Functional and Molecular Characterization of Hedgehog Signalling Regulation

Hedgehog (Hh) signalling plays a key role in controlling cell fate and patterning of tissues during development and also throughout adulthood for tissue homeostasis. The physiological importance of this pathway is further highlighted when mutations in Hh pathway components lead to developmental disorders and diseases: defects of the limbs, cranial structure or nervous system, and cancer in adults. Although the pathway is highly conserved between flies and humans, important divergences exist between them, including the necessity for Sufu to negatively regulate the Gli transcription factors, thought to mediate all Hedgehog transcriptional outputs. The first study presented in this thesis describes Zfp629, a new transcription factor first identified as a novel interactor of Sufu through a proteomics study. ChIP-seq analysis and validation confirmed that Zfp629 controlled the expression of Foxp2. Also a member of this pathway is Smoothened (Smo), a class F G-protein coupled receptor (GPCR). Several inhibitors have already been introduced to treat Hh-driven cancers such as medulloblastoma and basal cell carcinoma. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of SMO crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In the second study of this thesis, we docked 3.2 million available, lead-like molecules against SMO, looking for those with high physical complementarity to its structure; this represents the first such campaign against a class F GPCR. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize SMO with IC50 values better than 50 μM. A second screen for analogs revealed another six molecules, with IC50 values in the 2.3 μM-22.4 μM range. Importantly, one of the most active of the new Smo antagonists continued to be efficacious against the D473H mutant of SMO, which confers clinical resistance to the antagonist vismodegib in cancer treatment.Ph.D