Identification of α-Tocopherol succinate as an RFFL-substrate interaction inhibitor inducing peripheral CFTR stabilization and apoptosis.

The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.

Identification of a derivative of the alkaloid emetine as an inhibitor of the YAP-TEAD interaction and its potential as an anticancer agent.

TEAD is a transcription factor responsible for the output of the tumor suppressor Hippo pathway. The transcriptional activity of TEAD requires molecular interaction with its transcriptional coactivator, YAP. Aberrant activation of TEAD is deeply involved in tumorigenesis and is associated with poor prognosis, suggesting that inhibitors targeting the YAP-TEAD system are promising as antitumor agents. In this study, we identified NPD689, an analog of the natural product alkaloid emetine, as an inhibitor of the YAP-TEAD interaction. NPD689 suppressed the transcriptional activity of TEAD and reduced the viability of human malignant pleural mesothelioma and non-small cell lung cancer cells but not the viability of normal human mesothelial cells. Our results suggest that NPD689 is not only a new useful chemical tool for elucidating the biological role of the YAP-TEAD system but also has potential as a starting compound for developing a cancer therapeutic agent that targets the YAP-TEAD interaction.

N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine inhibits bladder cancer progression by suppressing YAP1/TAZ.

Bladder cancer (BlC) is the fourth most common cancer in males worldwide, but few systemic chemotherapy options for its effective treatment exist. The development of new molecularly-targeted agents against BlC is therefore an urgent issue. The Hippo signaling pathway, with its upstream LATS kinases and downstream transcriptional co-activators YAP1 and TAZ, plays a pivotal role in diverse cell functions, including cell proliferation. Recent studies have shown that overexpression of YAP1 occurs in advanced BlCs and is associated with poor patient prognosis. Accessing data from our previous screening of a chemical library of compounds targeting the Hippo pathway, we identified DMPCA (N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine) as an agent able to induce the phosphorylation of LATS1 and YAP1/TAZ in BlC cells, thereby suppressing their viability both in vitro and in mouse xenografts. Our data indicate that DMPCA has a potent anti-tumor effect, and raise the possibility that this agent may represent a new and effective therapeutic option for BlC.

Development of Low-Molecular-Weight Compounds Targeting the Cancer-Associated KLF5 Transcription Factor.

Krüppel-like factor 5 (KLF5) is a potential target for anticancer drugs. However, as an intrinsically disordered protein (IDP) whose tertiary structure cannot be solved, innovative strategies are needed. We focused on its hydrophobic α-helix structure, defined as an induced helical motif (IHM), which is a possible interface for protein-protein interaction. Using mathematical analyses predicting the α-helix's structure and hydrophobicity, a 4-amino-acid site (V-A-I-F) was identified as an IHM. Low-molecular-weight compounds that mimic the main chain conformation of the α-helix with the four side chains of V-A-I-F were synthesized using bicyclic pyrazinooxadiazine-4,7-dione. These compounds selectively suppressed the proliferation and survival of cancer cells but not noncancer cells and decreased the protein but not mRNA levels of KLF5 in addition to reducing proteins of Wnt signaling. The compounds further suppressed transplanted colorectal cancer cells in vivo without side effects. Our approach appears promising for developing drugs against key IDPs.

Identification of a Small Molecule That Enhances Ferroptosis via Inhibition of Ferroptosis Suppressor Protein 1 (FSP1).

Glutathione peroxidase 4 (GPX4) is an intracellular enzyme that oxidizes glutathione while reducing lipid peroxides and is a promising target for cancer therapy. To date, several GPX4 inhibitors have been reported to exhibit cytotoxicity against cancer cells. However, some cancer cells are less sensitive to the known GPX4 inhibitors. This study aimed to explore compounds showing synergistic effects with GPX4 inhibitors. We screened a chemical library and identified a compound named NPD4928, whose cytotoxicity was enhanced in the presence of a GPX4 inhibitor. Furthermore, we identified ferroptosis suppressor protein 1 as its target protein. The results indicate that NPD4928 enhanced the sensitivity of various cancer cells to GPX4 inhibitors, suggesting that the combination might have therapeutic potential via the induction of ferroptosis.

Mitochondrial complex I inhibitors suppress tumor growth through concomitant acidification of the intra- and extracellular environment.

The disruption of the tumor microenvironment (TME) is a promising anti-cancer strategy, but its effective targeting for solid tumors remains unknown. Here, we investigated the anti-cancer activity of the mitochondrial complex I inhibitor intervenolin (ITV), which modulates the TME independent of energy depletion. By modulating lactate metabolism, ITV induced the concomitant acidification of the intra- and extracellular environment, which synergistically suppressed S6K1 activity in cancer cells through protein phosphatase-2A-mediated dephosphorylation via G-protein-coupled receptor(s). Other complex I inhibitors including metformin and rotenone were also found to exert the same effect through an energy depletion-independent manner as ITV. In mouse and patient-derived xenograft models, ITV was found to suppress tumor growth and its mode of action was further confirmed. The TME is usually acidic owing to glycolytic cancer cell metabolism, and this condition is more susceptible to complex I inhibitors. Thus, we have demonstrated a potential treatment strategy for solid tumors.

Pharmacological inhibition of Mint3 attenuates tumour growth, metastasis, and endotoxic shock.

Hypoxia-inducible factor-1 (HIF-1) plays essential roles in human diseases, though its central role in oxygen homoeostasis hinders the development of direct HIF-1-targeted pharmacological approaches. Here, we surveyed small-molecule compounds that efficiently inhibit the transcriptional activity of HIF-1 without affecting body homoeostasis. We focused on Mint3, which activates HIF-1 transcriptional activity in limited types of cells, such as cancer cells and macrophages, by suppressing the factor inhibiting HIF-1 (FIH-1). We identified naphthofluorescein, which inhibited the Mint3-FIH-1 interaction in vitro and suppressed Mint3-dependent HIF-1 activity and glycolysis in cancer cells and macrophages without evidence of cytotoxicity in vitro. In vivo naphthofluorescein administration suppressed tumour growth and metastasis without adverse effects, similar to the genetic depletion of Mint3. Naphthofluorescein attenuated inflammatory cytokine production and endotoxic shock in mice. Thus, Mint3 inhibitors may present a new targeted therapeutic option for cancer and inflammatory diseases by avoiding severe adverse effects.

Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation.

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

Targeting Epigenetic and Posttranscriptional Gene Regulation by PSF Impairs Hormone Therapy-Refractory Cancer Growth.

RNA-binding protein PSF functions as an epigenetic modifier by interacting with long noncoding RNAs and the corepressor complex. PSF also promotes RNA splicing events to enhance oncogenic signals. In this study, we conducted an in vitro chemical array screen and identified multiple small molecules that interact with PSF. Several molecules inhibited RNA binding by PSF and decreased prostate cancer cell viability. Among these molecules and its derivatives was a promising molecule, No. 10-3 [7,8-dihydroxy-4-(4-methoxyphenyl)chromen-2-one], that was the most effective at blocking PSF RNA-binding ability and suppressing treatment-resistant prostate and breast cancer cell proliferation. Exposure to No. 10-3 inhibited PSF target gene expression at the mRNA level. Treatment with No. 10-3 reversed epigenetically repressed PSF downstream targets, such as cell-cycle inhibitors, at the transcriptional level. Chromatin immunoprecipitation sequencing in prostate cancer cells revealed that No. 10-3 enhances histone acetylation to induce expression of apoptosis as well as cell-cycle inhibitors. Furthermore, No. 10-3 exhibited antitumor efficacy in a hormone therapy-resistant prostate cancer xenograft mouse model, suppressing treatment-resistant tumor growth. Taken together, this study highlights the feasibility of targeting PSF-mediated epigenetic and RNA-splicing activities for the treatment of aggressive cancers. SIGNIFICANCE: This study identifies small molecules that target PSF-RNA interactions and suppress hormone therapy-refractory cancer growth, suggesting the potential of targeting PSF-mediated gene regulation for cancer treatment.

CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells.

The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial-mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.

Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Miclxin, a Novel MIC60 Inhibitor, Induces Apoptosis via Mitochondrial Stress in ß-Catenin Mutant Tumor Cells.

The Wnt signaling pathway regulates diverse cellular processes. ß-Catenin is one of the major components of this pathway, in which it plays a main role. Although it has been established that ß-catenin is mutated in a wide variety of tumors, there are currently no effective therapeutic agents that target ß-catenin. In this study, we searched for the compound that targets mutant ß-catenin and found DS37262926 (miclxin). Miclxin exhibited ß-catenin-dependent apoptosis in ß-catenin-mutated HCT116 cells and isogenic HCT116 (CTNNB1 Δ45/-) cells; however, this effect was not observed in isogenic HCT116 (CTNNB1 +/-) cells. Using miclxin-immobilized beads, MIC60, one of the major components of the mitochondrial contact site and cristae organizing system (MICOS) complex, was identified as a target protein of miclxin. We revealed that MIC60 dysfunction caused by miclxin induced a mitochondrial stress response in a mutant ß-catenin-dependent manner. Activation of the mitochondrial stress response was responsible for the downregulation of Bcl-2, leading to severe loss of mitochondrial membrane potential and subsequent apoptosis-inducing factor-dependent apoptosis. Our findings suggest that targeting MIC60 is a potential strategy with which tumor cells can be killed through induction of severe mitochondrial damage in a mutant ß-catenin-dependent manner.

Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry.

Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several other methods used for drug-target identification, such as biochemical fractionation or radioactive ligand binding assay, affinity purification is widely used for its straight-forward and easy approach. To identify the target protein of an inhibitor of cancer cell-accelerated fibroblast migration, we prepared the inhibitor-conjugated beads by photo-crosslinking. Proteins were pulled down from cell lysates by the compound beads and separated by SDS-PAGE, and a specifically pulled down protein was cut out, trypsin-digested, analyzed using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) and identified by peptide mass fingerprinting (PMF) method. Since the photo-crosslinking enables the immobilization of ligands on an affinity matrix in a functional group-independent manner, we do not have to determine the functional group of the compound to conjugate the matrix. In addition, as compared to other MS techniques such as electrospray ionization, MALDI offers a less complex sample preparation procedure and higher sensitivity, and thus is better suited for the rapid identification of proteins isolated by gel electrophoresis.

Mechanism of Action of Prethioviridamide, an Anticancer Ribosomally Synthesized and Post-Translationally Modified Peptide with a Polythioamide Structure.

Thioviridamide, prethioviridamide, and JBIR-140, which are ribosomally synthesized and post-translationally modified peptides (RiPPs) possessing five thioamide bonds, induce selective apoptosis in various cancer cells, especially those expressing the adenovirus oncogene E1A. However, the target protein of this unique family of bioactive compounds was previously unknown. To investigate the mechanism of action, we adopted a combined approach of genome-wide shRNA library screening, transcriptome profiling, and biochemical identification of prethioviridamide-binding proteins. An shRNA screen identified 63 genes involved in cell sensitivity to prethioviridamide, which included translation initiation factors, aminoacyl tRNA synthetases, and mitochondrial proteins. Transcriptome profiling and subsequent analysis revealed that prethioviridamide induces the integrated stress response (ISR) through the GCN2-ATF4 pathway, which is likely to cause cell death. Furthermore, we found that prethioviridamide binds and inhibits respiratory chain complex V (F1Fo-ATP synthase) in mitochondria, suggesting that inhibition of complex V leads to activation of the GCN2-ATF4 pathway. These results imply that the members of a unique family of RiPPs with polythioamide structure target mitochondria to induce the ISR.

Development of Mirror-Image Screening Systems for XIAP BIR3 Domain Inhibitors.

The X-linked inhibitor of apoptosis protein baculovirus IAP repeat (XIAP BIR3) domain is a promising therapeutic target for cancer treatment. For the mirror-image screening campaign to identify drug candidates from an unexplored mirror-image natural product library, a facile synthetic protocol for XIAP BIR3 domain synthesis was established by a native chemical ligation strategy using conserved cysteines present among BIR domains. The native and mirror-image XIAP BIR3 domains with an appropriate functional group for labeling were prepared using the established protocol. Taking advantage of the resulting synthetic proteins, several bioassay systems were developed to characterize inhibitors of the protein-protein interaction between the XIAP BIR3 domain and the second mitochondria-derived activator of caspases.

Identification of a chemical modulator of EZH2-mediated silencing by cell-based high-throughput screening assay.

Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.

A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell-accelerated fibroblast migration.

Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened ∼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity.

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

Identification of a small-molecule ligand of ß-arrestin1 as an inhibitor of stromal fibroblast cell migration accelerated by cancer cells.

Stromal fibroblasts, which occupy a major portion of the tumor microenvironment, play an important role in cancer metastasis. Thus, targeting of these fibroblasts activated by cancer cells (carcinoma-associated fibroblasts; CAFs) might aid in the improved treatment of cancer metastasis. NIH3T3 fibroblasts cocultured with MCF7 cells displayed enhanced migration compared to NIH3T3 fibroblasts cultured alone. We used this system to identify the small-molecule inhibitors responsible for their enhanced migration, a characteristic of CAFs. We selected ß-arrestin1, which showed high expression in cocultured cells, as a molecular target for such inhibitors. Cofilin, a protein downstream of ß-arrestin1, is activated/dephosphorylated in this condition. The small-molecule ligands of ß-arrestin1 obtained by chemical array were then examined using a wound healing coculture assay. RKN5755 was identified as a selective inhibitor of activated fibroblasts. RKN5755 inhibited the enhanced migration of fibroblasts cocultured with cancer cells by binding to ß-arrestin1 and interfering with ß-arrestin1-mediated cofilin signaling pathways. Therefore, these results demonstrate the role of ß-arrestin1 in the activation of fibroblasts and inhibiting this protein by small molecule inhibitor might be a potential therapeutic target for the stromal fibroblast activation (cancer-stroma interaction).

Investigation of the inhibitory mechanism of apomorphine against MDM2-p53 interaction.

Mirror-image screening using d-proteins is a powerful approach to provide mirror-image structures of chiral natural products for drug screening. During the course of our screening study for novel MDM2-p53 interaction inhibitors, we identified that NPD6878 (R-(-)-apomorphine) inhibited both the native l-MDM2-l-p53 interaction and the mirror-image d-MDM2-d-p53 interaction at equipotent doses. In addition, both enantiomers of apomorphine showed potent inhibitory activity against the native MDM2-p53 interaction. In this study, we investigated the inhibitory mechanism of both enantiomers of apomorphine against the MDM2-p53 interaction. Achiral oxoapomorphine, which was converted from chiral apomorphines under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2, leading to the inhibitory effect against the binding to p53.