Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Abstract

Affinity probes are useful tools for determining molecular targets and elucidating mechanism of action for novel, bioactive compounds. In the case of covalent inhibitors, activity based probes are particularly valuable for ensuring acceptable selectivity margins. However, there is a variety of bioorthogonalchemisty reactions available for modifying compounds of interest with clickable tags. Here, we describe a direct comparison of tetrazine ligation and strain promoted azide-alkyne cycloaddition using benzimidazole based probes to bind their known target, the gastric proton pump, ATP4A. This study validates the use of chemical probes for target identification and illustrates the superior efficiency of tetrazine ligation for copper-free click systems. In addition, we have identified several novel binding partners of benzimidazole probes: Isoform 2 of deleted in malignant brain tumors 1 protein (DMBT1) and three uncharacterized proteins.

Bioorthogonal Reactions in Activity-Based Protein Profiling

“…This non-specific reactivity, however, can be partially resolved by masking cysteine residues using, for example, iodoacetamide [ 63 ]. Nonspecific labeling by cyclooctynes was not observed in an ABPP study with probes targeting a gastric proton pump [ 64 ]. In this case, labeling was performed in membrane fractions, which likely have a lower abundance of free thiols than whole cell extracts.…”

“…A few studies have performed direct comparisons between iEDDA and other bioorthogonal ligation reactions for two-step ABPP. Comparing the efficiency of tetrazine-based iEDDA with that of SPAAC using a set of structurally similar benzimidazole probes, Paresi et al found that iEDDA showed superior results [ 64 ]. While Liu et al reported reduced selectivity of the tetrazine ligation as compared to CuAAC when using ibrutinib derivatives for labeling of Bruton’s tyrosine kinase [ 80 ], Willems et al found the opposite when targeting proteasome activity [ 43 ].…”