Design, Synthesis and Characterization of Covalent KDM5 Inhibitors

Abstract

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Keywords: KDM5; covalent inhibitors; epigenetics; lysine demethylase.

Figure 1

A) KDM alignment at positions C480 and C497 (KDM5B numbering). B) Reported KDM5 inhibitors CPI‐455, 1 and 2. C) Overlay of compound 2 docked in the X‐ray structure of 3 (green) in KDM5B (PDB ID 6EIN). D) PP compounds. E) PZ compounds. *IC₅₀ (μm).

Figure 2

A) Time‐dependent inhibition of covalent inhibitors 4 and 7. B,C) 2‐OG competition assay with non‐covalent variants (1 and 2) and covalent inhibitors (4 and 7).

Figure 3

A) Intact mass spectra of covalent binding by MS. B) Peptide mapping of KDM5B after treatment with compounds 3 and 7. Observed peptides for native protein (light red box), compound 3 adduct (green box) and compound 7 adduct (light blue box).

Figure 4

A) Cellular activity of compound 9 by NanoBRET assay. B) Distribution of H3K4me3 around transcriptional start sites (TSS). Densities of ChIP‐seq reads for H3K4me3 and input in HEK293 cells treated with DMSO and compounds 4, 7, 9 and non‐covalent reference compound KDOAM‐25.