Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

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 Förster resonance energy transfer (FRET)-active Hh precursor whose cholesterolysis can be monitored continuously in multi-well plates (dynamic range = 4, Z′ = 0.7), offering advantages in throughput over conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 substitution nucleophilic aromatic (S_NAr) mechanism.