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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.


Crystallography and mass spectrometry guided modifications of CP2.(a) Sites of modifications were made based on the crystal structure and MS degradation analysis. (b) MS analysis of degradation fragments of CP2(T13Z) observed on incubation with cell lysate. Cleavage sites are indicated as blue lines.
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f4: Crystallography and mass spectrometry guided modifications of CP2.(a) Sites of modifications were made based on the crystal structure and MS degradation analysis. (b) MS analysis of degradation fragments of CP2(T13Z) observed on incubation with cell lysate. Cleavage sites are indicated as blue lines.

Mentions: We then modified CP2 by backbone amide N-methylation, which is reported to improve peptide stability and cellular uptake.25 Guided by the CP2-KDM4A crystal structure (Fig. 2), we synthesized mono N-methylated variants at backbone amide positions not engaging in critical hydrogen bonding interactions with KDM4A and within the CP2 β-sheet. The selected residues, Cys14, Thr13, Val2 and Tyr1, were mainly in the non-interacting, thioether-linkage region of CP2 (Table 2 and Fig. 4a; peptides 13–16). Mass spectrometric (MS) analysis of CP2 degradation patterns after incubation in cell lysates confirmed that these sites are prone to proteolysis (Fig. 4b and Supplementary Fig. 9). D-Ala was also substituted for Gly8 of CP2 with the aim of improving proteolytic stability, as the crystal structure predicted this substitution would be tolerated and hydrolysis at this site was observed by MS (Fig. 4b). 4-Fluorophenylalanine was further incorporated with the aim of improving cellular uptake by increasing hydrophobicity (peptides 17 and 18). Although some of the CP2 modifications reduced activity, in general the structure-guided modifications were well tolerated. Combinations of tolerated modifications were prepared (named as CP2.1, CP2.2 and CP2.3, peptides 19–21, respectively) (Table 2, Fig. 5a and Supplementary Fig. 6); Potency and selectivity for KDM4A over other KDM families were maintained for these modified CPs with isolated enzymes. (Supplementary Table 1). Interestingly, the inhibitory effect of CP2.3 against KDM4B was much weaker than for CP2, but was retained for KDM4A/C. CP2.3 demonstrated greater stability in HeLa cell extracts (t1/2∼5 h) compared with CP2 (t1/2∼1 h), as determined by MS (Supplementary Fig. 10). Furthermore, the modified CP2.3 was able to stabilize FLAG-KDM4A as observed in CETSA, at concentrations lower than the unmodified CP2 (EC50<100 nM) (Fig. 5c), suggesting that the target engagement was maintained on modification and that peptide cellular stability was increased.


Highly selective inhibition of histone demethylases by de novo macrocyclic peptides
Crystallography and mass spectrometry guided modifications of CP2.(a) Sites of modifications were made based on the crystal structure and MS degradation analysis. (b) MS analysis of degradation fragments of CP2(T13Z) observed on incubation with cell lysate. Cleavage sites are indicated as blue lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Crystallography and mass spectrometry guided modifications of CP2.(a) Sites of modifications were made based on the crystal structure and MS degradation analysis. (b) MS analysis of degradation fragments of CP2(T13Z) observed on incubation with cell lysate. Cleavage sites are indicated as blue lines.
Mentions: We then modified CP2 by backbone amide N-methylation, which is reported to improve peptide stability and cellular uptake.25 Guided by the CP2-KDM4A crystal structure (Fig. 2), we synthesized mono N-methylated variants at backbone amide positions not engaging in critical hydrogen bonding interactions with KDM4A and within the CP2 β-sheet. The selected residues, Cys14, Thr13, Val2 and Tyr1, were mainly in the non-interacting, thioether-linkage region of CP2 (Table 2 and Fig. 4a; peptides 13–16). Mass spectrometric (MS) analysis of CP2 degradation patterns after incubation in cell lysates confirmed that these sites are prone to proteolysis (Fig. 4b and Supplementary Fig. 9). D-Ala was also substituted for Gly8 of CP2 with the aim of improving proteolytic stability, as the crystal structure predicted this substitution would be tolerated and hydrolysis at this site was observed by MS (Fig. 4b). 4-Fluorophenylalanine was further incorporated with the aim of improving cellular uptake by increasing hydrophobicity (peptides 17 and 18). Although some of the CP2 modifications reduced activity, in general the structure-guided modifications were well tolerated. Combinations of tolerated modifications were prepared (named as CP2.1, CP2.2 and CP2.3, peptides 19–21, respectively) (Table 2, Fig. 5a and Supplementary Fig. 6); Potency and selectivity for KDM4A over other KDM families were maintained for these modified CPs with isolated enzymes. (Supplementary Table 1). Interestingly, the inhibitory effect of CP2.3 against KDM4B was much weaker than for CP2, but was retained for KDM4A/C. CP2.3 demonstrated greater stability in HeLa cell extracts (t1/2∼5 h) compared with CP2 (t1/2∼1 h), as determined by MS (Supplementary Fig. 10). Furthermore, the modified CP2.3 was able to stabilize FLAG-KDM4A as observed in CETSA, at concentrations lower than the unmodified CP2 (EC50<100 nM) (Fig. 5c), suggesting that the target engagement was maintained on modification and that peptide cellular stability was increased.

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.