UCSF

Covalent inhibitors are versatile and powerful compounds, useful in their potential as drugs, tools for drug discovery, and probes for chemical biology. Traditionally, covalent inhibitors have targeted cysteine. As the most reactive nucleophilic amino acid at physiological pH, this allows for even weakly reactive electrophiles to exhibit good reaction kinetics towards their target residue. The ability to utilize weakly reactive electrophiles in addition to cysteines low abundance in the proteome reduces the risk of undesired, off-target effects. However, its low abundance comes with the trade-off that many potential protein targets do not contain a cysteine residue proximal to small molecule binding sites, rendering them unviable as targets for cysteine-targeted covalent inhibitors. This leads to the need to develop covalent inhibitors utilizing electrophiles that target non-cysteine amino acids, expanding the breadth of proteins targetable with covalent inhibitors and probes. We first focused on salicylaldehydes as reversible-covalent inhibitors to target lysines. While salicylaldehydes have previously been reported to form imine adducts with lysine residues on proteins, these adducts have only shown good stability when buried in a hydrophobic pocket where exclusion of water hinders hydrolysis. Whether salicylaldehydes can form highly stable adducts with surface lysines, where solvation makes rapid hydrolysis of the imine more likely, remains an open question. We developed a series of salicylaldehyde probes targeting the surface exposed lysine on the ATPase domain of Hsp90, the best of which engaged the target lysine in a quasi-irreversible manner. Furthermore, upon reduction of the reversible binding scaffold to a fragment-like size, salicylaldehyde compounds showed significant and sustained target engagement at low micromolar concentrations, demonstrating the potential of salicylaldehydes for use in covalent-fragment based drug discovery. Second, we explored histidine-targeting covalent probes. Sulfonyl fluorides have previously been shown to react with histidine residues on proteins, but their metabolic stability remains a challenge when developing inhibitors towards in cell applications, necessitating a switch towards similar, but less reactive and more stable electrophiles like fluorosulfates. This balance between compound stability and target modification rate remains a challenge in developing histidine-targeting covalent inhibitors. Utilizing a series of sulfonyl fluoride compounds with different linkers, we showed that conformational constraints are an additional tool for tuning the reaction kinetics of a sulfonyl fluoride with a target histidine outside of altering innate electrophile reactivity. Analysis of histidines proximal to ligands in 65,288 mammalian protein structures from the PDB found that 18% of proteins had at least one ligand-proximal histidine, exemplifying the breadth of proteins containing histidines targetable with covalent inhibitors.