Discovery of a Novel, Isothiazolonaphthoquinone-Based Small Molecule Activator of FOXO Nuclear-Cytoplasmic Shuttling.
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ABSTRACT:
SUBMITTER: Cautain B
PROVIDER: S-EPMC5147912 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.
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Project description:In the canonical Wnt pathway, beta-catenin acts as a key coactivator that stimulates target gene expression through interaction with Tcf/Lef transcription factors. Its nuclear accumulation is the hallmark of active Wnt signaling and is frequently associated with cancers. Chibby (Cby) is an evolutionarily conserved molecule that represses beta-catenin-dependent gene activation. Although Cby, in conjunction with 14-3-3 chaperones, controls beta-catenin distribution, its molecular nature remains largely unclear. Here, we provide compelling evidence that Cby harbors bona fide nuclear localization signal (NLS) and nuclear export signal (NES) motifs, and constitutively shuttles between the nucleus and cytoplasm. Efficient nuclear export of Cby requires a cooperative action of the intrinsic NES, 14-3-3, and the CRM1 nuclear export receptor. Notably, 14-3-3 docking provokes Cby binding to CRM1 while inhibiting its interaction with the nuclear import receptor importin-alpha, thereby promoting cytoplasmic compartmentalization of Cby at steady state. Importantly, the NLS- and NES-dependent shuttling of Cby modulates the dynamic intracellular localization of beta-catenin. In support of our model, short hairpin RNA-mediated knockdown of endogenous Cby results in nuclear accumulation of beta-catenin. Taken together, these findings unravel the molecular basis through which a combinatorial action of Cby and 14-3-3 proteins controls the dynamic nuclear-cytoplasmic trafficking of beta-catenin.
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Project description:Small heterodimer partner (SHP, NR0B2) is a nuclear orphan receptor without endogenous ligands. Due to its crucial inhibitory role in liver cancer, it is of importance to identify small molecule agonists of SHP. As such, we initiated a probe discovery effort to identify compounds capable of modulating SHP function. First, we performed binding assays using small molecule microarrays (SMM) and discovered 5-(diethylsulfamoyl)-3-hydroxynaphthalene-2-carboxylic acid (DSHN) as a novel activator of SHP. DSHN transcriptionally activated Shp mRNA, but also stabilized the SHP protein by preventing its ubiquitination and degradation. Second, we identified Ccl2 as a new SHP target gene by RNA-seq. We showed that activation of SHP by DSHN repressed Ccl2 expression and secretion by inhibiting p65 activation of CCL2 promoter activity, as demonstrated in vivo in Shp-/- mice and in vitro in HCC cells with SHP overexpression and knockdown. Third, we elucidated a strong inhibitory effect of SHP and DSHN on HCC cell migration and invasion by antagonizing the effect of CCL2. Lastly, by interrogating a publicly available database to retrieve SHP expression profiles from multiple types of human cancers, we established a negative association of SHP expression with human cancer metastasis and patient survival. In summary, the discovery of a novel small molecule activator of SHP provides a therapeutic perspective for future translational and preclinical studies to inhibit HCC metastasis by blocking Ccl2 signaling. Mol Cancer Ther; 15(10); 2294-301. ©2016 AACR.
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Project description:The adapter protein tumor necrosis factor receptor (TNFR)1-associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.
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Project description:UnlabelledThe nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication.ImportanceIt is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.
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Project description:The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.
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Project description:UnlabelledBackgroundCaspase-3 inhibition has been demonstrated to be therapeutically effective in moderating excessive programmed cell death. Interest in caspase-3 as a therapeutic target has led many to pursue the development of inhibitors. To date, only a few series of non-peptide inhibitors have been described, and these have limitations on their drug-like properties.MethodsHere, we report the screening of 70 novel small molecules against the caspase-3 enzyme which belongs to four different series (indole fluoromethylketone, indole difluoro and tetrafluorophenoxymethylketone, and oxalamide). Selected molecules were subjected for counter-screening, cell-based, ADME/PK assays in order to understand the potency and drug-like properties.ResultsThe screening yielded series of hits with IC50 values ranging from 0.11 to 10 ?M with reasonable SAR, irreversible mode of inhibition, and reasonable selectivity against other proteases including caspase-1, cathepsin B and D, and thrombin. On the basis of in vitro profile, the selected molecules were evaluated for their drug-like properties. Among the compounds evaluated, compound 3D exhibited good solubility, low permeability, interaction with efflux pump, and low potential for CYP450 drug-drug interaction. After intravenous administration, compound 3D showed low clearance (588 ml/hr/kg), medium volume of distribution, and good oral bioavailability (90%).ConclusionsThese results support further advancement of compound 3D in different apoptotic models to develop as a new anti-apoptotic agent in relevant disease conditions.
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Project description:We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.
| S-EPMC4007828 | biostudies-other