A chemoprobe tracks its target

Abstract

Small-molecule inhibitors of histone-modifying enzymes have significant clinical utility for managing diseases such as cancer. These inhibitors are usually identified and monitored through their effects on the gain or loss of specific histone marks. In cells, multiple related enzymes can place or remove a specific mark; therefore, relying on an indirect measure of inhibitor engagement can be misleading. Mascaró et al. describe a luminescence-based ELISA approach that directly monitors binding of inhibitors to the histone lysine demethylase KDM1A.

Figure 1.

An adaptable approach to measure direct binding of inhibitors to their target proteins. A, KDM1A is a lysine demethylase that removes mono- and dimethyl marks on lysine 4 of histone H3. B, the sensitive AlphaPlex assay developed by Mascaró et al. to monitor singlet oxygen (¹O₂) channeling from the mAb mAb1-bound bead (blue) to second antibody (mAb2)-bound bead (yellow) and to the chemoprobe (OG-881)-bound streptavidin bead (orange). C, pretreatment of purified KDM1A in vitro or in vivo (cells or animals) with covalent or noncovalent (SP2509) inhibitors of KDM1A, followed by subsequent treatment with the chemoprobe and measurement of total KDM1A (signal between the blue and yellow pair) versus the percentage of KDM1A accessible to the chemoprobe (signal between blue and orange probes) provides a measure of direct target engagement by each inhibitor.