated their apoptosis. Furthermore, compound A2 also manifested a robust anticancer effect in HCCLM3 xenograft mouse models, with no apparent toxic effects. Our findings in this study provide an effective approach to target NAD+ metabolism for HCC treatment.

Therapeutic targeting of BET bromodomain and other epigenetic acetylrecognition domain-containing factors.

Development of cancer therapies targeting chromatin modifiers and transcriptional regulatory factors is rapidly expanding to include new targets and novel targeting strategies. At the same time, basic molecular research continues to refine our understanding of the epigenetic mechanisms regulating transcription, gene expression, and oncogenesis. This mini-review focuses on cancer therapies targeting the chromatin-associated factors that recognize histone lysine acetylation. Recently reported safety and efficacy are discussed for inhibitors targeting the bromodomains of bromodomain and extraterminal domain (BET) family proteins. In light of recent results indicating that the transcriptional regulator BRD4-PTEFb can function independently of BRD4's bromodomains, the clinical trial performance of these BET inhibitors is placed in a broader context of existing and potential strategies for targeting BRD4-PTEFb. Recently developed therapies targeting bromodomain-containing factors within the SWI/SNF (BAF) family of chromatin remodeling complexes are discussed, as is the potential for targeting the bromodomain-containing transcription factor TAF1 and the YEATS acetylrecognition domain-containing factor GAS41. Recent findings regarding the selectivity and combinatorial specificity of acetylrecognition are highlighted. In conclusion, the potential for further development is discussed with a focus on proximity-based therapies targeting this class of epigenetic factors.

BRD2-specific inhibitor, BBC0403, inhibits the progression of osteoarthritis pathogenesis in osteoarthritis-induced C57BL/6 male mice.

BACKGROUND AND PURPOSE: The discovery of new bromo- and extra-terminal inhibitors presents new drugs to treat osteoarthritis (OA). EXPERIMENTAL APPROACH: The new drug, BBC0403, was identified in the DNA-encoded library screening system by searching for compounds that target BRD (bromodomain-containing) proteins. The binding force with BRD proteins was evaluated using time-resolved fluorescence energy transfer (TR-FRET) and binding kinetics assays. Subsequently, in vitro and ex vivo analyses demonstrated the effects of the BRD2 inhibitor, BBC0403, on OA. For animal experiments, medial meniscus destabilization was performed to create a 12-week-old male C57BL/6 mouse model, and intra-articular (i.a.) injections were administered. Histological and immunohistochemical analyses were then performed. The underlying mechanism was confirmed by gene set enrichment analysis (GSEA) using RNA-seq. KEY RESULTS: TR-FRET and binding kinetics assays revealed that BBC0403 exhibited higher binding specificity for BRD2 compared to BRD3 and BRD4. The anti-OA effects of BBC0403 were tested at concentrations of 5, 10 and 20 µM (no cell toxicity in the range tested). The expression of catabolic factors, prostaglandin E2 (PGE2) production and extracellular matrix (ECM) degradation was reduced. Additionally, the i.a. injection of BBC0403 prevented OA cartilage degradation in mice. Finally, BBC0403 was demonstrated to suppress NF-κB and MAPK signalling pathways. CONCLUSION AND IMPLICATIONS: This study demonstrated that BBC0403 is a novel BRD2-specific inhibitor and a potential i.a.-injectable therapeutic agent to treat OA.

Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.