title: Label-Free Bottom-Up Proteomic Workflow for Simultaneously Assessing the Target Specificity of Covalent Drug Candidates and Their Off-Target Reactivity to Selected Proteins.
abstract: 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.
authors.affiliation: Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
authors.forename: Yanou
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
authors.forename: Yue-Zhong
authors.initials: YZ
authors.lastname: Shu
authors.affiliation: Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
authors.forename: W Griffith
authors.initials: WG
authors.lastname: Humphreys
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D006801:Humans; D015394:Molecular Structure; D058990:Molecular Targeted Therapy; D010880:Piperidines; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D011720:Pyrazoles / Q000737:chemistry* / Q000494:pharmacology*; D011743:Pyrimidines / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010880:Piperidines; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; C551803:ibrutinib; D011505:Protein-Tyrosine Kinases; D000225:Adenine