György M. Keserű
Covalent fragment approaches in drug discovery
Medicinal Chemistry, Research Center for Natural Sciences, Budapest, Hungary
Covalent fragments defined as fragment sized electrophilic compounds might realize the advantages of fragment-based approaches in covalent drug discovery. Consequently, screening electrophilic fragments emerged as a promising alternative to discover and validate novel targets and to generate viable chemical starting points even for challenging targets. [1,2]
Recently our group developed specific covalent fragment libraries. In target discovery settings we aimed identifying druggable cysteines experimentally and mapped the reactivity and accessibility of these residues using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. [3] Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against targets and anti-targets enables the experimental investigation of the accessibility and reactivity of the targeted cysteines and help to identify suitable warheads for selective covalent inhibitors. [4,5]
For hit finding applications we developed the covalent version of ultra-low-molecular-weight ligands (MiniFrags) introduced by Astex for X-ray screening. Covalent MiniFrags were compiled from small electrophilic heterocycles [6] and were screened against challenging targets such as KRAS G12C, the main protease of SARS-Cov-2 [7] ant HDAC8 [8]. Since electron-withdrawing heterocycles activate their small (max. 1–2 atoms) electrophilic substituents the fragments bind to cysteine nucleophiles that makes them readily detectable by MS, X-ray or NMR. Hits identified by screening covalent MiniFrags can be considered as viable starting point for covalent FBDD programs, and they might provide targeted covalent inhibitors by replacing the heterocyclic scaffold of non-covalent ligands. This approach would allow the precise positioning of the reactive group toward a catalytic/non-catalytic protein nucleophile in the proximity of the binding site while maintaining the key non-covalent interactions.
Finally, we discuss a new screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection [9]. We have designed, synthesized, and screened 100 diazirine-tagged small molecule fragments against protein-protein interactions represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several new fragment hits against all these targets and identified their binding sites via enzymatic digestion, structural studies and modelling. These results revealed that the protocol outperforms screening traditional PPI targeted libraries in better exploration of the available binding sites and higher hit rates observed for even difficult targets.
References: [1] Drug Disc. Today 2020, 25, 983; [2] Trends Pharmacol Sci 2023 , 44, 802; [3] Chembiochem. 2020 22, 743; [4] Eur J Med Chem. 2020, 207, 112836; [5] Eur J Med Chem. 2022 231, 114163; [6] Medchemcomm. 2018, 10, 263; [7] Nature Comm. 2020, 11, 5047; [8] Keserű GM. et al. J Med Chem 2024, 67, 572; [9] Nature Comm. 2021, 12, 3201; [10] Comms Chem 2024, 7, 168.
