Hedgehog (Hh) proteins function in cell/cell signaling processes linked to human embryo development and the progression of several types of cancer. Here we describe an optical assay of hedgehog cholesterolysis, a unique autoprocessing event critical for Hh function. The assay uses a recombinant FRET-active hedgehog precursor whose cholesterolysis can be monitored continuously in multi-well plates (dynamic range, 4; Z’, 0.7), offering advantages in throughput over conventional SDS-PAGE assays. Application of the optical assay in a pilot small molecule screen produced a novel cholesterolysis inhibitor (apparent IC50, 5×10−6 M) that appears to inactivate hedgehog covalently by a SNAr mechanism.

Hedgehog proteins, signaling molecules implicated in human embryo development and cancer, can be inhibited at the stage of autoprocessing by the trivalent arsenical phenyl arsine oxide (PhAsIII). The interaction (apparent Ki, 4×10−7M) is characterized by an optical binding assay and by NMR spectroscopy. PhAsIII appears to be the first validated inhibitor of hedgehog autoprocessing, which is unique to hedgehog proteins and essential for biological activity.

A series of tolyl 2-azido-2-deoxy-thio-glucoside donors with different combinations of protecting groups were prepared. These donors were used in glycosylation reactions to test the correlations between the stereoselectivity and the pattern of the protecting groups. Acetyl groups showed a position dependent stereo-directing effect. A remote participating mechanism is proposed to explain the observed results.

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