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Efficient selection platform enables discovery of novel lysine-targeting covalent inhibitors

In a study in Angewandte Chemie International Edition, a research team integrated activity-based protein profiling (ABPP) data with the covalent DNA-encoded chemical library (CoDEL) technology and identified structurally novel lysine-targeting covalent inhibitors with diverse mechanisms of action.

The team was led by Lu Xiaojie from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, collaborating with Zhou Lu from Fudan University and Sun Yi from the Zhejiang University School of Medicine.

Covalent drugs function by forming covalent bonds with specific amino acid residues, enabling sustained modulation of target proteins. Compared with cysteine-targeting strategies, lysine serves as an alternative covalent binding site that circumvents the limitation of cysteine scarcity in ligand-binding pockets and broadens the landscape of druggable targets. In recent years, structure-based drug design has facilitated the development of lysine-targeting covalent inhibitors.

The CoDEL technology is rapidly emerging as a key platform for covalent drug discovery. Lu Xiaojie's team, along with the collaborators, has applied this technology to discover novel cysteine-targeting covalent inhibitors in proteins, and has developed an integrated ABPP-CoDEL strategy to identify tyrosine-targeting covalent inhibitors.

However, a systematic CoDEL selection platform specifically designed for lysine-targeting inhibitors has yet to be established. In addition, the widespread distribution of lysine across the human proteome makes random target selection inefficient for screening.

By integrating compound-based and warhead-based ABPP datasets, the researchers constructed a protein dataset enriched with lysine residues exhibiting both high reactivity and ligandability, thereby facilitating rational target selection for screening. They then incorporated eight lysine-targeting covalent warheads with distinct reaction mechanisms to synthesize CoDELs comprising 10.7 million compounds.

Covalent selection identified lysine-targeting covalent inhibitors against phosphoglycerate mutase 1 (PGAM1), bromodomain (BRD) family proteins, and ubiquitin-conjugating enzyme E2 N (UBE2N).

Among them, Compound 1 functioned as a photo-covalent probe for the active site of PGAM1, while Compound 4 formed a reversible covalent bond with a previously unexplored site within the bromodomain of BRD family proteins.

Notably, Compound 9 irreversibly bound to UBE2N, induced conformational changes in the UBE2N/UBE2V2 complex, disrupted polyubiquitin chain formation, and impaired its downstream functional activity. This novel mechanism provided a new strategy for regulating the ubiquitination pathway.

This study establishes an efficient selection platform for lysine-targeting covalent inhibitors by integrating proteomic data with CoDEL technology. This strategy not only expands the applicability of covalent drugs in target selection but also provides technical support for the rational design of covalent inhibitors.