A Selective Inhibitor and Probe of the Cellular Functions of Jumonji C Domain-Containing Histone Demethylases

Abstract

Histone methylations are important chromatin marks that regulate gene expression, genomic stability, DNA repair, and genomic imprinting. Histone demethylases are the most recent family of histone-modifying enzymes discovered. Here, we report the characterization of a small-molecule inhibitor of Jumonji C domain-containing histone demethylases. The inhibitor derives from a structure-based design and preferentially inhibits the sub-family of trimethyl lysine demethylases. Its methyl ester prodrug, methylstat, selectively inhibits Jumonji C domain-containing his-tone demethylases in cells and may be a useful small-molecule probe of chromatin and its role in epigenetics.

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4‐pyridine‐dicarboxylic acid as an in vitro and in cell inhibitor

“…The K m value for ccKDM5B was more than 60-fold lower compared with the trimethylated substrate H3 (7)(8)(9)(10)(11)(12)(13)(14)K9me3 at KDM4A-D [25], 100-fold lower compared with the trimethylated substrate H3(20-34)K27me3 at KDM6A [22] and 250-fold lower compared with the dimethylated substrate H3(1-24)K9me2 at PHF8 [26]. Only KDM7A (KIAA1718) has been reported to have a K m value in a similar range as ccKDM5B, of < 1.2 lm for its dimethylated substrate H3(1-24)K9me2 [26].…”

“…However, demethylation of the dimethylated substrate was more efficient with ccKDM5B compared with KDM4A-D, with 2-to 10-fold higher k cat ⁄ K app m values. The K app m value for the dimethylated substrate at ccKDM5B is 13-to 20-fold higher compared with the K m values of H3 (7)(8)(9)(10)(11)(12)(13)(14)K9me2 substrates at KDM4A-D. KDM5B demethylase activity towards H3K4me1 has previously been reported; however, the results have been contradictory [2,3,5,28]. To examine the catalytic activity of ccKDM5B towards monomethylated H3K4, an H3(1-15)K4me1 peptide was tested as a substrate.…”

“…However, kinetic parameters could not be determined under the assay conditions tested, as the turnover rate for H3(1-15)K4me1 was too slow to be detected in the FDH assay. Instead, MALDI-TOF analysis of H3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) K4me1 in the presence of ccKDM5B was performed and showed trace amounts of H3(1-15) (Fig. S2).…”

“…Such mechanisms, referred to as chromatin cross-talk, have been suggested to be involved in fine-tuning the transcription and regulation of silenced genes (reviewed in [7][8][9]). In this context, a regulatory role of domains such as the PHD and tudor domains has been suggested.…”

Identification of catechols as histone–lysine demethylase inhibitors

“…A number of HDM inhibitors have been reported [9], including N-oxalyl-glycine (NOG) based derivatives with increased affinity and selectivity [10], inhibitors that are based on 2,4-pyridine di-carboxylic acid (2,4-PDCA) [11], hydroxamic acids [12,13], and recently, a KDM4C selective substrate-based inhibitor [14]. In addition to the reported synthesis of de-novo compounds, screening of a number of different compound libraries against KDM4E have been reported [15][16][17].…”