Aims
Peroxiredoxins are important for the ability of cells to sense and respond to oxidative stress. The catalytic unit of 2-cys peroxiredoxins is a homodimer, yet they assemble into decameric rings that are proposed to influence biological activity. Peroxiredoxins are overexpressed in some cancers, and we have shown point mutations that disrupt decamer formation impact cancer cell growth. Our goal is to find small molecules that mimic these mutations.
Methods
We designed a multi-phased method for high throughput screening of a library of 2,700 FDA-approved small molecules. The drug library was screened using differential scanning fluorimetry (DSF) as a high throughput method of selecting potential drug candidates. Drug binding interactions were validated using saturation transfer difference (STD)-NMR, and size exclusion chromatography (SEC) allowed for analysis of structural disruption.
Results
The fluorescent response of recombinant peroxiredoxins in DSF varies distinctly based on the structural status of the protein. Several compounds were identified that modulate the DSF profile, and their binding and structural disruption were validated in STD-NMR and SEC. A drug compound was identified that strongly binds peroxiredoxin 2 and significantly decreases the relative proportion of protein in the decameric state. Preliminary studies suggest inhibition of cell proliferation. Further work is required to determine if this is attributable to disruption of peroxiredoxin decamers in cells.
Conclusions
We have established a methodology for the rapid screening of large drug libraries and validation through complementary methods of protein analysis. A lead compound has been identified that disrupts peroxiredoxin decamers and inhibits the growth of cancer cells.