biostudies-literatureWang SDeutsche Forschungsgemeinschaft13295-13320https://www.ebi.ac.uk/biostudies/studies/S-EPMC12257510biostudies-literatureUnknown68(13)Lysine methyltransferase 9 (KMT9), an obligate heterodimer (KMT9α/KMT9β), belongs to the few described Rossmann-fold histone lysine methyltransferases and monomethylates histone H4 at lysine 12 (H4K12me1). KMT9 depletion or inhibition impairs the proliferation of tumors, including prostate, lung, colon, and bladder cancer cells, underscoring its therapeutic potential. Here, we show the development of branched cofactor analogues with a methionine side chain as highly potent KMT9 inhibitors. Through structure-guided design, a basic nitrogen and 4-chlorophenoxy-2-fluorobenzene in the substrate branch contribute most to the high potency and selectivity. Due to the zwitterionic methionine side chain, the inhibitors did not show cellular activity. Importantly, an ethyl ester prodrug <b>8</b> exhJournal of medicinal chemistryStructure-Guided Design of a KMT9 Inhibitor Prodrug with Cellular Activity.PMC12257510SFB 992-192904750Schu688/15-1DKTK FR01-374EXC-2189-390939984SFB 1381-403222702Regenass PStaudt MZhang LHazai VISarraf DSchule RSum MWang SGunther SWalz JKlein SOKummel PMishra PBreit BBarthes NPFUrban SWarstat REinsle OBerlin CRuprecht JHeller NMetzger EPappert TBacher JJung MPeng LfalseStructure-Guided Design of a KMT9 Inhibitor Prodrug with Cellular Activity.Lysine methyltransferase 9 (KMT9), an obligate heterodimer (KMT9α/KMT9β), belongs to the few described Rossmann-fold histone lysine methyltransferases and monomethylates histone H4 at lysine 12 (H4K12me1). KMT9 depletion or inhibition impairs the proliferation of tumors, including prostate, lung, colon, and bladder cancer cells, underscoring its therapeutic potential. Here, we show the development of branched cofactor analogues with a methionine side chain as highly potent KMT9 inhibitors. Through structure-guided design, a basic nitrogen and 4-chlorophenoxy-2-fluorobenzene in the substrate branch contribute most to the high potency and selectivity. Due to the zwitterionic methionine side chain, the inhibitors did not show cellular activity. Importantly, an ethyl ester prodrug <b>8</b> exh2025-01-01T00:00:00Z2025 Jul2026-07-15T12:39:33.242Z2026-07-04T03:14:34.452ZS-EPMC122575104052692710.1021/acs.jmedchem.4c02953