The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-β-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-β signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-β1 at a concentration of 0.5 or 5 ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-β1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-β1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-β1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-β1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-β1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-β1. EdU staining showed that high concentration of TGF-β1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-β1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-β exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-β activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-β activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.

2018-06-01·Drug Metabolism and Disposition2区 · 医学

Refinement of In Vitro Methods for Identification of Aldehyde Oxidase Substrates Reveals Metabolites of Kinase Inhibitors

2区 · 医学

Article

作者: Dick, Ryan A

To identify aldehyde oxidase (AO) substrates, an assay procedure was developed that leverages the capabilities of high-resolution mass spectrometry to simultaneously monitor parent loss and formation of hydroxylated metabolite over time in incubations with liver cytosol. By incorporating metabolite monitoring, false positives resulting from metabolism by other cytosolic enzymes or processes were decreased. A diverse set of 34 kinase inhibitors containing AO-substrate motifs was screened, and 35% of the compounds were identified as human AO substrates. Confirmation was obtained through determination of the site of metabolism. Human AO substrates identified contained unsubstituted diazanaphthalene moieties (A77-01, INCB 28060, ML-347, LDN-193189, and SB-525334), 4-aminoquinazoline cores (lapatinib, lapatinib M1, and CL-387785), and terminal pyridine and pyrimidine groups (imatinib, bafetinib, and AMG 900). Rat and cynomolgus monkey AO displayed substrate specificities that overlapped moderately with human; rates of metabolism were often higher and lower for cynomolgus monkey and rat, respectively, compared with human. A subset of novel AO substrates identified in this study was also subjected to two other methods for AO substrate determination: comparison of human liver microsome and hepatocyte stability, and the effect of hydralazine, an AO-specific inhibitor, on hepatocyte stability. These methods appeared to correlate and be capable of identifying AO substrates when more than one-third of metabolism in hepatocytes was AO-mediated; however, significant limitations exist. Considering the sensitivity, efficiency, and definitiveness of the cytosol assay with metabolite monitoring, its use is recommended as a primary screen for AO substrates.

2013-06-01·Bioorganic & Medicinal Chemistry Letters4区 · 医学

Synthesis and structure–activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of Dorsomorphin: The discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe

4区 · 医学

Article

作者: Darren W. Engers ; Craig W. Lindsley ; Corey R. Hopkins ; Charles C. Hong ; Audrey Y. Frist

A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.

100 项与 ML-347 相关的药物交易

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