Identificatie van een nieuwe verbinding die ectopische staartvorming induceert, indicatief voor modificaties in het Wnt/PCP signaalpad, door middel van een chemical genetics screening in de zebravis voor BMP en wnt signaalpad modulatoren

Over the last decade, zebrafish have proven increasingly useful as an animal model in theearly drug discovery process. As an alternative to target-based drug discovery approaches,chemical genetics in zebrafish enables the identification of bioactive small molecules basedon their ability to induce specific developmental dysmorphologies or behaviors. These in vivophenotypes can be associated with the modulation of key signaling pathways and ultimatelylead to the elucidation of the compound’s biological target.Because of multiple genetic screens carried out to date, a wide range of mutant phenotypesin zebrafish have been thoroughly characterized and reported, including genes and pathwaycomponents of the Bone Morphogenetic Protein (BMP) and Wingless/Int (Wnt) signalingpathways for which the biochemistry and/or mechanisms of action have been well-studied.Dorsoventral (DV) and anterior-posterior (AP) axis defects as well as ectopic tail formationhave been reported in BMP/Wnt mutant or transgenic zebrafish. The recent identification ofdorsomorphin, a novel small-molecule inhibitor of BMP receptor signaling, proved theusefulness of phenotype-based compound screening in zebrafish embryos. This moleculeinduced severe dorsalization (as a result of DV patterning defects) in developing embryos. Todate, this molecule and some of its derivatives have been extensively studied as modifiers ofiron homeostasis, bone formation, metabolism and stem cell differentiation.Molecules that can modulate BMP or Wnt signaling pathways are of pharmacologicalinterest, as these conserved pathways are not only crucial for embryonic development, butare also important in adult homeostasis, tissue repair and disease. Aberrant signaling islinked with several major diseases such as cancer and arthritic diseases. New drug-like leadcompounds that target and modulate BMP or Wnt signaling could therefore be oftherapeutic utility.A zebrafish-based phenotypic screen for BMP and Wnt signaling modulators was carried outusing the Strathclyde Natural Products Library as a potential source of novel, bioactivecompounds. The library consists of 5000 methanolic plant extracts representing 90% of plantfamilies worldwide. One of the bioactive plant extracts identified in this screen – Jasminumgilgianum, an Oleaceae species native to Papua New Guinea – induced ectopic tails duringzebrafish embryonic development. As ectopic tail formation occurs when BMP or noncanonicalWnt signaling is inhibited during the tail protrusion process, we suspected aconstituent of this extract to act as a modulator of either one or both of these pathways. Abioassay-guided isolation was carried out on the basis of this zebrafish phenotype,identifying para-coumaric acid methyl ester (pCAME) as the active compound.We performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos,including a tissue-specific marker analysis of the secondary tails. We found pCAME tosynergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails,and cause convergence-extension defects in compound-treated embryos. Taken together,our results indicated that pCAME may interfere with non-canonical Wnt signaling.Structurally-related compounds of pCAME were reported to inhibit both MAP kinases andtyrosine kinases. Therefore, we looked for possible kinase targets present in the Wnt/PCPpathway. C-jun N-terminal kinase (Jnk) belongs to the family of MAPKs, and is downstreamof the receptor complex of the Wnt/PCP pathway. Antisense morpholino knockdown of jnk2and pulsing of embryos with the small-molecule inhibitor SP600125, suggested pCAME totarget Jnk kinase. Although SP600125 is generally used as specific Jnk inhibitor thecompound is able to inhibit more kinases than only Jnk isoforms. Immunoblottingexperiments however, revealed that pCAME does not directly inhibit Jnk-mediatedphosphorylation of C-jun. These results suggested the modulation of other targets/pathwaysin ectopic tail formation by pCAME. Further investigation of pCAME’s mechanism of actionwill help determine this compound’s pharmacological utility.status: publishe