Development of a differential scanning fluorimetry based high throughput screening assay for the discovery of affinity binders against an anthrax protein

The anthrax protein protective antigen (PA) is responsible lot cell-sin face recognition and aids the delivery of the toxic anthrax enzymes into host cells By targeting PA and preventing it from binding to host cells, it is hoped that the delivery of toxins into the cell will be inhibited The cui rent assay reported for PA is a low throughput functional assay Here, the high throughput so ceiling method using differential scanning fluorimetry (DSF) was developed and optimized to screen a number of libraries from various sources including a selection of FDA-approved drugs as well as hits selected by a virtual soul-ling campaign DSF is a rapid technique that uses fluorescence to monitor the thermal unfolding of proteins using a standard QPCR instrument A positive shift in the calculated melting temperature (T-m), of the protein in the presence of a compound, relative to the T-m of the unbound protein, indicates that stabilization of the protein by ligand binding may have occurred. Optimization of the melting assay showed SYPRO Orange to be an ideal dye as a marker and lead to the reduction of DMSO concentiation to <1%(v/v) in the final assay The final assay volume was minimized to 25 mu L with 5 mu g protein per well of 96-well plate In addition, a buffer, salt and additive screen lead to the selection of 10 mM HEPES-NaOH pH 7 5, 100 mM NaCl as the assay buffer This method has been shown here to be useful as a primary method for the detection of small-molecule PA ligands, giving a hit rate of similar to 7% These ligands can then be stitched further using PA functional assays to confirm then biological activities before being selected as lead compounds for the treatment of anthrax