Identification of 2,6-Disubstituted 3H-Imidazo[4,5-b]pyridines as Therapeutic Agents for Dysferlinopathies through Phenotypic Screening on Patient-Derived Induced Pluripotent Stem Cells.

Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.

Effects of 4(1H)-quinolinone derivative, a novel non-nucleotide allosteric purinergic P2Y 2 agonist, on cardiomyocytes in neonatal rats.

Purinergic P2Y 2 receptors, G-protein coupled receptors that primarily couple with Gαq/11-proteins, are activated equipotently by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate. Evidence suggests that P2Y 2 agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y 2 agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y 2 agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y 2 agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gαq/11-coupled human P2Y (hP2Y) receptors and one representative Gαi/o-coupled hP2Y12 receptor. Its P2Y 2 agonistic effect on mouse P2Y 2 receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca2+ mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y2 activation in various cardiovascular diseases.

APOA-1 is a novel marker of erythroid cell maturation from hematopoietic stem cells in mice and humans.

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.

Construction of a high-performance human fetal liver-derived lentiviral cDNA library.

The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10(7) individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.

Cloning of the feline GADD45 cDNA and analysis of its mutation in feline lymphoma cell lines.

Growth arrest and DNA damage-inducible protein 45 (GADD45) plays an important role in suppressing multistep carcinogenesis. In this report, we describe the isolation of the complete wild-type feline GADD45 cDNA from feline tissues. Expression of feline GADD45 mRNA was detected in the liver, spleen, kidney, lung, and testis. The predicted amino acid sequences encoded by the full-length feline GADD45 cDNA display sequence homology with those from other vertebrates, and as in the case of human GADD45, cell growth suppression was observed by ectopic expression of feline GADD45. However, no mutations were detected by sequence analysis of feline GADD45 in several feline lymphoma cell lines, indicating that the GADD45 mutation might be uncommon in feline oncogenesis.

Transcriptional control of BubR1 by p53 and suppression of centrosome amplification by BubR1.

Elimination of the regulatory mechanism underlying numeral homeostasis of centrosomes, as seen in cells lacking p53, results in abnormal amplification of centrosomes, which increases the frequency of chromosome segregation errors, and thus contributes to the chromosome instability frequently observed in cancer cells. We have previously reported that p53(-/-) mouse cells in prolonged culture undergo genomic convergence similar to that observed during tumor progression; early-passage p53(-/-) cells are karyotypically heterogeneous due to extensive chromosome instability associated with centrosome amplification, while late-passage p53(-/-) cells are aneuploid yet karyotypically homogeneous and chromosomally stable. Moreover, they contain numerically normal centrosomes. Through the microarray analysis of early- and late-passage p53(-/-) cells, we identified the BubR1 spindle checkpoint protein, which plays a critical role in suppression of centrosome amplification and stabilization of chromosomes in late-passage p53(-/-) cells. Up-regulation of BubR1 augments the checkpoint function, which effectively senses the spindle/chromosome aberrations associated with centrosome amplification. We further found that BubR1 transcription is largely controlled by p53. In early-passage p53(-/-) cells, BubR1 expression is low and the checkpoint function in response to microtubule toxin is considerably compromised. In late-passage cells, however, regaining of BubR1 expression restores the checkpoint function to mitotic aberrations caused by microtubule toxin. Our studies demonstrate the molecular aspect of genomic convergence in cultured cells, providing critical information for understanding the stepwise progression of tumors.

Centrosome amplification and chromosomal instability in feline lymphoma cell lines.

To evaluate the presence of centrosome amplification and the resulting chromosomal instability in cat tumors, a newly established feline lymphoma cell line and four already established feline lymphoma cell lines were examined using immunohistochemical analysis of centrosomes. The number of chromosomes were subsequently counted by metaphase spread. Moreover, to explore whether mutational inactivation of the p53 gene or inactivation of the P53 protein caused by mdm2 gene overexpression, occurred in the feline lymphoma cell lines, mutational analysis of the feline p53 gene was carried out. The expression of feline mdm2 mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Centrosome amplification and chromosomal instability was observed in three out of the five feline lymphoma cell lines. Of these three feline lymphoma cell lines, one had aberrations in the P53 amino-acid sequence, whereas the others had none. There was no significant difference in the expression of mdm2 mRNA between peripheral blood mononuclear cells (PBMC) obtained from a normal cat and that of the five feline lymphoma cell lines. These findings indicate that centrosome amplification also occurs in cat tumors and is strongly correlated with chromosomal instability, suggesting that the immunostaining of centrosomes could be an alternative method for the examination of the chromosomal instability. Furthermore, this study suggests the presence of unknown mechanism that leads to the centrosome amplification in feline lymphomas.

Detection of the anti-P53 antibodies in dogs with tumors.

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.