3-Amino-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carbonitrile: A fluorescent molecule that induces differentiation in PC12 cells.

Neural differentiation is triggered by the activation of multiple signaling pathways initiated by various neurotrophic factors. An elucidation of these mechanisms is anticipated to facilitate the prevention of diseases and the development of novel therapeutic approaches. Alternative small-molecule inducers for neuroscience studies are required instead of protein-based reagents for more efficient and convenient experiments. We demonstrated that small molecules of thieno[2,3-b]pyridine derivatives that induce neural differentiation, compounds 3a and 9a in particular, exhibited significant neuritogenic activity in rat pheochromocytoma (PC12) cells. Moreover, 3a displayed pronounced fluorescence and a discernible Stokes shift. Furthermore, the outcome of the experiment conducted on the NGF-insensitive clones of rat PC12 cells, and the results of the intercellular uptake analyses suggested that the 3a-mediated activation of neural differentiation occurred independently of the TrkA receptor. Therefore, 3a portrays potential applicability both as a small molecule reagent to replace novel neurotrophic factors and as a potent fluorescent reagent for various techniques, including bioimaging.

Structure-activity relationship study of the anti-obesity natural product yoshinone A.

Yoshinone A was derived from marine algae and shown to inhibit adipogenic differentiation. The natural compound is composed of a γ-pyrone ring and a side chain and that contains two asymmetric carbons. Although their absolute configuration has been determined, there is no information available on the stereoisomers and their bioactivities. To address this question, we synthesized all four stereoisomers and measured their activities. We also prepared three more derivatives of yoshinone A and found that the stereo-configuration inside the side chain, the γ-pyrone ring, and bulkiness of the side chain all played important roles in its activity. Our findings should help to elucidate the mechanism of action of yoshinone A.

Small-molecule-induced clustering of heparan sulfate promotes cell adhesion.

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy.

Deactivation of STAT6 through serine 707 phosphorylation by JNK.

Signal transducer and activator of transcription 6 (STAT6), which plays a critical role in immune responses, is activated by interleukin-4 (IL-4). Activity of STAT family members is regulated primarily by tyrosine phosphorylations and possibly also by serine phosphorylations. Here, we report a previously undescribed serine phosphorylation of STAT6, which is activated by cell stress or by the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Our analyses suggest that Ser-707 is phosphorylated by c-Jun N-terminal kinase (JNK). Phosphorylation decreases the DNA binding ability of IL-4-stimulated STAT6, thereby inhibiting the transcription of STAT6-responsive genes. Inactivation of STAT6 by JNK-dependent Ser-707 phosphorylation may be one mechanism of controlling the balance between IL-1ß and IL-4 signals.

Translational repression stabilizes messenger RNA of autophagy-related genes.

In response to amino acid starvation, autophagy mediates the lysosome-dependent turnover of cytosolic components via autophagosome formation. Despite advances in understanding the molecular basis of autophagy process, the regulatory mechanism remains unclear. Here, we show that repression of protein synthesis stabilizes the messenger RNAs of specific autophagy-related (ATG) genes, increasing their respective half-lives. Further analysis indicated that the stabilization process is attributable to the coding region of the mRNAs. The results suggest a novel mechanism of autophagy regulation by post-transcriptional mRNA stabilization, in which repression of protein synthesis plays a direct role to sustain the autophagy process.

Analysis of the Structural Aspects of Tannin-Based Adhesives by 2D-NMR.

We developed non-toxic, harmless adhesives composed of all-natural and renewable resources, of which one was composed of tannin and gelatin, which unfortunately was lacking water resistance, and the other of tannin and ε-poly-l-lysine. In this study, we analyzed the chemical structures of these adhesives by two-dimensional nuclear magnetic resonance (2D-NMR) to explain the difference in water-resistance of the two glues. The results showed that only one proton was left in the benzene ring of tannin after mixing. This suggests that the amino group of the protein was directly attached to the benzene ring by a Michael addition-type reaction, and not to the hydroxyl group. In addition, the heteronuclear multiple bond correlation spectrum of the tannin-poly-l-lysine compound indicated that the hydroxyl groups of the tannin oxidized, suggesting the improvement of its water resistance.

Drug Leads Derived from Japanese Marine Organisms.

Many natural products with extraordinary chemical structures and brilliant biological activities have been obtained from marine organisms. We have investigated such fascinating bioactive molecules, exemplified by the potent marine toxin palytoxin and the antitumor molecule halichondrin B, which has been developed as the anticancer drug Halaven®, to explore novel frontiers in organic chemistry and bioscience. Working within the traditional discipline, we have sought to acquire a deeper understanding of biological phenomena. We introduce here our major work along with up-todate topics. We isolated yoshinone A from marine cyanobacteria and completed a gram-scale synthesis. Yoshinone A is a novel polyketide that inhibited the differentiation of 3T3-L1 cells into adipocytes without significant cytotoxicity. The detailed mechanisms of action will be elucidated via further experiments in vitro and in vivo. In this study, we explore the true producers of okadaic acid and halichondrin B by immunostaining of Halichondria okadai with an antibody that was prepared using these natural products as an antigen. We will analyze isolated symbionts and reveal biosynthetic pathways.

Identification of the hypertension drug niflumic acid as a glycine receptor inhibitor.

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl- conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Chemical genetic identification of the histamine H1 receptor as a stimulator of insulin-induced adipogenesis.

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.

A chemical probe that labels human pluripotent stem cells.

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Small-molecule fluorescent probes for specific RNA targets.

A method was developed that uses small molecules as fluorescent probes to detect specific mRNAs. In this approach, the fluorescence of fluorophore-quencher conjugates is restored by the binding of an mRNA aptamer tag to the quencher segment of the molecules. The method allows real-time detection of mRNA transcripts in vitro.

A novel screen using the Reck tumor suppressor gene promoter detects both conventional and metastasis-suppressing anticancer drugs.

The membrane-anchored matrix metalloproteinase-regulator RECK is often downregulated in various types of cancers; the levels of residual RECK in resected tumors often correlate with better prognosis. Forced expression of RECK in cancer cells suppresses tumor angiogenesis, invasion, and metastasis in xenograft models. RECK is therefore a promising marker for benignancy and a potential effector in cancer therapy. We established a cell line containing two transgene systems: (1) the secreted alkaline phosphatase (SEAP) gene fused to Reck promoter and (2) the HRAS(12V) oncogene driven by the Tet-off promoter system. This cell line exhibits transformed phenotype in regular medium and flat morphology with increased SEAP activity in the presence of doxycycline, allowing the assessment of RECK-inducing activity of chemicals in the contexts of both transformed and untransformed cells. Our pilot experiments with 880 known bioactive compounds detected 34 compounds that activate RECK promoter; among these, 10 were authentic anticancer drugs. Four selected compounds up-regulated endogenous RECK protein in several human cancer cell lines. The top-ranking compound, disulfiram, strongly suppressed spontaneous lung-metastasis of human fibrosarcoma cells in nude mice. Our data demonstrate the value of this screen in discovering effective cancer therapeutics.

A small molecule that blocks fat synthesis by inhibiting the activation of SREBP.

Sterol regulatory element binding proteins (SREBPs) are transcription factors that activate transcription of the genes involved in cholesterol and fatty acid biosynthesis. In the present study, we show that a small synthetic molecule we previously discovered to block adipogenesis is an inhibitor of the SREBP activation. The diarylthiazole derivative, now called fatostatin, impairs the activation process of SREBPs, thereby decreasing the transcription of lipogenic genes in cells. Our analysis suggests that fatostatin inhibits the ER-Golgi translocation of SREBPs through binding to their escort protein, the SREBP cleavage-activating protein (SCAP), at a distinct site from the sterol-binding domain. Fatostatin blocked increases in body weight, blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled food intake. Fatostatin may serve as a tool for gaining further insights into the regulation of SREBP.

Polyproline-rod approach to isolating protein targets of bioactive small molecules: isolation of a new target of indomethacin.

Identification of protein targets of bioactive small molecules has been a technical hurdle of chemical genetics. Here we report a polyproline-rod approach to isolating protein targets of small molecules from cell lysates. The results indicate that insertion of a long, rigid polyproline helix between a small-molecule bait and a biotin tag boosts the capacity of affinity purification and thereby permits isolation of low-abundance or low-affinity proteins. In the course of the proof-of-concept experiments, we isolated glyoxalase 1 (GLO1) as a new target of indomethacin, a widely used antiinflammatory drug. Molecular biological experiments suggest that inhibition of GLO1 enzyme activity is related to the clinically recognized beneficial side effects of the indomethacin family of nonsteroidal antiinflammatory drugs.

Synthesis of synthetic small molecule transcription factors (STF).

External control of gene expression by small synthetic molecules represents a challenge in chemistry. Here we discuss the synthetic aspects of small molecule transcription factor mimics including a molecule we call STF1 (1). STF1 stimulates the transcription of a reporter gene controlled by specific DNA elements and behaves just as a naturally occurring transcription factor. However, STF1 had limited cell permeability, even though the hairpin-polyamide-FITC conjugate and the wrenchnolol (2) are cell permeable as separate compounds. Systematic synthetic exploration of STF1 analogs (STFs), by optimizing the physical properties of the molecule, is expected to increase its cell permeability for biological studies.

Identification of bioactive molecules by adipogenesis profiling of organic compounds.

An important step in the postgenomic drug discovery is the construction of high quality chemical libraries that generate bioactive molecules at high rates. Here we report a cell-based approach to composing a focused library of biologically active compounds. A collection of bioactive non-cytotoxic chemicals was identified from a divergent library through the effects on the insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic and sensitive morphological alterations in cultured mammalian cells. The resulting focused library amply contained unique compounds with a broad range of pharmacological effects, including glucose-uptake enhancement, cytokine inhibition, osteogenesis stimulation, and selective suppression of cancer cells. Adipogenesis profiling of organic compounds generates a focused chemical library for multiple biological effects that are seemingly unrelated to adipogenesis, just as genetic screens with the morphology of fly eyes identify oncogenes and neurodegenerative genes.