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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.


KDM4A can demethylate methylated lysine and arginine containing non-histone sequences.(a) Overlay of views from crystal structures of KDM4A with CP2 (red) and CP2(R6Kme3).NOG (cyan). It is noteworthy that the binding site of NOG, an inactive 2OG analogue, is distinct from the binding site of CPs. (b) CP2(R6Kme3) (peptide 9) and (c) CP2(R6me2a) (peptide 10) are substrates of KDM4A. KDM4A1-359 (2 μM) was incubated with CP2 variant (10 μM) in the presence of 2OG (100 μM), Fe(II) (10 μM) and ascorbate (100 μM) for 2 h at 37 °C. The reaction product was analysed using matrix-assisted laser desorption/ionization–time of flight MS. Reactions containing enzymes are in red, no enzyme peptide controls are in black.
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f3: KDM4A can demethylate methylated lysine and arginine containing non-histone sequences.(a) Overlay of views from crystal structures of KDM4A with CP2 (red) and CP2(R6Kme3).NOG (cyan). It is noteworthy that the binding site of NOG, an inactive 2OG analogue, is distinct from the binding site of CPs. (b) CP2(R6Kme3) (peptide 9) and (c) CP2(R6me2a) (peptide 10) are substrates of KDM4A. KDM4A1-359 (2 μM) was incubated with CP2 variant (10 μM) in the presence of 2OG (100 μM), Fe(II) (10 μM) and ascorbate (100 μM) for 2 h at 37 °C. The reaction product was analysed using matrix-assisted laser desorption/ionization–time of flight MS. Reactions containing enzymes are in red, no enzyme peptide controls are in black.

Mentions: To investigate the role of Arg6 of CP2, which binds in a similar manner to the H3Kme(n) residue, we substituted it with other residues (Table 2 and Supplementary Fig. 6); substitution with alanine (peptide 4) or with hydrophobic/uncharged side chains including N-ɛ-acetyl lysine and citrulline (peptides 5, 6 and 7) leads to loss of potency (IC50>2,400 nM for KDM4A). Inhibition of activity is regained upon substitution with positively charged residues (lysine (peptide 8)/N-ɛ-trimethyllysine (peptide 9), IC50=24 and 12 nM, respectively, for KDM4A). The relevance of the crystallographically observed orientation of Arg6 in solution was explored using the trimethyl-lysine CP2 variant (peptide 9). Despite the lack of similarity between CP2 and the histone substrates, N-ɛ-trimethyl-lysine-CP2 (R6Kme3) was efficiently demethylated by KDM4A to Kme2/Kme1/Kme0 (Fig. 3b). A co-crystal structure of CP2(R6Kme3) in complex with N-oxalylglycine (NOG, an inactive form of 2OG) and KDM4A supports the productive binding conformation of the CP2(R6Kme3) (Fig. 3a); the acceptance of CP2(R6Kme3) as a KDM4A substrate unequivocally demonstrates that the CP2 series does not compete with 2OG. Notably, KDM4A was also able to demethylate methylated Arg6 (CP2(R6me2a), peptide 10) to the non-methylated, inhibitory CP2 sequence (Fig. 3c). The different sequences and binding modes between the well-established histone substrates (H3K9me3/K36me3) and CP2 suggests that KDM4s might be more promiscuous in their substrate selectivity and binding mode than presently perceived.


Highly selective inhibition of histone demethylases by de novo macrocyclic peptides
KDM4A can demethylate methylated lysine and arginine containing non-histone sequences.(a) Overlay of views from crystal structures of KDM4A with CP2 (red) and CP2(R6Kme3).NOG (cyan). It is noteworthy that the binding site of NOG, an inactive 2OG analogue, is distinct from the binding site of CPs. (b) CP2(R6Kme3) (peptide 9) and (c) CP2(R6me2a) (peptide 10) are substrates of KDM4A. KDM4A1-359 (2 μM) was incubated with CP2 variant (10 μM) in the presence of 2OG (100 μM), Fe(II) (10 μM) and ascorbate (100 μM) for 2 h at 37 °C. The reaction product was analysed using matrix-assisted laser desorption/ionization–time of flight MS. Reactions containing enzymes are in red, no enzyme peptide controls are in black.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384220&req=5

f3: KDM4A can demethylate methylated lysine and arginine containing non-histone sequences.(a) Overlay of views from crystal structures of KDM4A with CP2 (red) and CP2(R6Kme3).NOG (cyan). It is noteworthy that the binding site of NOG, an inactive 2OG analogue, is distinct from the binding site of CPs. (b) CP2(R6Kme3) (peptide 9) and (c) CP2(R6me2a) (peptide 10) are substrates of KDM4A. KDM4A1-359 (2 μM) was incubated with CP2 variant (10 μM) in the presence of 2OG (100 μM), Fe(II) (10 μM) and ascorbate (100 μM) for 2 h at 37 °C. The reaction product was analysed using matrix-assisted laser desorption/ionization–time of flight MS. Reactions containing enzymes are in red, no enzyme peptide controls are in black.
Mentions: To investigate the role of Arg6 of CP2, which binds in a similar manner to the H3Kme(n) residue, we substituted it with other residues (Table 2 and Supplementary Fig. 6); substitution with alanine (peptide 4) or with hydrophobic/uncharged side chains including N-ɛ-acetyl lysine and citrulline (peptides 5, 6 and 7) leads to loss of potency (IC50>2,400 nM for KDM4A). Inhibition of activity is regained upon substitution with positively charged residues (lysine (peptide 8)/N-ɛ-trimethyllysine (peptide 9), IC50=24 and 12 nM, respectively, for KDM4A). The relevance of the crystallographically observed orientation of Arg6 in solution was explored using the trimethyl-lysine CP2 variant (peptide 9). Despite the lack of similarity between CP2 and the histone substrates, N-ɛ-trimethyl-lysine-CP2 (R6Kme3) was efficiently demethylated by KDM4A to Kme2/Kme1/Kme0 (Fig. 3b). A co-crystal structure of CP2(R6Kme3) in complex with N-oxalylglycine (NOG, an inactive form of 2OG) and KDM4A supports the productive binding conformation of the CP2(R6Kme3) (Fig. 3a); the acceptance of CP2(R6Kme3) as a KDM4A substrate unequivocally demonstrates that the CP2 series does not compete with 2OG. Notably, KDM4A was also able to demethylate methylated Arg6 (CP2(R6me2a), peptide 10) to the non-methylated, inhibitory CP2 sequence (Fig. 3c). The different sequences and binding modes between the well-established histone substrates (H3K9me3/K36me3) and CP2 suggests that KDM4s might be more promiscuous in their substrate selectivity and binding mode than presently perceived.

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.