Search

CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine. However, the stem cell selectivity mechanism of CDy1 has not been fully understood. Here, we report ALDH2 and ABCB1 as the molecular targets of CDy1, elucidated by live-cell affinity-matrix and ABC transporter CRISPRa library screening. The two unique orthogonal mechanisms provide the potential of multi-demensional cellular distinction of specific cell types., CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, but a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain (RC) was fully functional, both in human embryonic kidney (HEK) 293 cells and primary skin fibroblasts. This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes, such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at or slightly above 50 °C. In view of their potential consequences, these observations need to be further validated and explored by independent methods. Our study prompts a critical re-examination of the literature on mitochondria., Author summary To ensure a stable internal temperature, endothermic species make use of the heat released during the final steps of food burning by the mitochondria present in all cells of the organism. Indeed, only a fraction of the energy released by the oxidation of respiratory substrates is used to generate ATP, while a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured the temperature of active mitochondria in cultured intact human cells. Mitochondria were found to be more than 10 °C warmer when the respiratory chain was functional. This differential was abolished in cells depleted of mitochondrial DNA or by respiratory inhibitors but preserved or enhanced by the expression of thermogenic enzymes such as Ciona alternative oxidase or by uncoupling protein 1. The activity of various respiratory chain enzymes was found to be maximal near 50 °C. Note that in view of their potential consequences, the observations reported here need to be validated and explored further by independent methods.

Synthetic molecules that modulate and probe biological events are critical tools in chemical biology. Utilizing combinatorial and diversity-oriented synthetic strategies, access to large numbers of small molecules is becoming more and more feasible, and research groups in this field can take advantage of the power of chemical diversity. Since the majority of early studies were focused on the discovery of compounds that perturb protein functions, diversity-based approaches are often considered as therapeutic lead discovery tactics. However, the diversity-oriented approach can also be applied to advance distinct aims, such as target protein identification, or the development of imaging probes and sensors. This review provides a personal perspective of the chemical-diversity-based approach and how this principle can be adapted to various chemical biology studies.

Summary Glycolytic enzymes are attractive anticancer targets. They also carry out numerous, nonglycolytic "moonlighting" functions in cells. In this study, we investigated the anticancer activity of the triazine small molecule, GAPDS, that targets the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDS showed greater toxicity against cancer cells compared to a known GAPDH enzyme inhibitor. GAPDS also selectively inhibited cell migration and invasion. Our analysis showed that GAPDS treatment reduced GAPDH levels in the cytoplasm, which would modulate the secondary, moonlighting functions of this enzyme. We then used GAPDS as a probe to demonstrate that a moonlighting function of GAPDH is tubulin regulation, which may explain its anti-invasive properties. We also observed that GAPDS has potent anticancer activity in vivo. Our study indicates that strategies to target the secondary functions of anticancer candidates may yield potent therapeutics and useful chemical probes.

Type 2 diabetes mellitus (T2DM) significantly impacts on human health and patient numbers are predicted to rise. Discovering novel drugs and targets for treating T2DM is a research priority. In this study, we investigated targeting of the glycolysis enzyme, enolase, using the small molecule ENOblock, which binds enolase and modulates its non-glycolytic ‘moonlighting’ functions. In insulin-responsive cells ENOblock induced enolase nuclear translocation, where this enzyme acts as a transcriptional repressor. In a mammalian model of T2DM, ENOblock treatment reduced hyperglycemia and hyperlipidemia. Liver and kidney tissue of ENOblock-treated mice showed down-regulation of known enolase target genes and reduced enolase enzyme activity. Indicators of secondary diabetic complications, such as tissue apoptosis, inflammatory markers and fibrosis were inhibited by ENOblock treatment. Compared to the well-characterized anti-diabetes drug, rosiglitazone, ENOblock produced greater beneficial effects on lipid homeostasis, fibrosis, inflammatory markers, nephrotoxicity and cardiac hypertrophy. ENOblock treatment was associated with the down-regulation of phosphoenolpyruvate carboxykinase and sterol regulatory element-binding protein-1, which are known to produce anti-diabetic effects. In summary, these findings indicate that ENOblock has potential for therapeutic development to treat T2DM. Previously considered as a ‘boring’ housekeeping gene, these results also implicate enolase as a novel drug target for T2DM.

Enolase is a component of the glycolysis pathway and a "moonlighting" protein, with important roles in diverse cellular processes that are not related to its function in glycolysis. However, small molecule tools to probe enolase function have been restricted to crystallography or enzymology. In this study, we report the discovery of the small molecule "ENOblock", which is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity. ENOblock was isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. Further analysis revealed that ENOblock can inhibit cancer cell metastasis in vivo. Moreover, an unexpected role for enolase in glucose homeostasis was revealed by in vivo analysis. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.

Alzheimer's disease is the major cause of senile dementia with the hallmark of beta-amyloid deposition in neurons. Although the main cause(s) of this deposition is not fully understood, however, the wealth of the present literature data supports the pivotal role of reactive oxygen and nitrogen species in both the initiation and progression of beta-amyloid aggregation and deposition. In the present study, we were interested to evaluate the free-radical protecting effect of AA3E2, a triazine derivative with a beta-amyloid-breaking activity, among SK-N-MC neuroblastoma cells exposed to hydrogen peroxide (H(2)O(2)) as an exogenous source of free radicals. Exposure of the cells to different doses of AA3E2 (1-16 microM) for 3h followed by subsequent exposure to a single dose of H(2)O(2) (mainly 150 microM) attenuated the extent of superoxide dismutase (SOD) and catalase (CAT) inhibition by H(2)O(2), in a dose dependent manner. Furthermore, significant reduction was observed in the extent of cellular lactate dehydrogenase release, intracellular ROS and the extent of apoptosis among the cells pre-treated with AA3E2. Based on these data, an antioxidant mode of action is proposed for AA3E2 besides its previously beta-amyloid-breaking activity.

Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid beta peptide (Abeta), the so-called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Abeta from its precursor (beta- and gamma-secretases) or interrupt and/or reverse Abeta aggregation. To identify potent inhibitors of Abeta aggregation, we have developed a high-throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42-residue AD pathogenesis effecter Abeta42. Specifically, a tripartite fusion between the Tat-dependent export signal ssTorA, the Abeta42 peptide and the beta-lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Abeta42 moiety. Here, we reasoned that small, cell-permeable molecules that inhibited Abeta42 aggregation would render the ssTorA-Abeta42-Bla chimera competent for Tat export to the periplasm where Bla is active against beta-lactam antibiotics such as ampicillin. Using a fluorescence-based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell-permeable compounds that promoted efficient Tat-dependent export of ssTorA-Abeta42-Bla. Each of these was subsequently shown to be a bona fide inhibitor of Abeta42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.

Novel heterocyclic ring-substituted pyrimidines have been designed as inhibitors of glycogen synthase kinase-3beta (GSK-3beta) from the modification of known inhibitors. Several potent inhibitors exhibiting nanomolar activities were discovered against GSK-3beta kinase as well as in an NF-kappaB reporter gene assay. Based on the results from in vitro TNF-alpha release inhibition and in vivo endotoxima, these inhibitors are expected to be useful candidates for treatment of inflammation-related diseases.

Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, beta-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both beta-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.

Intracellular thermometry at the microscopic level is currently a hot topic. Herein we describe a small molecule fluorescent thermometer targeting mitochondria (Mito thermo yellow). Mito thermo yellow successfully demonstrates the ability to monitor the intracellular temperature gradient, generated by exogenous heating, in various cells.

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action. Citation Format: Yi Yang, Young Hyun Yu, Hyung-Ho Ha, Kyung-Hwa Lee, Kyung-Sub Moon, Kyung Keun Kim, Hangun Kim. Inhibitory activity of lichen secondary metabolite, physciosporin, against lung cancer cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2017-4209

Abnormal tau aggregates are presumed to be neurotoxic and are an important therapeutic target for multiple neurodegenerative disorders including Alzheimer’s disease. Growing evidence has shown that tau intermolecular disulfide cross-linking is critical in generating tau oligomers that serve as a building block for higher-order aggregates. Here we report that a small molecule inhibitor prevents tau aggregation by blocking the generation of disulfide cross-linked tau oligomers. Among the compounds tested, a rosamine derivative bearing mild thiol reactivity selectively labeled tau and effectively inhibited oligomerization and fibrillization processes in vitro. Our data suggest that controlling tau oxidation status could be a new therapeutic strategy for prevention of abnormal tau aggregation.

We report here a novel fluorescent chemical probe CDy5 which stains distinct neural stem/progenitor cells (NSPCs) by binding to acid ceramidase in mouse neurospheres. CDy5 is distributed evenly or unevenly to the daughter cells during multiple mitoses enabling the live imaging of symmetric and asymmetric divisions of isolated NSPCs.

Herein we report a parallel solid-phase synthesis of 1,3,5-triazine based nucleoside analogues by a three-step substitution, starting from 2,4,6-trichloro-1,3,5-triazine. A library of 80 galactosyl-1,3,5-triazine compounds was prepared in high purity without extensive reaction conditions or tedious purification, suggesting the generality of this method.

Fluorescent small-molecules become extensively used for live-cell imaging, but mainly in the context of labeling conjugates for other protein-binding motifs, such as antibodies.[1] As most fluorescent molecules are flat and hydrophobic, it has generally been believed that these fluorophores may bind to many hydrophobic proteins in cells, without any specificity.[2] This conventional wisdom, however, has not been tested systemically due to the lack of sufficiently diverse dye sources. Recently, we developed a diversity-oriented fluorescence library approach (DOFLA) to use fluorescent dyes to distinguish directly cellular components such as GTP,[3] DNA,[4] RNA,[5] heparin,[6] and organelles.[7] In this system, the diverse structural motifs of each dye molecule in the library endowed target selectivity. From these results, we envisioned that sufficient structural modifications of fluorophore scaffold could lead us to develop probes that label specific proteins from whole proteomes.[8] In addition to our recent finding of a fluorescein derivative labeling glutathione s-transferase,[9] here we report a rosamine derivative that labels tubulin in vitro and a mitochondrial protein in live cells. Previously a fluorescent small molecule capable of detecting differentiated myotubes was discovered from mitochondria-targeted rosamine library.[10, 11, 12] The hallmark of muscle differentiation is the fusion of mono-nucleated myoblasts to multi-nucleated myotubes.[13] During murine C2C12 myogenesis, the fluorescence intensity of one rosamine compound, E26, increased significantly. This myotube selectivity may be achieved by binding to one of the differentiation markers expressed more highly in myotubes; alternatively, the probe may detect other physiological changes after differentiation. When subjected for the further investigation, unfortunately, E26 showed photo-instability under strong and continuous light irradiation (Supplementary Fig. 1 online). The rosamine library compounds were retested under long-term light exposure and we selected two compounds (I25 and I31; Fig. 1) based on high photo-stability and fluorescence response after differentiation (I25: 2.4 ± 0.2, I31: 3.0 ± 0.5 fold increase, N = 3; Supplementary Fig. 2 online). Figure 1 Probes for myogenic differentiation In order to identify protein binders of these compounds, affinity matrices were prepared based on careful SAR studies (Fig. 2a). Affinity pull-down assay is the most conventional method for identification of small-molecule binding proteins.[14] Affinity resins were incubated with myotube lysates and washed with buffer to get rid of non-specific binders. Then, resin-bound proteins were separated by SDS-PAGE and stained with Coomassie blue (Fig. 2b). One enriched protein band at approximately 54 kDa was observed along with several other bands. To determine the specificity of protein binders to these compounds, competition assay was followed. Myotube cell lysate was pre-incubated with 100 µM of I25, I31, rhodamine 123, or rhodamine B before affinity pull-down. Figure 2 Identification of protein-binders in vitro vs. in living cells The strongest band at 54 kDa completely disappeared upon competition with unmodified I25 or I31, but not with rhodamine 123 or rhodamine B, which were included as structurally similar controls. Thus, we concluded that the 54 kDa band was the most convincing binding target protein of the compounds. The band was excised, sequenced, and identified to be tubulin (Supplementary Note 1 online). While affinity pull-down assay identified the major binder to be tubulin, a well-known cytosolic protein, our compounds appeared to be localized to mitochondria in live cells. Affinity-based isolation greatly depends on protein abundace as well as protein binding affinity. Since tubulin is a highly abundant protein in cells (10 ~ 20 µM),[15] despite the intrinsic affinity, its isolation might be an artifact. Therefore, we further explored the endogenus binding protein in the context of live cells. For live-cell investigation, we synthesized a cell-permeable chemical affinity derivative, which has a thiol reactive chloroacetyl group, to enable covalent binding to target proteins (Fig. 2c). The compound is named as CDy2, Compound of Designation yellow 2, following the biological convention of Cluster of Designation (CD) for cell-surface markers. The benefit of the chemical affinity probe is that once it forms a covalent bond with its targets in live cells. Those labeled proteins can be visualized by scanning the SDS-PAGE gel with a fluorescence scanner, even though the proteins are denatured. When applied to myoblasts and myotubes, CDy2 showed a 2.3-fold increase in fluorescence intensity after differentiation (2.3 ± 0.4 fold increase, N = 3; Fig. 2d), which is comparable to the increases observed with I25 and I31. To unveil the endogenus binding protein(s), myoblasts or myotubes were incubated with CDy2 for 1 hour. Labeled lysates were separated by SDS-PAGE and analyzed with a fluorescence scanner (Fig. 2d). Again, a unique fluorescently labeled band was observed around 54 kDa (Fig. 2e). Also, pre-treatment of myotubes with I31 reduced the intensity of CDy2-labeled protein band, indicating effective competition of CDy2 with I31 in live cells (Supplementary Fig. 3b online). To determine the identity of the labeled protein, cell lysates were separated by 2D-gel electrophoresis, and fluorescently labeled spots around 54 kDa were excised and sequenced (Fig. 2f; Supplementary Note 2 online). To our surprise, the major spots were found to be mitochondrial aldehyde dehydrogenase (ALDH2), not tubulin. To validate the binding in live cells, firstly ALDH2 or tubulin was overexpressed in HEK cells. Each protein was tagged with green fluorescent protein (GFP) to distinguish those from the endogenous proteins. Forty-eight hours after transfection, cells were labeled with CDy2 and each lysate was subjected to SDS-PAGE analysis (Fig. 3a); the HEK293 cell line was chosen because of its relatively high transfection efficiency. The result shows that CDy2 labeled ALDH2-GFP, but not tubulin-GFP. Secondly, ALDH2 expression was suppressed by siRNA (Fig. 3b). Upon ALDH2 knock-down, the CDy2-labeled band (54 kDa) was dramatically reduced. These siRNA and overexpression experiments clearly indicate that CDy2 selectively binds to ALDH2 in living cells. However, when treated to cell lysates, CDy2 labeled tubulin instead of ALDH2 (Supplementary Fig. 4d online). Altogether, these results suggest that CDy2 binds to two distinct proteins depending on the experimental environment. Figure 3 Validation of labeled protein identity in living cells In-gel fluorescence analysis showed that CDy2 labels ALDH2 stronger in myotubes over myoblast (Fig. 2e). Interestingly, however, the total amount of ALDH2 remained unchanged before and after differentiation. Thus it was necessary to determine the mechanism of CDy2 selectivity for myotubes. For example, the mitochondrial membrane potential of skeletal muscle is quite high, possibly due to increased energy requirements for muscle contractions;[13] this elevated membrane potential may cause the myotube-selectivity of CDy2. In fact, when cells were fixed with formaldehyde, CDy2 lost its mitochondrial preference, as well as its selectivity for myotubes (Supplementary Fig. 4a online). Further, the mitochondrial membrane potential was disrupted by treating cells with the mitochondrial uncoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone).[16] Upon pre-treatment with CCCP, the amount of fluorescently labeled protein was significantly reduced (Supplementary Fig. 4c online). These results support the notion that an increase in the mitochondrial membrane potential as a result of myogenesis gives rise to the selectivity of CDy2 for myotubes. Rosamine compounds are derivatives of rhodamine, which have long been used as mitochondrial probes. Their aromatic and cationic properties direct them to mitochondria due to the membrane potential across its bilayer.[17] The rosamine probes are sensitive to the incresed membrane potential after myogenic differentiation. Once localized in mitochondria, they labeled ALDH2 selectively. Although ALDH2 itself is not a differentiation marker, the selectivity to rosamine probes deserves careful consideration. Up until now, it has been generally believed that rhodamine dyes stain the mitochondrial membrane without showing specific interactions with mitochondrial proteins.[18] A cell is a highly ordered structure,[19] where small-molecule localization is precisely controlled based on chemical properties. Once a small molecule is sequestered in an organelle such as mitochondria, its interaction with proteins will be greatly limited within the cellular compartment. In this study, CDy2 showed an apparent binding affinity to tubulin in vitro, but resulted in binding to ALDH2 in live cells. This implies that CDy2 molecules are sequestered in mitochondria rapidly before they have a chance to react with tubulin in the cytoplasm. Elucidating small molecule’s binding protein is the most challenging part of chemical genetics work. Our observations cast important warning that the binding partner should be carefully evaluated in the context of the environmental factors.

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(II) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.

During muscle differentiation, mitochondria undergo dramatic changes in their morphology and distribution to prepare for the higher rate of energy consumption. By applying a mitochondria-targeted rosamine library in C2C12 myogenesis, we discovered one compound that controls muscle differentiation. When treated to undifferentiated myoblasts, our selected compound, B25, inhibited myotube formation, and when treated to fully differentiated myotubes, it induced fission of multinucleated myotubes into mononucleated fragments. Compared to myoseverin, which is known for inducing myotube fission by destabilizing microtubules, B25 affects neither microtubule stability nor cell cycle. Further investigation identified that B25 induces myotube fission through the activation of NF-kappaB, which is one of the important signaling pathways linked to skeletal muscle differentiation. So far, the use of small-molecule fluorophores is limited in the discovery of labeling agents or sensors. In addition to their potential as a sensor, here we show the application of fluorescent small molecules in the discovery of a bioactive probe that induces a specific cellular response.

Imaging a specific protein of interest in live cell has versatile applications in biological research. Recently, diverse chemical tags have been developed to overcome the limits of autofluorescence protein (FP) tags. However, current chemical methods still need to be progressed to compete with FPs in the scope of specificity and convenience in staining procedure. We report a novel protein tagging method that provides a convenient and specific fluorescent labeling of membrane proteins. Ω tag is developed by employing a mammalian enzyme glutathione sulfur-transferase omega 1 (GSTO1) and its partner dye, 5-bromomethyl fluorescein (BMF). Ω-tagged membrane proteins were labeled by BMF efficiently for live cell imaging and in-gel analysis. Endocytosis of epidermal growth factor receptor (EGFR) was successfully visualized by using this Ω tagging system. Ω tag will provide a convenient tool for the physiological study of membrane proteins in live cells.

SummaryCarcinoma-associated fibroblasts (CAFs) promote tumor invasion by secreting soluble factors. A tagged triazine library was screened in our novel transwell coculture model of CAF and oral squamous cell carcinoma (OSCC). We discovered compound S06, which reduced OSCC invasion by inhibiting secretion of CAF-derived proinvasive chemokines. The N-terminus of Hsp90 was found to be the cellular target of S06. Importantly, S06 did not induce hepatic toxicity, a side effect associated with well-known Hsp90 inhibitors. Moreover, S06 inhibited tumor cell migration in a zebrafish xenograft model. Our results demonstrate that Hsp90 is a novel target for stromal-based therapy to modulate proinvasive molecular crosstalk within the tumor microenvironment. Furthermore, S06 represents a new class of Hsp90 inhibitor and is an attractive candidate for anticancer drug development.