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

ABSTRACT: 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 (SNAr) mechanism.

SUBMITTER: Owen TS

PROVIDER: S-EPMC4591182 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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

Owen Timothy S TS Ngoje George G Lageman Travis J TJ Bordeau Brandon M BM Belfort Marlene M Callahan Brian P BP

Analytical biochemistry 20150618

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 con ...[more]

PMID: 26095399

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