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 microM 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.
Augustine-Rauch
Karen A
KA
Department of Reproductive Toxicology, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, USA. karen.augustine@bms.com
Zhang
Qin J
QJ
Leonard
Jennifer L
JL
Chadderton
Antony
A
Welsh
Michael J
MJ
Rami
Harshad K
HK
Thompson
Mervyn
M
Gaster
Laramie
L
Wier
Patrick J
PJ
D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D019521:Body Patterning / Q000187:drug effects*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D051542:Hepatocyte Nuclear Factor 3-beta; D017403:In Situ Hybridization; D007211:Indoles / Q000633:toxicity*; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics; D011247:Pregnancy; D011725:Pyridines / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012702:Serotonin Antagonists / Q000633:toxicity*; D013723:Teratogens / Q000633:toxicity*; D014157:Transcription Factors / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; C496504:Foxa2 protein, rat; D007211:Indoles; D009687:Nuclear Proteins; D011725:Pyridines; C121558:SB 236057; D012702:Serotonin Antagonists; D013723:Teratogens; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta