Structure-Activity Relations of Successful Pharmacologic Chaperones for Rescue of Naturally Occurring and Manufactured Mutants of the Gonadotropin-Releasing Hormone Receptor

Abstract

We expressed a test system of wild-type (WT) rat (r) and human (h) gonadotropin-releasing hormone (GnRH) receptors (GnRHRs), including naturally occurring (13) and manufactured (five) "loss-of-function" mutants of the GnRHR. These were used to assess the ability of different GnRH peptidomimetics to rescue defective GnRHR mutants and determine their effect on the level of membrane expression of the WT receptors. Among the manufactured mutants were the shortest rGnRHR C-terminal truncation mutant that resulted in receptor lossof-function (des [325][326][327] -rGnRHR), two nonfunctional deletion mutants (des [237][238][239][240][241] -rGnRHR and des 260 -265

Refolding of misfolded mutant GPCR: Post-translational pharmacoperone action in vitro

“…Cells were treated with pharmacoperones as previously reported (Janovick et al, 2001;Janovick et al, 2003), where indicated. Cells were washed then "pre-loaded" for 6 hours (unless otherwise indicated) with 4 μCi/ml myo-[2-3 H(N)]-inositol in 0.25 ml DMEM (prepared without inositol; Brothers et al, 2004).…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…It is also worth remarking on the observation that chemical structures, which bind to the active site (or at least compete with the ability of agonists to do so), also serve as pharmacoperones, causing misfolded proteins to fold in a way that makes them fold correctly and escape the QCS (Morello et al, 2000;Janovick et al, 2003a;Noorwez et al, 2004;Hawtin, 2006). This observation suggests that there may be a relation between proper construction of the active site and proper overall construction of the receptor.…”

“…For that reason they may come from diverse chemical classes. In the case of the GnRHR, pharmacoperones coming from classes as diverse as erythromycin macrolides, indoles, and quinolones have been identified (Janovick et al, 2003a).…”

“…Information condensed as follows: rhodopsin (Stojanovic and Hwa, 2002;Mendes et al, 2005;Tao, 2006); V2R (Bernier et al, 2004a,b;Fujiwara and Bichet, 2005;Bichet, 2006;Boson et al, 2006;Robben and Deen, 2007;Robben et al, 2006); GnRHR (Beranova et al, 2001;Janovick et al, 2003a;Ulloa-Aguirre et al, 2004a,b); CaR (Brown, 2007;Huang and Breitwieser, 2007); LHR (Gromoll et al, 2002;Martens et al, 2002;Huhtaniemi and Themmen, 2005;Piersma et al, 2007); FSHR (Rannikko et al, 2002;Meduri et al, 2003;Huhtaniemi and Themmen, 2005); TSHR (Biebermann et al, 1997;Costagliola et al, 1999;Tonacchera et al, 2000Tonacchera et al, , 2004Calebiro et al, 2005;Davies et al, 2005); E-BR (Tanaka et al, 1998;Fuchs et al, 2001;Chen et al, 2006;Tao, 2006); MC1R-4R (Beaumont et al, 2005;Clark et al, 2005;Govaerts et al, 2005;Tao, 2005Tao, , 2006Farooqi and O´Rahilly, 2006;Hinney et al, 2006;Lubrano-Berthelier et al, 2006;Alharbi et al, 2007;Lin et al, 2007); and CCR5 receptor (Lede...…”

“…1, A and B) and their own chemically distinct pharmacoperones (Janovick et al, 2003a;Bernier et al, 2004a), some commonalities that may provide important clues about how to identify systems that are especially amenable to this rescue approach are evident: 1) Both the GnRHR and the V2R recognize small peptide ligands, a decapeptide and nonapeptide sequence, respectively; 2) each ligand is only slightly larger than 1000 Da in mass (Fig. 1,A and B), and both peptide ligands are somewhat hydrophobic in nature; and 3) because these are small molecules, it is likely that the compartments for docking in their corresponding receptor are also relatively small, and both receptors are believed to bind ligand mainly in their TM sections, consonant with their hydrophobic nature (Czaplewski et al, 1998a,b;Millar et al, 2004).…”