Abstract
Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases. However, inhibition of histone lysine modifiers often leads to local rather than total changes in histone marks. Also, because histone modifications can be modulated by more than one cellular enzyme, it is not always clear whether changes in histone marks are a direct or indirect consequence of the inhibitor treatment applied. Direct assessment of target occupation can provide a useful tool to quantify the fraction of an epigenetic modifier that is bound to a pharmacological inhibitor (target engagement). Here, we developed and used a novel chemoprobe-based immunoassay to quantify target engagement in cells. Quantification of the fraction of free KDM1A was made possible, in an immune-based assay, by coupling a biotinylated chemoprobe to a warhead capable of selectively and irreversibly binding to the free active form of KDM1A. The results obtained confirmed that this approach is able to determine the degree of target engagement in a dose-dependent manner. Furthermore, the assay can be also used on tissue extracts to analyze the in vivo pharmacokinetics and pharmacodynamics relationship of KDM1A inhibitors, as has been exemplified with ORY-1001 (iadademstat), a potent and irreversible inhibitor of KDM1A. The principle of this assay may be applied to other targets, and the KDM1A probe may be employed in chemoproteomic analyses.
Highlights
Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases
Formation of the trimeric complex between streptavidin, OG-861, and KDM1A was demonstrated by surface plasmon resonance (SPR)
Given the sensitivity of the luminescent chemoprobe immunosorbent assays and wide use of the corresponding equipment, we selected this methodology for further experiments and analyzed the KDM1A target engagement response in acute myeloid leukemia cells treated with ORY-1001
Summary
To develop an assay to quantify KDM1A occupation, we used ORY-1001 (IC50 ϭ 18 nM; Fig. 1A) [9] as a chemical starting point to design biotinylated probes that would bind the FAD co-factor of KDM1A in a similar way as ORY-1001 and have an IC50 maximum 10-fold higher than that of ORY-1001. Critical factors in the design of biotinylated KDM1A probes include the potency of the substructure that will react covalently with the FAD cofactor as well as the proper orientation and the length of the linker. We used a variety of spacers and PEG chains of different lengths to couple ORY-1001
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