Functionally distinct Gata3/Chd4 complexes coordinately establish T helper 2 (Th2) cell identity.

GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4-nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.

Identification of phosphorylated proteins involved in the oncogenesis of prostate cancer via Pin1-proteomic analysis.

BACKGROUND: The peptidyl-prolyl isomerase Pin1 regulates a subset of phosphorylated proteins by catalyzing the cis-trans isomerization of their specific phosphorylated Ser/Thr-Pro motifs. Although Pin1 has been shown to be involved in cell transformation and the maintenance of the malignant phenotype in prostate cancer, its specific substrates during these processes have not yet been determined. METHODS: Cancer-specific phosphorylated proteins were isolated from two human prostate cancer cell lines (PC-3, LNCaP) and the Dunning rat prostate cancer cell lines by GST-pull down analysis with recombinant GST-Pin1 protein. These proteins were then identified by the LC-MS/MS analysis using a Q-Tof micro mass spectrometer and processed for further functional analysis. RESULTS: We newly identified five prostate cancer-specific Pin1 binding proteins (PINBPs) in this screen. Among these, TRK-fused gene (TFG) was found to be preferentially up-regulated in prostate cancer cell lines and tissues. The targeted inhibition of TFG by specific siRNA resulted in the reduced cell proliferation and the induction of premature senescence in PC3 prostate cancer cells. We further found that TFG can facilitate the cell signaling mediated by NF-kappaB and androgen receptor (AR). Tissue micro-dissection based quantitative RT-PCR analysis of prostate cancer tissues following radical prostatectomy further revealed that TFG expression is closely associated with both a higher probability and shorter period of tumor recurrence following surgery. CONCLUSIONS: Pin1-based proteomics analysis is a useful tool for the identification of prostate cancer-specific phosphorylated proteins. TFG could be a potential diagnostic and/or prognostic marker and therapeutic target in prostate cancer.

Synthesis and discovery of N-carbonylpyrrolidine- or N-sulfonylpyrrolidine-containing uracil derivatives as potent human deoxyuridine triphosphatase inhibitors.

Recently, deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure-activity relationship (SAR) studies of uracil derivatives. N-Carbonylpyrrolidine- and N-sulfonylpyrrolidine-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (12k) having excellent potencies (IC(50) = 0.15 µM). The X-ray structure of a complex of 16a and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = 0.27-0.30 µM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors.

1,2,3-Triazole-containing uracil derivatives with excellent pharmacokinetics as a novel class of potent human deoxyuridine triphosphatase inhibitors.

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing uracil derivatives. Compound 45a, which possesses 1,5-disubstituted 1,2,3-triazole moiety that mimics the amide bond of tert-amide-containing inhibitor 6b locked in a cis conformation showed potent inhibitory activity, and its structure-activity relationship studies led us to the discovery of highly potent inhibitors 48c and 50c (IC(50) = ~0.029 µM). These derivatives dramatically enhanced the growth inhibition activity of 5-fluoro-2'-deoxyuridine against HeLa S3 cells in vitro (EC(50) = ~0.05 µM). In addition, compound 50c exhibited a markedly improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that 50c is a promising candidate for combination cancer chemotherapies with TS inhibitors.

Discovery of highly potent human deoxyuridine triphosphatase inhibitors based on the conformation restriction strategy.

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound 7, we designed and synthesized two conformation restricted analogues, i.e., compounds 8 and 9. These compounds exhibited increased in vitro potency compared with the parent compound 7. Further structure-activity relationship (SAR) studies identified a compound 43 with the highest in vitro potency (IC(50) = 39 nM, EC(50) = 66 nM). Furthermore, compound 43 had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.

Discovery of a novel class of potent human deoxyuridine triphosphatase inhibitors remarkably enhancing the antitumor activity of thymidylate synthase inhibitors.

Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure-activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor 7 (IC(50) = 100 µM), we developed compound 26, which is the most potent human dUTPase inhibitor (IC(50) = 0.021 µM) reported to date. Not only does compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2'-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC(50) = 0.075 µM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising candidate for clinical development.