Evidence for a molecular mechanism of teratogenicity of SB-236057, a 5-HT1B receptor inverse agonist that alters axial formation

BACKGROUND: SB-236057 is a potent skeletal teratogen in rodents and rabbits, producing axial and posterior somite malformations in cultured rat embryos. The compound shares some structural similarity to cyclopamine. METHODS: M13 phage display was used to identify amino acid motifs with binding affinity to SB-236057. A 10 μM SB-236057 solution was administered to cultured day 9 postcoitus rat embryos and real-time PCR was conducted at 6 hr posttreatment to evaluate early transcriptional response of axial development genes. Whole-mount in situ hybridization of selected transcripts was conducted on embryos at 48 hr post-compound administration. The rat-enhancer of split protein 1 (r-esp1) expression-functional characterization was done by transcriptional expression and morpholino antisense approaches. RESULTS: We identified several amino acid motifs that had high binding affinity to SB-236057-biotin conjugates, one with 100% sequence homology to a region of r-esp1, one of the Groucho homologs transcribed by the enhancer of split complex (En[spl]C). SB-236057 repressed expression of r-esp1 and members of the Notch-En[spl]C pathway. Goosecoid and HNF3-beta, both suspected to associate with Groucho proteins, were also responsive, although expression of another putative binding protein, engrailed-1 (en-1), and other en-1 pathway members was not affected. R-esp1 mRNA was localized along the axis and antisense inhibition produced similar somite malformations as SB-236057 did. At 48 hr post-SB-236057 or post-r-esp1 antisense administration, affected embryos demonstrated unchanged sonic hedgehog (shh) expression, however HNF3-beta expression was either absent, altered, or reduced. CONCLUSIONS: We present experimental evidence that the mechanism of SB-236057 teratogenicity includes transcriptional alterations to the Notch1-En[spl] pathway. In addition, alterations in HNF3-beta expression were similar to those induced by cyclopamine. The relationships between r-esp1 with Notch1 and shh signaling pathways and potential mechanisms of SB-236057 teratogenicity are also discussed.