Targeting the hedgehog pathway in disease: a structural biology approach

The Hedgehog (Hh) pathway is a morphogen pathway essential for proper embryonic development of all bilaterians that remains active in adults where it contributes to tissue homoeostasis. Malfunction of the pathway results in developmental defect, while constitutive activation was correlated with multiple cancers. The Hh pathway is a validated cancer drug target with two FDA approved inhibitors targeting the Hh GPCR signal transducer Smoothened. This thesis focuses on two alternative Hh targets, the Sonic Hh (ShhN) morphogen and its acyltransferase Skinny Hh. In Chapter 2, the aim was to structurally characterise a complex between ShhN and its only small-molecule inhibitor Robotnikinin using in silico docking and crystallography. The results revealed that the most likely binding site of Robotnikinin is the pseudo-active site of ShhN and that the interactions involve the His134-Ser136 loop. In Chapter 3, the aim was to identify novel ShhN inhibitors using crystallographic fragment-based screening of 1,200 unique fragments. The screening resulted in 13 potential hits, 5 of which were found within the ion binding cleft. In Chapter 4, the aim was to validate the obtained hits and conduct preliminary structure activity relationship (SAR) assays. Based on the results, FRG 155 was identified as the preferred hit that bound to the the ShhN pseudo-active site with low micromolar affinity. Preliminary SAR identified positive and negative features of the fragment. In Chapter 5, the aim was to structurally and functionally characterise Skinny Hh. Protocols for successful expression and purification of milligram quantities of thermostable and active Skinny Hh were developed, providing a platform for crystallisation and drug screening. The first luminal loop of Skinny Hh was found to be essential for the activity of Skinny Hh. In summary, alternative Hh pathway targets, ShhN and Skinny Hh, were characterised using structural biology approaches and will contribute to the development of novel inhibitors with the application in Hh ligand-dependent tumours.