System-Wide Profiling by Proteome Integral Solubility Alteration Assay of Drug Residence Times for Target Characterization

Most drugs are used
in the clinic and drug candidate
target multiple
proteins, and thus detailed characterization of their efficacy targets
is required. While current methods rely on quantitative measurements
at thermodynamic equilibrium, kinetic parameters such as the residence
time of a drug on its target provide a better proxy for efficacy in vivo. Here, we present a residence time proteome integral
solubility alteration (ResT-PISA) assay, which facilitates monitoring
temporal protein solubility profiles after drug removal (“off-curve”)
in cell lysates or intact cells, quantifying the lifetime of drug–target
interaction. A compressed version of the assay measures the integral
under the off-curve enabling the multiplexing of binding affinity
and residence time assessments into a single proteomic analysis. We
introduce a combined scoring system for three parametric dimensions
to improve prioritization of targets. By providing complementary information
to other characteristics of drug–target interaction, the ResT-PISA
approach will be useful in drug development and precision medicine.