Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription

Abstract

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Structure–Activity Relationship Studies on (R)‐PFI‐2 Analogues as Inhibitors of Histone Lysine Methyltransferase SETD7

“…Clinically approved antihistaminic cyproheptadine was found to inhibit SETD7‐mediated methylation of H3K4 (IC 50 of 3.4 μ m ). The substrate in this study was ERα, and in vivo inhibition led to blocking of estrogen‐dependent breast cancer cell growth in MCF7 cells . A structure–activity relationship study was performed, however, none of the analogues proved to be more potent than cyproheptadine .…”

Development of Novel Inhibitors for Histone Methyltransferase SET7/9 based on Cyproheptadine

“…Compound 2 n binds to SET7/9 at the same position and with the same orientation as cyproheptadine (Figure ). As in the SET7/9–SAM–cyproheptadine complex, the N ‐methylpiperidine nitrogen atom of 2 n forms a 2.6 Å hydrogen bond with the carbonyl oxygen atom of Thr266, and the methylpiperidine ring makes hydrophobic contacts with the side chain of Tyr335. Importantly, the hydroxy group of 2 n forms 2.6 and 3.0 Å hydrogen bonds with the side‐chain oxygen atom and main‐chain nitrogen atom of Asp338, respectively, and they contribute to the increased affinity for SET7/9 (Figures b and ).…”

“…Interestingly, the side chain of Tyr337, which occupies the cyproheptadine‐binding site to form the lysine access channel if bound with a substrate peptide, is completely flipped outward (Figures b and ). In the SET7/9–SAM–cyproheptadine complex, the electron density of Tyr337 is absent, and only weak electron density of a short stretch in the loop region is visible (Figure ) . On the other hand, the interactions between compound 2 n and Asp338 are likely to restrain the conformation of the flexible loop, which thereby makes Tyr337 and the subsequent residues clearly visible in the electron density map.…”

“…A SET7/9 methyltransferase domain (residues 111–366) containing an N‐terminal GSSGSSG sequence was prepared as previously described . Briefly, a solution of 8 mg mL −1 SET7/9 protein in 20 m m Tris⋅HCl (pH 7.5) buffer solution containing 200 m m NaCl and 5 m m dithiothreitol was mixed with 1.5 m m coenzyme analogue, Sinefungin, which was used in place of SAM.…”

“…However, structure–activity studies of these inhibitors are quite limited, and no compound has yet been approved for clinical use. We also identified cyproheptadine ( 1 , Figure ), a clinically used serotonin receptor antagonist and histamine receptor (H1) antagonist, as a SET7/9 inhibitor . Cyproheptadine ( 1 ) blocks the ubiquitination of ERα by methylation of lysine (Lys302) and induces the digestion of ERα, so it could be a candidate therapeutic agent for diseases associated with upregulation of ERα, such as breast cancer.…”