文摘
Although designed covalent inhibitors as drug candidates offer several unique advantages over conventional reversible inhibitors, including high potency and the potential for less frequent dosing, there is a general tendency to avoid the covalent mode of action in drug discovery programs due to concerns regarding immune-mediated toxicity that can arise from indiscriminate reactivity with off-target proteins. Therefore, the ability to assess off-target reactivity relative to target specificity is desirable for optimizing covalent drug candidates in the early discovery stage. One concern with current surrogate nucleophile trapping approaches is that they employ a simplistic model nucleophile such as glutathione, which may not reliably reflect the covalent interactions with cellular or extracellular proteins. One way to get a more relevant reactivity assessment is to directly measure the ability of an inhibitor to covalently modify nucelophilic amino acids on biologically relevant proteins, both on- and off-target. In this article, we describe a label-free bottom-up proteomic workflow for simultaneous evaluation of target binding and off-target reactivity of covalent drug candidates to selected proteins at the peptide level. Ibrutinib, a covalent drug targeting the active site of BTK protein, was used as a model compound to demonstrate the feasibility of the workflow. The compound was incubated with a mixture of target protein, Bruton’s tyrosine kinase (BTK), and two abundant proteins in blood, hemoglobin (Hb) and human serum albumin (HSA), and then the ibrutinib modification sites were determined utilizing a bottom-up proteomic approach. A non-BTK specific model compound (1) known to modify cysteine residues was also included. By comparing the extent of off-target modifications to the targeted BTK C481 binding in a wide compound concentration range, we were able to determine the concentration where maximum target binding was achieved with minimal off-target reactivity. The generic label-free bottom-up proteomics workflow described in this article should be useful in the rank order assessment of off-target reactivity vs on-target reactivity of covalent drug candidates in the early drug discovery stage.