direct-measurement-kdm1a-target-engagement-using-chemoprobe-based

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

The covalent modification of histones is closely associated with regulation of gene transcription. Chromatin modifications have been suggested to represent an epigenetic code that is dynamically 'written' and 'erased' by specialized proteins, and 'read', or interpreted, by proteins that translate the code into gene expression changes. Initially thought to be an irreversible process, histone methylation is now known to be reversed by demethylases, FAD dependent amineoxidases and by iron(II)-alpha-ketoglutarate dependent deoxygenases of the Jumonji family. Altered histone demethylase activities have been associated with human disease, including cancer. The first wave of novel investigational drugs directed against KDM1A has recently entered the clinic, and the first specific inhibitor targeting a Jumonji KDM is advancing in preclinical regulatory studies. 

Advances in the development of histone lysine demethylase inhibitors.

The lysine-specific demethylase KDM1A is a key regulator of stem cell potential in acute myeloid leukemia (AML). ORY-1001 is a highly potent and selective KDM1A inhibitor that induces H3K4me2 accumulation on KDM1A target genes, blast differentiation, and reduction of leukemic stem cell capacity in AML. ORY-1001 exhibits potent synergy with standard-of-care drugs and selective epigenetic inhibitors, reduces growth of an AML xenograft model, and extends survival in a mouse PDX (patient-derived xenograft) model of T cell acute leukemia. Surrogate pharmacodynamic biomarkers developed based on expression changes in leukemia cell lines were translated to samples from patients treated with ORY-1001. ORY-1001 is a selective KDM1A inhibitor in clinical trials and is currently being evaluated in patients with leukemia and solid tumors.

ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia.

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  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.

Article	Year
Advances in the development of histone lysine demethylase inhibitors. Current opinion in pharmacology\| PubMed ID: 26057211	2015
ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia. Cancer cell\| PubMed ID: 29502954	2018
Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors. The Journal of biological chemistry\| PubMed ID: 30804215	2019

Tamara Maes

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