High-throughput screening identifies a novel small-molecule modulator of Hsp70 that selectively enhances ubiquitination and degradation of misfolded neuronal NO synthase.

The Hsp90 and Hsp70 chaperones act as a protein quality control system for several hundred client proteins, including many implicated in neurodegenerative disorders. Hsp90 and Hsp70 are widely thought to be important drug targets. Although many structurally distinct compounds have been developed to target Hsp90, relatively few are known to target Hsp70 and even fewer have been tested in protein quality control systems. To address this, we describe a high-throughput thermal shift-based screen to find compounds that bind and stabilize Hsp70 and then employ assays with misfolded forms of a well-established client protein, neuronal NO synthase (nNOS), to identify compounds that enhance ubiquitination of client proteins. The ubiquitination assay employed a quantitative ELISA method to measure Hsp70:CHIP-dependent ubiquitination of heme-deficient nNOS, which is a model of a misfolded client, in reaction mixtures containing purified E1, E2, Hsp70, CHIP, and ubiquitin. We screened 44,447 molecules from the Maybridge and ChemDiv libraries and found one compound, protein folding disease compound 15 (PFD-15), that enhanced in vitro nNOS ubiquitination with an EC