Adding a Lysine Mimic in the Design of Potent Inhibitors of Histone Lysine Methyltransferases

Abstract

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers) and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein (GLP) by including a 5-aminopentyloxy moiety, which is inserted into the target lysinebinding channel and becomes methylated by GLP, albeit slowly. The compound enhanced its potency in vitro and reduced cell toxicity in vivo. We suggest that adding a lysine or methyllysine mimic should be considered in the design of small molecule inhibitors for other methyl-lysine writers, erasers and readers. Author Contributions Y.C. performed SET-domain enzyme purifications, mass spectrometry-based inhibition assays, ITC measurements, crystallization, and participated in X-ray data collection; T.G. performed compound design, chemical synthesis, and wrote the method of chemical synthesis; A.K.U. performed Jumonji demethylation assays; J.R.H. collected X-ray data, determined structures and performed structural refinements; A.S. and M.T.B. performed Fas reactivation; J.L. performed Glide docking and contributed to compound design; A.S. assisted with compound design; X.Z developed and optimized mass spectrometry-based assay; Y.S. performed cell toxicity assay; J.P.S. coordinated synthesis and modeling activities, contributed to compound design, and wrote method of molecular modeling and legend of supplementary figure S6; X.C. organized and designed the scope of the study, participated in designing compounds with T.G., and wrote the manuscript; all were involved in analyzing data and helped in revising the manuscript. PDB accession numbersThe coordinates and structure factor for human GLP catalytic domain bound with E72, E67, and E11, respectively, and AdoHcy have been deposited with accession numbers3MO5, 3MO2, and 3MO0.

Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms inPlasmodium falciparum

“…These results indicate that BIX-01294 does indeed exert an effect on parasite histone methylation levels, and this effect is not simply a consequence of parasites dying. Together with its significant precedence as an HKMT inhibitor (33)(34)(35)(36)(37)(38), this result is highly suggestive of inhibition of parasite histone methyltransferase activity.…”

“…BIX-01294 was shown to be noncompetitive for the methyl donor S-adenosylmethionine and instead competitive for the histone substrate (30,37). In light of the high precedence of this class of compounds to serve as mammalian HKMT inhibitors (33)(34)(35)(36)(37)(38), focused BIX-01294 derivatives were explored for the development of parasitespecific histone methyltransferase inhibitors in our study. Our discovery that BIX-01294-treated and TM2-115-treated P. falciparum parasites exhibit greatly reduced H3K4me3 levels in BIX-01294-treated parasites, while maintaining a distinct profile compared with human cell lines treated with BIX-01294, is highly supportive of our compounds acting as parasite histone methyltransferase inhibitors.…”

“…S1) have resulted only in very low enzymatic activity. There is evidence that this small-molecule scaffold can be tuned to effect HKMT specificity (33)(34)(35)(36)(37)(38), in particular altering activity against HKMTs other than G9a. It is highly likely that focused medicinal chemistry efforts will enable further specificity of these compounds toward their parasite targets.…”

“…BIX-01294 and analogs were synthesized according to well-established procedures (33)(34)(35)(36)(37)(38). See SI Compound Characterization Data for characterization data for BIX-01294, TM2-115, and -119.…”

“…1) was discovered in a high-throughput screen and was shown to be an inhibitor of the HKMTs G9a/GLP (30). BIX-01294 has also been used successfully in stem cell modulation (31,32), and subsequent medicinal chemistry studies have shown the potential of this scaffold in the discovery of compounds with increased potency, selectivity, and cellular permeability (33)(34)(35)(36)(37)(38). In this work, we identified two compounds that target histone methylation in P. falciparum.…”