Department of Developmental Biology, University of Pittsburgh, BST3, 3501 5th Avenue, Pittsburgh, PA 15213, USA.
The University of Pittsburgh Drug Discovery Institute, 200 Lothrop Street, Pittsburgh, PA 15260, USA.
Molecules. 2018 Jul 11;23(7):1691. doi: 10.3390/molecules23071691.
Zebrafish is the preferred vertebrate model for high throughput chemical screens to discover modulators of complex biological pathways. We adapted a transgenic zebrafish line, , which reports on fibroblast growth factor (Fgf)/Ras/Mapk activity, into a quantitative, high-content chemical screen to identify novel Fgf hyperactivators as chemical probes for zebrafish heart development and regeneration. We screened 10,000 compounds from the TimTec ActiProbe library, and identified several structurally distinct classes of molecules that enhanced Fgf/Ras/Mapk signaling. We chose three agents-ST020101, ST011282, and ST006994-for confirmatory and functional studies based on potency, repeatability with repurchased material, favorable whole organism toxicity, and evidence of structure⁻activity relationships. Functional follow-up assays confirmed that all three compounds induced the expression of Fgf target genes during zebrafish embryonic development. Moreover, these compounds increased cardiac progenitor populations by effecting a fate change from endothelial to cardiac progenitors that translated into increased numbers of cardiomyocytes. Interestingly, ST006994 augmented Fgf/Ras/Mapk signaling without increasing Erk phosphorylation, suggesting a molecular mechanism of action downstream of Erk. We posit that the ST006994 pharmacophore could become a unique chemical probe to uncover novel mechanisms of Fgf signaling during heart development and regeneration downstream of the Mapk signaling node.
斑马鱼是高通量化学筛选以发现复杂生物途径调节剂的首选脊椎动物模型。我们将一种报告成纤维细胞生长因子 (Fgf)/Ras/Mapk 活性的转基因斑马鱼系 ，改编为一种定量、高内涵化学筛选方法，以鉴定新型 Fgf 激活剂作为研究斑马鱼心脏发育和再生的化学探针。我们从 TimTec ActiProbe 文库中筛选了 10000 种化合物，并鉴定出几种结构不同的分子类别，它们增强了 Fgf/Ras/Mapk 信号。我们选择了三种试剂-ST020101、ST011282 和 ST006994-用于基于效力、重复购买材料的可重复性、有利的整体毒性和结构-活性关系的证据进行确认和功能研究。功能后续分析证实，这三种化合物都在斑马鱼胚胎发育过程中诱导了 Fgf 靶基因的表达。此外，这些化合物通过诱导内皮细胞向心脏祖细胞的命运转变，增加了心脏祖细胞的数量，从而增加了心肌细胞的数量。有趣的是，ST006994 增强了 Fgf/Ras/Mapk 信号，而没有增加 Erk 磷酸化，这表明其作用机制可能位于 Erk 下游。我们假设 ST006994 的药效团可能成为一种独特的化学探针，以揭示心脏发育和再生过程中 Fgf 信号的新机制，该机制位于 Mapk 信号节点的下游。
