A cell-based Sonic Hedgehog signaling transduction system to identify additive and synergistic chemical interactions

Chemical co-exposures are important contributors to adverse biological responses yet remain poorly understood, especially in the context of prenatal development. Sonic Hedgehog (Shh) signaling is an essential developmental pathway that is sensitive to small-molecule disruption and directly linked to common and etiologically complex human birth defects. Numerous mechanistically diverse small-molecule Shh pathway antagonists have been identified, but their interactions in pathway disruption have received minimal attention. We established a tractable co-culture model in which autonomous SHH ligand production initiates this complex inter- and intracellular signal transduction cascade and culminates in activation of a GLI-responsive luminescent reporter. Compounds reported to target SHH ligand processing (RU-SKI 43, AY 9944, U18666A), SMO-mediated signal transduction (cyclopamine, vismodegib, piperonyl butoxide, cannabidiol), and GLI transcription factors (GANT 61, arsenic trioxide) reduced Shh pathway-driven reporter activity with AC50 values in the low micromolar range or below. We then evaluated chemical interactions among Shh pathway inhibitors using isobolographic analysis. Co-exposure assays revealed additive interactions from combined SMO and GLI inhibition, whereas disruption of SMO and cholesterol dynamics synergistically decreased Shh pathway activity. Unexpectedly, piperonyl butoxide synergized with other SMO inhibitors, and further characterization of piperonyl butoxide's impacts on Shh signaling supported an additional mechanism of inhibition independent of SMO. In zebrafish embryos, combined exposure to piperonyl butoxide and cyclopamine also produced a synergistic increase in craniofacial dysmorphogenesis. These findings demonstrate the importance of tractable models that recapitulate complex signal transduction pathways to empirically test for additive and synergistic chemical interactions in risk assessment.