Discovery of the first tight-binding reversible antagonists of Hedgehog protein autoprocessing.

Hedgehog (Hh) signaling ligands (HhN) are expressed as precursors with an adjacent C-terminal autoprocessing domain, HhC. Autoprocessing activity of HhC releases and cholesterylates the Hh ligand thereby activating the HhN for full biological activity. Inhibitors of HhC activity are of interest as probes of the Hh signaling pathway, and HhC inhibitors may also offer new therapeutic approaches to suppress oncogenic signaling activity of HhN. The intramolecular nature of the HhN/HhC system poses a challenge for reversible inhibitors, and to date most antagonists of HhC bind the active site covalently. Here we report we report the discovery and optimization of the first tight-binding reversible inhibitor of HhC. Using a light-to-dark FRET activity assay for Drosophila melanogaster HhC, we screened a focused library of 1187 sterol-like analogs in search of HhC inhibitors. Four structurally related hits were identified and further validated by dose-response assays. The most effective inhibitor from the screen had an IC value of 2x10 M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT-HBT, a derivative with a sub-micromolar IC of 3x10 M. Inhibition appears to be mediated by a previously unidentified allosteric binding site on HhC. Kinetic analysis, photoaffinity labeling, computational modeling, and point mutagenesis studies suggest that the binding site is nearby, but not overlapping with the cholesterol binding site of HhC.