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Highly selective inhibition of histone demethylases by de novo macrocyclic peptides

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Development of KDM4A cyclic peptide inhibitors.Cyclic peptide binders of KDM4A were selected using the RaPID system (Supplementary Fig. 1). The hit peptide sequences (CP1–CP5) were synthesized and further tested in enzymatic assays. Peptides were cyclized by a thioether formation.
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f1: Development of KDM4A cyclic peptide inhibitors.Cyclic peptide binders of KDM4A were selected using the RaPID system (Supplementary Fig. 1). The hit peptide sequences (CP1–CP5) were synthesized and further tested in enzymatic assays. Peptides were cyclized by a thioether formation.

Mentions: A messenger RNA template library was designed with the general form AUG-(NNK)4–12-UGC, where the AUG start codon was reassigned from Met to either N-chloroacetyl-L-Tyr or N-chloroacetyl-D-Tyr, which induce cyclization of the translated peptides through reaction with an appropriately positioned cysteine (UGC) downstream of the random sequence to yield lantibiotic-like thioether rings1617. The selection used two thioether-macrocycle libraries (LTyr and DTyr libraries)12 with screening against the catalytic domain of His-tagged KDM4A (residues 1–359) (Fig. 1 and Supplementary Fig. 1a). Enriched complementary DNA pools from both libraries were cloned and analysed. Out of 21 (DTyr-library) and 23 (LTyr-library) clones sequenced (Supplementary Fig. 1b,c), five cyclic sequences (CP1–5, 11–14 residues in length) were selected for further analysis based on hit frequency and similarity; these were synthesized by solid-phase peptide synthesis. Catalytic inhibition of KDM4A/C by the selected peptides was tested using a luminescence-based AlphaScreen activity assay18. From the KDM4A screen, two cyclic peptides (CP2 and CP4) demonstrated potencies of half-maximal inhibitory concentration (IC50)<50 nM against KDM4A (Table 1); CP5 was less potent (IC50<500 nM). The other two hits (CP1 and CP3) were much less effective (IC50>100 μM).


Highly selective inhibition of histone demethylases by de novo macrocyclic peptides
Development of KDM4A cyclic peptide inhibitors.Cyclic peptide binders of KDM4A were selected using the RaPID system (Supplementary Fig. 1). The hit peptide sequences (CP1–CP5) were synthesized and further tested in enzymatic assays. Peptides were cyclized by a thioether formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384220&req=5

f1: Development of KDM4A cyclic peptide inhibitors.Cyclic peptide binders of KDM4A were selected using the RaPID system (Supplementary Fig. 1). The hit peptide sequences (CP1–CP5) were synthesized and further tested in enzymatic assays. Peptides were cyclized by a thioether formation.
Mentions: A messenger RNA template library was designed with the general form AUG-(NNK)4–12-UGC, where the AUG start codon was reassigned from Met to either N-chloroacetyl-L-Tyr or N-chloroacetyl-D-Tyr, which induce cyclization of the translated peptides through reaction with an appropriately positioned cysteine (UGC) downstream of the random sequence to yield lantibiotic-like thioether rings1617. The selection used two thioether-macrocycle libraries (LTyr and DTyr libraries)12 with screening against the catalytic domain of His-tagged KDM4A (residues 1–359) (Fig. 1 and Supplementary Fig. 1a). Enriched complementary DNA pools from both libraries were cloned and analysed. Out of 21 (DTyr-library) and 23 (LTyr-library) clones sequenced (Supplementary Fig. 1b,c), five cyclic sequences (CP1–5, 11–14 residues in length) were selected for further analysis based on hit frequency and similarity; these were synthesized by solid-phase peptide synthesis. Catalytic inhibition of KDM4A/C by the selected peptides was tested using a luminescence-based AlphaScreen activity assay18. From the KDM4A screen, two cyclic peptides (CP2 and CP4) demonstrated potencies of half-maximal inhibitory concentration (IC50)<50 nM against KDM4A (Table 1); CP5 was less potent (IC50<500 nM). The other two hits (CP1 and CP3) were much less effective (IC50>100 μM).

View Article: PubMed Central - PubMed

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-&#603;-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.

No MeSH data available.