Identification of pharmacological chaperones for misfolded proteins

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DESCRIPTION (provided by applicant): Compounds that stabilize a protein's native fold, termed pharmacological chaperones, hold great promise as therapeutics for treating diseases such as amyloid diseases, cystic fibrosis, and forms of cancer, that have etiological origins in protein misfolding. A major obstacle in the search for such compounds is the lack of generic high-throughput assays to identify stabilizing molecules. The lack of an appropriate assay is often an insurmountable obstacle for proteins that do not have easily assayable phenotypes. Described here is the development of three independent assays that can be used in high-throughput screens to identify potential pharmacological chaperones. Two of these assays are generic: that is, they are designed to be used with any protein and do not require knowledge of the protein's function. Together the assays constitute a unique platform not only for the identification of candidate chaperones, but also for dissecting their mode of action. Work is described to test the capabilities of each assay, first examining potential for functioning with a wide range of proteins, second carrying out chemical screens with model misfolded, disease protein variants, and finally examining the biological roles of hits obtained in these model studies. Importantly, the assays are expected to be rapid, straightforward, and transferable, and will provide basic methodologies for identifying pharmacological chaperones that interact with a wide variety of protein targets. The specific aims are: Aim 1. Establishment of yeast-based assays for rescue of misfolded proteins. (a) Complementation assay. In this assay, human disease protein complements a yeast strain such that the strain grows robustly if the protein is functional, but shows temperature-sensitive growth with a misfolded variant. The assay will be developed and tested with human AGT in high-throughput screens of 11,000 compounds. (b) DHFR-based protein misfolding sensor. In this assay, an unstable protein is coupled to the essential enzyme dihydrofolate reductase (DHFR), which acts as a reporter. The generality of this approach will be tested with several human proteins, including AGT and 1-galactosidase A, in high-throughout screening as in (a). Aim 2. Evaluation of a mass-spectrometry-based SUPREX assay for ligand-induced protein stabilization. This approach uses hydrogen-deuterium exchange and MALDI mass-spectrometry to report ligand-induced stability changes. We will assess the efficacy and generality of this assay for identification of pharmacological chaperones using disease proteins including AGT, 1-galactosidase A, and 2-glucosidase. Disease variants will be screened in high-throughput chemical screens for stabilizing ligands. Aim 3. Evaluation of relevance of hits to protein misfolding. Data from the cell-based and mass- spectrometry based screens will be compared. Hits that are common to the cell- and mass-spectrometry based screens will be subject to Kd value measurements; hits that are exclusive to the cell-based assays will be investigated for potential effects on general folding and/or quality control pathways within the cell. Misfolded proteins are responsible for many human diseases, yet few generic assays exist for efficiently identifying compounds or molecules that may correct defective protein folding. In this proposal, we describe the development of three independent assays that can be used in high-throughput screens for factors that enhance protein folding. As these assays are robust, generalizable, and can be extended to any misfolded protein of interest, they are expected to greatly accelerate identification of therapeutic compounds for misfolding disease.