Hsp70 molecular chaperones bind and release polypeptide substrates concomitant with ATP dissociation and association. Based on this attribute, members of this abundant and conserved family of chaperones catalyze a diverse number of essential processes, including protein maturation and degradation, and prevention of protein aggregation. Because aberrations in these processes may lead to human diseases, there is much interest in modulating HspTO function and/or expression. Specifically, Hsp70s are known to inhibit apoptosis and maintain the transformed state in a wide range of malignancies. Moreover, Hsp70s help """"""""correct"""""""" the conformations of mis-folded proteins and destroy potentially cytotoxic unfolded polypeptides. Unfortunately, few Hsp70 modulators have been identified, in part due to the lack of tools amenable to high throughput screens (HTS). Thus, the goal of the current effort is to create and optimize assays to identify both inactivators and activators of the inducible Hsp70. These tools can be used to conduct HTS and will be made available to the scientific community. The compounds identified from such efforts will represent important leads to gain insight into the rational translation of Hsp70 modulators in the treatment of cancers, neurodegenerative diseases, inflammation, ischemia and viral infections. To these ends, two aims are proposed in this grant application. First, a phenotypic cell-based assay will be developed, optimized and validated to identify modulators of inducible Hsp70 expression. The assay employs a miniaturized, cell-based immunodetection system amenable to HTS. Second, a fluorescence polarization assay will be developed, optimized and validated to identify modulators of Hsp70 function. This assay is based on the competition of a fluorescently (FITC or red-shifted) labeled peptide to bind recombinant Hsp70. Overall, given the importance of Hsp70 function and activation in human disease, the time is ripe to design and perform a large-scale screening regimen to identify Hsp70 modulators. Surprisingly, this has not yet been accomplished. To develop the requisite tools to screen for Hsp70 modulators, this proposal co-opts the combined expertise of the collaborating Pis in chaperone biology, biochemistry and drug discovery, and their abilities and proven track-records in the design and development of screening regimens for small molecule chaperone modulators. ? ?
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