Covalent fragment-based ligand screening approaches for identification of novel ubiquitin proteasome system modulators.

Ubiquitination is a key regulatory mechanism vital for maintenance of cellular homeostasis. Protein degradation is induced by E3 ligases via attachment of ubiquitin chains to substrates. Pharmacological exploitation of this phenomenon via targeted protein degradation (TPD) can be achieved with molecular glues or bifunctional molecules facilitating the formation of ternary complexes between an E3 ligase and a given protein of interest (POI), resulting in ubiquitination of the substrate and subsequent proteolysis by the proteasome. Recently, the development of novel covalent fragment screening approaches has enabled the identification of first-in-class ligands for E3 ligases and deubiquitinases revealing so far unexplored binding sites which highlights the potential of these methods to uncover and expand druggable space for new target classes.

Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes.

Covalent inhibitors are emerging as a promising therapeutic means for efficient and sustained targeting of key disease-driving proteins. As for classic non-covalent inhibitors, understanding target engagement and selectivity is essential for determining optimal dosing and limiting potential on- or off-target toxicity. Here, we present a complementary activity-based protein profiling (ABPP) strategy for unbiased proteome-wide profiling of cysteine-reactive inhibitors based on two orthogonal approaches. We illustrate the use of clickable alkyne probes for in-gel fluorescence and mass spectrometry studies using a series of therapeutic XPO1 inhibitors as an example.