This proposal is a resubmission in response to PAR-14-284, ?High Throughput Screening (HTS) to Discover Chemical Probes (R01). This funding opportunity supports collaboration between academic, nonprofit, or commercial HTS screening facilities that have the requisite expertise and experience to implement an HTS-ready assay for the discovery and development of small molecule chemical probes. The work proposed will be conducted by collaboration between the Conn laboratory that developed the concept of pharmacoperones (and the assay) and the laboratories at Scripps Research Institute with extensive experience in HTS Drug Discovery, Medicinal Chemistry, and Drug Metabolism and Pharmacokinetics (DMPK). We will use an existing and well-validated high throughput assay to identify pharmacoperones of the gonadotropin releasing hormone (GnRH) receptor (GnRHR). Pharmacoperones are target-specific and small molecules that diffuse into cells, rescue misfolded protein mutants and restore them to function. Rescue is based on a newly appreciated mechanism: correcting the cellular routing of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function. The efficacy of these drugs has been demonstrated both in cell cultures and in vivo. The assay will be applied to screen a library of more than 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the GnRHR in diseases of reproduction. One such disease is hypothalamic hypogonadism (HH). HH is a family of disorders, a subset of which is caused by mutations in the GnRHR which result in retention in the endoplasmic reticulum. In addition to increasing the expression of mutants at the plasma membrane, pharmacoperones of this receptor increase plasma membrane expression of WT GnRHR. Only 50% of the synthesized human GnRHR is normally expressed at the plasma membrane and this percent can be increased by pharmacoperones. Accordingly, these drugs may be used in treatment of disorders in which this receptor is sub optimally expressed and cancers in which GnRH is a therapeutic target. Because of the newness of this type of screen, this project will also serve as a prototype for the identification of pharmacoperones present in large chemical libraries. Accordingly, the proposed approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this technique offers an untapped opportunity for use of the HTS approach. Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking may already be present in existing libraries, but have not been identified using existing screens. Preliminary data show that the assay was successfully transferred to the HTS facility and early screening results show that the proposed approach is likely to be successful in identification of tractable hits.
We will use an existing and well-validated high throughput assay to screen for pharmacoperones of the GnRH receptor. Pharmacoperone drugs correct the folding of misfolded protein mutants and restore protein function; the utility of this approach has been shown in vivo. This assay will be applied to screen a library of over 640,000 individual compounds and will identify probe molecules for the potential treatment of disease caused by misrouting of mutants of the hGnRHR or the suboptimal expression of the WT receptor at the plasma membrane. A hit validation protocol, the means to confirm mechanism of action and an in vivo test system are in place.