# Proteome-Wide Monitoring of Drug Action in Living Cells Using a Novel Label-Free Solvent-Based Shift Assay

## TL;DR

A new method called SPICE uses organic solvents to study drug effects on proteins in live cells, revealing drug targets and biological impacts.

## Contribution

SPICE is a novel label-free assay for proteome-wide drug action monitoring in living cells, detecting drug targets and downstream effects.

## Key findings

- SPICE detects drug-target interactions and secondary effects in live cells, unlike lysate-based methods.
- SPICE complements CETSA by providing distinct insights into drug mechanisms and target engagement.
- SPICE can identify degrader drug targets even when targets are being degraded.

## Abstract

Biophysical proteomics assays allow for proteome-wide, label-free monitoring of ligand-induced changes in protein structure and stability, offering insights into protein-ligand interactions and modulation of biophysical properties of cellular proteins. These assays exploit the principle that compound-induced alterations in structure or stability of proteins can be detected through changes in their susceptibility to denaturation. Here, we introduce solvent proteome profiling in cells (SPICE), which employs solvent-based denaturation of proteins under otherwise physiological conditions in intact cells. We characterized solvent-induced denaturation of proteins inside cells as distinct from that in cell extracts and validated SPICE by detecting known drug-target interactions for multiple compound classes. Our results indicate that SPICE, unlike experiments in cell extracts, also detects secondary compound-induced effects such as target profiles of drug metabolites, modulation of protein-protein interactions, and downstream signaling events. We further demonstrate complementarity of SPICE and cellular thermal shift assay, which both robustly detect the designated targets of well-characterized drugs and individually provide biologically meaningful and interpretable results. Finally, we show that SPICE can detect covalent drug-targets, compound-induced target-destabilization and stabilization of degrader drug targets despite their concurrent degradation.

•Organic solvents allow for protein denaturation in live cells.•SPICE allows for drug target identification in live cells.•SPICE allows to infer biological downstream effects of inhibition of a drugs target.•SPICE is complementary to CETSA.•Modified format detects degrader target engagement despite concurrent degradation.

Organic solvents allow for protein denaturation in live cells.

SPICE allows for drug target identification in live cells.

SPICE allows to infer biological downstream effects of inhibition of a drugs target.

SPICE is complementary to CETSA.

Modified format detects degrader target engagement despite concurrent degradation.

A method for solvent proteome profiling in cells has been developed. Protein denaturation by organic solvents in live cells was characterized and intracellular target engagement was detected for multiple compound classes. In contrast to lysate-based experiments, the method allowed to infer biological downstream effects of drug target inhibition. Denaturation profiling, target identification, and mechanism-of-action deconvolution provided complementary insights to the cellular thermal shift assay. Modification of the method enabled detection of intracellular target engagement despite concurrent degradation of target proteins.