Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein–Drug Interactions

Described
here is a mass spectrometry-based proteomics approach
for the large-scale analysis of protein–drug interactions.
The approach involves the evaluation of ligand-induced protein folding
free energy changes (ΔΔGf)
using chemical denaturation and protein precipitation (CPP) to identify
the protein targets of drugs and to quantify protein–drug binding
affinities. This is accomplished in a chemical denaturant-induced
unfolding experiment where the folded and unfolded protein fractions
in each denaturant containing buffer are quantified by the amount
of soluble or precipitated protein (respectively) that forms upon
abrupt dilution of the chemical denaturant and subsequent centrifugation
of the sample. In the proof-of-principle studies performed here, the
CPP technique was able to identify the well-known protein targets
of cyclosporin A and geldanamycin in a yeast. The technique was also
used to identify protein targets of sinefungin, a broad-based methyltransferase
inhibitor, in a human MCF-7 cell lysate. The CPP technique also yielded
dissociation constant (Kd) measurements
for these well-studied drugs that were in general agreement with previously
reported Kd or IC50 values.
In comparison to a similar energetics-based technique, termed stability
of proteins from rates of oxidation (SPROX), the CPP technique yielded
significantly better (∼50% higher) proteomic coverage and a
largely reduced false discovery rate.