Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale

Abstract

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug–protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDU­FV1 and NDU­FAB1 were identified for the first time, and the NDU­FV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug–target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

Recent advances in proteome‐wide label‐free target deconvolution for bioactive small molecules

“…Although the proteome coverage is poor, there were still four kinases showing significant stabilization only in SIP but not in the initial staurosporine MS‐CETSA study which was performed in K562 cell extract, suggesting some extent of complementarity of the different approaches. However, we note these kinases could possibly exist in different forms in the different cell lines (293T vs. K562) 28,46 …”

“…Recently, Zhang et al 28 developed a new approach named SIP, which is based on the observation that compared to free proteins, ligand‐bound proteins are more tolerant to organic SIP. More specifically, the cell lysate aliquots in the presence and absence of ligand of interest are equilibrated with a series of concentrations of a mixed organic solvent (acetone/ethanol/acetic acid = 50:50:0.1) to initialize protein denaturation and precipitation.…”

“…In the past decade or so, several label‐free target deconvolution approaches have emerged for evaluating the direct interactions between drugs and protein targets in a physiologically relevant environment such as whole cell lysate or intact cells. These methods include the cellular thermal shift assay (CETSA), 22 pulse proteolysis (PP), 23 chemical denaturant and protein precipitation (CPP), 24 stability of proteins from rates of oxidation (SPROX), 25 drug affinity responsive target stability (DARTS), 26 limited proteolysis (LiP), 27 and solvent‐induced protein precipitation (SIP) 28 . These approaches are largely based on the biophysical principle that proteins are thermodynamically stabilized or conformationally modulated upon ligand binding, and hence, such changes can be precisely probed by their differential responses to perturbations such as heating, proteolysis, and chemical denaturants.…”

Assessing target engagement using proteome-wide solvent shift assays

“…Various agents, including salt, acid, organic solvents, and some chemical denaturants, also disrupt protein folding leading to aggregation and precipitation (Chan et al, 1986; Meng et al, 2018; Shih et al, 1992; Wingfield, 1998). Recently, it was demonstrated that increasing concentrations of an organic solvent mixture composed of 50% acetone, 50% ethanol, and 0.1% acetic acid (AEA) induces precipitation of protein lysates and, more importantly, that this property can be exploited to identify ligand binding events (Zhang et al, 2020). This method, termed solvent-induced precipitation (SIP), provided the basic framework for a robust target engagement approach.…”

“…This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al, 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves.…”