Neural crest stem/progenitor cells (NCSCs) populate a variety of tissues, and their dysregulation is implicated in several human diseases including craniosynostosis and neuroblastoma. We hypothesised that small molecules that inhibit NCSC induction or differentiation may represent potential therapeutically relevant drugs in these disorders. We screened 640 FDA-approved compounds currently in clinical use for other conditions to identify those which disrupt development of NCSC-derived skeletal elements that form the zebrafish jaw. In the primary screen, we used heterozygous transgenic sox10:gfp zebrafish to directly visualise NCSC-derived jaw cartilage. We noted partial toxicity of this transgene in relation to jaw patterning, suggesting that our primary screen was sensitised for NCSC defects, and we confirmed 10 novel, 4 previously reported, and 2 functional analogue drug hits in wild-type embryos. Of these drugs, 9/14 and 7/14, respectively, are known to target pathways implicated in osteoarthritis pathogenesis or to cause reduced bone mineral density/increased fracture risk as side effects in patients treated for other conditions, suggesting that our screen enriched for pathways targeting skeletal tissue homeostasis. We selected one drug that inhibited NCSC induction and one drug that inhibits bone mineralisation for further detailed analyses which reflect our initial hypotheses. These drugs were leflunomide and cyclosporin A, respectively, and their functional analogues, teriflunomide and FK506 (tacrolimus). We identified their critical developmental windows of activity, showing that the severity of defects observed related to the timing, duration, and dose of treatment. While leflunomide has previously been shown to inhibit NCSC induction, we demonstrate additional later roles in cartilage remodelling. Both drugs altered expression of extracellular matrix metalloproteinases. As proof-of-concept, we also tested drug treatment of disease-relevant mammalian cells. While leflunomide treatment inhibited the viability of several human NCSC-derived neuroblastoma cell lines coincident with altered expression of genes involved in ribosome biogenesis and transcription, FK506 enhanced murine calvarial osteoblast differentiation and prevented fusion of the coronal suture in calvarial explants taken from Crouzon syndrome mice.

中文翻译：

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FDA 批准的影响颅面骨骼发育和颅缝早闭化合物的药物筛选。

神经嵴干/祖细胞（NCSC）分布在多种组织中，它们的失调与多种人类疾病有关，包括颅缝早闭和神经母细胞瘤。我们假设抑制 NCSC 诱导或分化的小分子可能代表这些疾病的潜在治疗相关药物。我们筛选了目前临床上用于其他病症的 640 种经 FDA 批准的化合物，以确定那些会破坏形成斑马鱼下颌的 NCSC 衍生骨骼元素发育的化合物。在初级筛选中，我们使用杂合转基因 sox10:gfp 斑马鱼来直接可视化 NCSC 来源的颌软骨。我们注意到该转基因与下颌图案相关的部分毒性，表明我们的初级筛选对 NCSC 缺陷敏感，并且我们在野生型胚胎中确认了 10 种新颖药物、4 种先前报道的药物和 2 种功能类似药物。在这些药物中，已知 9/14 和 7/14 分别针对与骨关节炎发病机制有关的途径，或导致骨矿物质密度降低/骨折风险增加（作为接受其他疾病治疗的患者的副作用），这表明我们的筛选丰富了针对骨骼组织稳态的途径。我们选择了一种抑制 NCSC 诱导的药物和一种抑制骨矿化的药物进行进一步的详细分析，这反映了我们最初的假设。这些药物分别是来氟米特和环孢菌素 A，以及它们的功能类似物特立氟胺和 FK506（他克莫司）。我们确定了它们的关键发育活动窗口，表明观察到的缺陷的严重程度与治疗的时间、持续时间和剂量有关。虽然来氟米特之前已被证明可以抑制 NCSC 诱导，但我们随后证明了它在软骨重塑中的其他作用。两种药物都改变细胞外基质金属蛋白酶的表达。作为概念验证，我们还测试了疾病相关哺乳动物细胞的药物治疗。虽然来氟米特治疗抑制了几种人 NCSC 衍生的神经母细胞瘤细胞系的活力，同时与核糖体生物发生和转录相关基因的表达改变相一致，但 FK506 增强了小鼠颅骨成骨细胞的分化，并阻止取自克鲁宗综合征小鼠的颅骨外植体中冠状缝的融合。