Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of beyond-cysteine covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of beyond-cysteine covalent inhibitors.

Automated summary

Sulfur(VI) fluorides (SFs) have been used as valuable electrophiles for designing covalent inhibitors that go beyond cysteine and have the potential to broaden the range of liganded proteome. By targeting nucleophilic amino acids, SFs provide a method for covalent protein modification without the need for a nearby cysteine residue. Libraries of reactive fragments containing SFs offer an innovative approach for ligand and tool discovery through mass spectrometry analysis. This study presents a screening approach that utilizes the unique properties of SFs, successfully identifying hit compounds for CAII and BCL6 through a direct-to-biology workflow. Further characterization of the most promising hits includes determining covalent modification sites, kinetics, and target engagement in cells, as well as a detailed molecular understanding through crystallography. This screening protocol holds potential for accelerated discovery of covalent inhibitors beyond cysteine.