title: Förster 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.
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Owen
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: George
authors.initials: G
authors.lastname: Ngoje
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Lageman
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Bordeau
authors.affiliation: Department of Biological Sciences and The RNA Institute, University at Albany, State University of New York, Albany, NY 12222, USA.
authors.forename: Marlene
authors.initials: M
authors.lastname: Belfort
authors.affiliation: Department of Chemistry, Binghamton University, Binghamton, NY 13902, USA. Electronic address: callahan@binghamton.edu.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Callahan
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism*; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010865:Pilot Projects; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D059748:Proteolysis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D054852:Small Molecule Libraries; D013777:Tetrazoles / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C509245:DHH protein, human; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D008164:Luminescent Proteins; D009579:Nitrobenzoates; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C509239:SHH protein, human; C000608527:ST044643; C578287:Shh protein, Drosophila; D054852:Small Molecule Libraries; D013777:Tetrazoles; D002784:Cholesterol
keywords: Cancer; FRET; Fluorescence; Hedgehog protein; Protein engineering; Small molecule screening