Discovery of proteolysis-targeting chimera targeting undruggable proteins using a covalent ligand screening approach.

Targeted protein degradation (TPD) technology, such as proteolysis-targeting chimera (PROTAC), has become a new therapeutic modality. However, the degradation of undruggable proteins, such as those involved in protein-protein interactions (PPIs), using PROTAC is still limited owing to the difficulties in finding small-molecule binders of these proteins. To identify new chemical moieties that bind to the target sites of the protein of interest (POI), we conducted a site-specific and fragment-based covalent ligand screening using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To apply the selected hits to the PROTAC approach, two-dimensional (2D) nuclear magnetic resonance (NMR) experiments were performed to evaluate the reversible binding of their analogs without covalent warheads. To proof the proposed approach, human mouse double minute (MDM)2 was selected as a model system since it is involved in PPIs and is known to be a degradable target protein. Western blot analysis showed that newly synthesized PROTACs, incorporated reversible analogs of screening hits, affected degradation in a dose- and time-dependent manner. This methodology makes it possible to use PROTAC technology to exploit previously undruggable proteins for TPD.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Young Ho Jeon has patent #Pharmaceutical composition for preventing or treating hyper-proliferative disease including new small molecules inhibiting MDM2 protein pending to Korea University Sejong Campus. H.L., J.Y.L., H.J., H.Y.C., M.K., S.H.B., J.J., J.Y.K., and Y.H.J. have filled a provisional patent in the Korea related to this work.
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