Pharmacological folding chaperones act as allosteric ligands of Frizzled4

Abstract

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-β-catenin pathway and for drug discovery.

Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

“…sites that do not produce antagonism or agonism) exist on GPCRs that produce conformational changes. We (26) and other laboratories (27–30) have similar findings. In fact, there are data to show that the entire surface of the GPCR can be considered a potential binding site for ligands, since conformational changes occur (31–34).…”

Photoluminescent Peptide‐Based Nanostructures as FRET Donor for Fluorophore Dye

“…Fluorescence microscopy : NBD/PEG 8 ‐F 6 solution (15 μL; 10 mg mL −1 ), prepared 24 h before, was deposited on a clean coverslip glass, dried, and imaged with fluorescence microscopy. Immunofluorescence images were taken with a Leica DFC320 video camera (Leica, Milan, Italy) connected to a Leica DMRB microscope equipped with 10 X and 40 X objectives, and the Image J Software (National Institutes of Health, Bethesda, MD) was used for analysis …”

“…Fluorescence microscopy:N BD/PEG 8 -F 6 solution (15 mL; 10 mg mL À1 ), prepared 24 hb efore, was deposited on ac lean coverslip glass, dried, and imaged with fluorescence microscopy.I mmunofluorescence images were taken with aL eica DFC320 video camera (Leica, Milan, Italy) connected to aL eica DMRB microscope equipped with 10 Xa nd 40 Xo bjectives, and the Image JS oftware (National Institutes of Health, Bethesda, MD) was used for analysis. [27] WAXS and SAXS:F iber diffraction WAXS and SAXS patterns were recorded from dried fibers prepared by the stretch-frame method. [22] Briefly,adroplet (10 mL) of peptide aqueous solution (3 wt %) was suspended between the ends of aw ax-coated capillary (spaced 2mma part).…”

Novel Allosteric Modulators of G Protein-coupled Receptors

“…For example, a series of small molecules that were originally designed to act as pharmacological chaperones for a misfolded mutant of Frizzled4 were subsequently identified as novel allosteric modulators of the wild-type form of Frizzled4; the binding site for these compounds was proposed to be located in the vicinity of intracellular loop 3 of the receptor (77). The recent crystal structure of the Smoothened receptor bound to the allosteric modulator, Sant1, also revealed an allosteric pocket that is located deep within the transmembrane-spanning cavity of the receptor, toward the cytosolic end; this is in contrast to the binding site that is closer to the extracellular entrance and utilized by canonical ligands of this receptor (78).…”