MSY000444_suppl. T01x.xlsx (64.97 kB)

Supplementary Material for: An FDA-Approved Drug Screen for Compounds Influencing Craniofacial Skeletal Development and Craniosynostosis

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posted on 24.07.2018, 11:32 by Seda M., Geerlings M., Lim P., Jeyabalan-Srikaran J., Cichon A.-C., Scambler P.J., Beales P.L., Hernandez-Hernandez V., Stoker A.W., Jenkins D.
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.