Highly selective inhibition of histone demethylases by de novo macrocyclic peptides

Abstract

The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.

Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity

“…Having such a structure(s), we should be able to further improve not only its potency, but also its selectivity toward TET1 over isoforms. Moreover, such a structure will guide us to generate a membrane‐permeable macrocyclic peptide, like we performed for the anti‐KDM4A macrocyclic peptide inhibitor, leading us to test if macrocyclic peptides can inhibit cellular full‐length TET1. These attempts are underway in our laboratory.…”

“…Thus, there is strong demand to discover molecules that selectively and potently inhibit TET1 catalysis . We have utilized the random nonstandard peptide integrated discovery (RaPID) system, in which affinity‐based selection was conducted to isolate thioether macrocyclic peptide ligands against the TET1CCD from a mass library consisting of trillions of diverse members (Figure B and Figure S2 in the Supporting Information) . This unique selection campaign by means of the RaPID system has yielded a TET1 inhibitor selective over the TET2 isoform and 2OG‐dependent oxygenases tested.…”

Max‐Bergmann award lecture:A RaPID way to discover bioactive nonstandard peptides assisted by the flexizyme and FIT systems

“…Artificial agonists based on ligands against a receptor tyrosine kinase (cMET) were successfully devised . We also found antagonists against human intracellular enzymes, a ubiquitin ligase (E6AP), a kinase (AKT2), and histone deacetylase and demethylase (SIRT2 and KDM4A). We recently reported antagonists against a pathogenic microorganism enzyme, a cofactor‐independent phosphoglycerate mutase (iPGM), and an inhibitor against Zaire Ebola virus protein 24 (VP24) disrupting the protein‐protein interaction with and human karyopherin alpha 5‐KPNA5 .…”

“…The above thioether‐macrocyclic peptides discovered by the RaPID system displayed remarkable potencies, for which K D values and IC 50 values were in the range of low to sub‐nM in most cases. Their mode of actions turned out to be quite diverse, eg, competing with the substrates in the binding site (human α‐amylase, AKT2, SIRT2, and KDM4A), clogging the transporter site (MATE), targeting allosteric or allosteric‐like site (CmABCB1, PlexB1, and VP24), binding a unique remote‐inhibitory site (iPGM), and dimerizing the intercellular kinase domain via dimerization of the extracellular domain (cMET).…”

Recent Advances in In Vitro Translation and Selection of Bioactive Nonstandard Peptides

“…A library of these ternary complexes is then subjected to repeated rounds of affinity selection, reverse-transcription, and amplification processes. 10 It turned out that the inhibitory activities of the selected cyclic peptides showed a remarkable selectivity for the intra-subfamily KDM4A-C over the other KDM families. The extreme diversity (>10 12 ) of randomized mRNA library accounts for the exceptionally high hit identification rate of RaPID screening.…”