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 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.

The Mechanism of Cholesterol Modification of Hedgehog Ligand

“…It should therefore be considered a placeholder structure that simply provides a tentative scaffold, positioning the cholesterol molecule in an orientation conducive to the chemical reaction. It is therefore just a starting point requiring further verification using experimental approaches, such as distance restraints derived from targeted mutagenesis combined with cholesterol‐addition assays …”

“…The protonation states of individual ionizable residues in the precursor were set according to their sidechains at neutral pH, that is, Asp and Glu sidechains were deprotonated; Lys, Arg, Tyr, and Cys sidechains were protonated, and His sidechains were neutral with a proton at the δ nitrogen atom. This is a reasonable starting point since pH dependence of Hh autoprocessing does not seem to have been specifically studied but is assumed to occur at a pH of 7.1 in the endoplasmic reticulum, and in vitro cholesterol‐addition assays also use this pH value for their buffers . The histidine residues occurring in the vicinity of the chemical reaction, that is, His329 and His450, are both solvent exposed and do not have any specific interactions that would obviously favor a particular protonation state.…”

“…There is no experimental information available yet on SRR structure, how cholesterol is recruited from the membrane into the SRR, and how SRR is attached or oriented with respect to the membrane. HH autoprocessing can, however, be replicated in vitro using cholesterol solubilization by detergents such as Triton X‐100, suggesting that a specific membrane environment may not be necessary for the autoprocessing reaction . The issue of membrane attachment of SRR is therefore not addressed in this study but certainly deserves attention in future experimental and computational efforts.…”

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer

“…We used an activity assay to evaluate interactions of A and G with the cholesterolysis‐active HhC segment from Drosophila melanogaster . The assay continuously monitors activity by changes in the fluorescence of FRET‐active proteins attached to HhC . Cyan fluorescent protein, serving as the FRET donor, is fused to the N terminus of HhC, replacing the signaling ligand; yellow fluorescent protein, the FRET acceptor, is fused to C terminus of HhC (Figure A).…”

Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling

“…More recently, Owen et al described an optical cholesterolysis assay that employs Förster resonance energy transfer (FRET) [ 126 ]. An engineered fluorescent Hh precursor is again utilized, except that the labels are soluble recombinant proteins.…”