(800.4) Discovery of the first tight-binding reversible antagonists of Hedgehog protein autoprocessing

Poster Board Number: A198

Andrew Wagner (Binghamton University, State University of New York ), Robert Stagnitta (Binghamton University, State University of New York), Zihan Xu (Binghamton University, State University of New York), John Pezzullo (SUNY-ESF Syracuse), Nabin Kandel (Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute), José-Luis Giner (SUNY-ESF Syracuse), Douglas Covey (Taylor Family Institute for Innovative Psychiatric Research), Chunyu Wang (Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute), Brian Callahan (Binghamton University, State University of New York)

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 IC50 value of 2x10-6 M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT-HBT, a derivative with a sub-micromolar IC50 of 3x10-7 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.

National Cancer Institute (Grant R01 CA206592); Department of Defense (Grant W81XWH-14-1-0155); NSF grant CHE-1048516 and NIH grant S10 OD012254.
Support: We acknowledge generous support from the NIH: CA206592 to BPC and CW; GM143714 to JLG; and HL067773 to DFC.