title: Therapeutic rescue of misfolded mutants: validation of primary high throughput screens for identification of pharmacoperone drugs.
abstract: BACKGROUND
  Functional rescue of misfolded mutant receptors by small non-peptide molecules has been demonstrated. These small, target-specific molecules (pharmacological chaperones or "pharmacoperones") serve as molecular templates, promote correct folding and allow otherwise misfolded mutants to pass the scrutiny of the cellular quality control system (QCS) and be expressed at the plasma membrane (PM) where they function similarly to wild type (WT) proteins. In the case of the gonadotropin releasing hormone receptor (GnRHR), drugs that rescue one mutant typically rescue many mutants, even if the mutations are located at distant sites (extracellular loops, intracellular loops, transmembrane helices). This increases the value of these drugs. These drugs are typically identified, post hoc, from "hits" in screens designed to detect antagonists or agonists. The therapeutic utility of pharmacoperones has been limited due to the absence of screens that enable identification of pharmacoperones per se.
  
  
  RESULTS
  We describe a generalizable primary screening approach for pharmacoperone drugs based on measurement of gain of activity in stable HeLa cells stably expressing the mutants of two different model G-protein coupled receptors (GPCRs) (hGnRHR[E(90)K] or hV2R[L(83)Q]). These cells turn off expression of the receptor mutant gene of interest in the presence of tetracycline and its analogs, which provides a convenient means to identify false positives.
  
  
  CONCLUSIONS
  The methods described and characterized here provide the basis of novel primary screens for pharmacoperones that detect drugs that rescue GPCR mutants of specific receptors. This approach will identify structures that would have been missed in screens that were designed to select only agonists or antagonists. Non-antagonistic pharmacoperones have a therapeutic advantage since they will not compete for endogenous agonists and may not have to be washed out once rescue has occurred and before activation by endogenous or exogenous agonists.
authors.affiliation: Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, Oregon, United States of America.
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Janovick
authors.forename: Byung S
authors.initials: BS
authors.lastname: Park
authors.forename: P Michael
authors.initials: PM
authors.lastname: Conn
mesh_terms: D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D016014:Linear Models; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000032:analysis*; D017510:Protein Folding; D011966:Receptors, LHRH / Q000737:chemistry / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D050505:Mutant Proteins; D004364:Pharmaceutical Preparations; D011966:Receptors, LHRH; D017483:Receptors, Vasopressin