Development of covalent inhibitors of Jumonji-C histone lysine demethylases

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In eukaryotes, chromosomal DNA is wrapped around histone proteins; post-translational histone modifications are important to regulate gene activation and silencing ('epigenetics'), for example, reversible methylations at N(Epsilon)-lysine amino groups and at arginine guanidinium groups of histone proteins. Histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs) have complementary functions in the regulation of histone methylation-demethylation homeostasis in healthy cells. The dysregulation of these enzymes is linked to diseases such as cancer, rendering KDMs important therapeutic targets to develop anti-cancer therapeutics. KDMs are divided into two subfamilies depending upon their sequence homologies and mechanisms. The first family, that is KDM1, uses flavin adenine dinucleotide (FAD) as its co-substrate, while the other class, that is the Jumonji-C (JmjC) enzymes KDM2/7, KDM3, KDM4, KDM5, and KDM6, use 2-oxoglutarate (2-OG) and dioxygen as co-substrates as well as non-heme Fe(II) as cofactor (Figure 1). Several KDM1A inhibitors like tranylcypromine, ladademstat and others, are undergoing clinical trials for cancer therapy.5 However, only few potent and selective inhibitors of the JmjC KDMs have been reported that show effects in cells, most of them inhibiting via a non-covalent mode. The objective of this project is to develop potent and selective inhibitors for KDM4A-C, KDM5B and KDM6B which are potential targets for the treatment of, for example, cervical, gastric and breast cancer; the inhibitors will be designed to allow for covalent inhibition. The results will inform on the inhibitors of other types of metalloenzymes, including those involved in antimicrobial resistance. JmjC KDM inhibitor development programs have focused on small-molecules which inhibit through non-covalent interactions, while small-molecules which inhibit via covalent modification of JmjC KDMs have been less well explored. Covalent inhibitors can be superior over non-covalent inhibitors as the protein-ligand interactions persist over a longer time period and may be irreversible, depending on the functional group chosen. Recent work in Oxford has afforded proof-of-principle evidence that sulfonylfluorides inhibit the JmjC KDM related 2OG oxygenases FIH and AspH with some level of selectivity. Considering the similarity of KDM6B with FIH (Figure 2), the design of sulfonylfluoride-bearing small-molecules is a promising start for development of novel inhibitors and was therefore chosen as a target (Figure 3). Structure activity relationship studies will be performed on sulfonylfluorides to inform on their potency and selectivity for KDM6B inhibition; for example, side chains will be incorporated into the small-molecules that are part of reported KDM6B inhibitors. Modelling, mass spectrometric, and crystallographic studies will guide the structure activity relationship studies and inform on the position and stoichiometry of the covalent modification. Other covalently reacting groups will also be explored. This project falls within the 'EPSRC healthcare technologies' research area.