Photon Factory Activity Report 2018 #36 (2019)

AR-NW12A/2008S2-001, BL-17A/2015G595

Structural Analysis of SET7/9 in Complex with Cyproheptadine and its DerivativeeHideaki NIWA1, Shin SATO1, and Takashi UMEHARA1,*Laboratory for Epigenetics Drug Discovery, RIKEN Center for Biosystems Dynamics Research,
1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan

1 Introduction
SET7/9 is a lysine methyltransferase that transfers themethyl group of the coenzyme S-adenosylmethionine(SAM) to a specific lysine residue in a substrate protein.
The substrate proteins of SET7/9 include histone H3 andother non-histone proteins, such as estrogen receptor α(ERα). SET7/9 is implicated in diverse biologicalprocesses. Specific SET7/9 inhibitors are regarded as a
useful chemical probe to investigate the biological functionof protein methylation. In addition, because ERα isoverexpressed in many breast cancers, small-molecule
SET7/9 inhibitors are expected to become potential
anticancer agents.
Prof. Minoru Yoshida (the University of Tokyo andRIKEN) and Prof. Akihiro Ito (Tokyo University ofPharmacy and Life Sciences and RIKEN) performed achemical library screening for SET7/9 inhibitors andidentified cyproheptadine (Fig. 1; hereafter referred to as1) as an inhibitor. Compound 1 is an antagonist of thehistamine receptor (H1) and serotonin receptor (5-HT2A),
and has already been used as an anti-allergic agent.
Compound 1 inhibited enzymatic activity of SET7/9 withan IC50 value of 1.0 μM in vitro. It also destabilized ERα
and suppressed the estrogen-dependent breast cancer cell(MCF7) growth [1]. We determined the crystal structure ofSET7/9 in complex with 1 and elucidated its binding mode
[1].
Based on the structure, Prof. Tomoya Hirano (TokyoMedical and Dental University) and his colleagues
developed a more potent SET7/9 inhibitor, 2hydroxycyproheptadine (Fig. 1; hereafter referred to as 2),
which inhibited SET7/9 with an IC50 value of 0.41 μM. We
determine the structure of SET7/9 in complex with 2 [2].
In this short report, we summarize the structural featuresof SET7/9 in complex with 1 and 2.

Fig. 1: Chemical structures of the inhibitors

2 Experiment
The binary complex of SET7/9–SAM were crystallized
by the handing-drop method at 277K with a reservoir
solution containing 0.1M Tris-HCl (pH 8.5) and 31% PEG
6000. The crystals of the ternary complex of SET7/9–
SAM–1 were obtained by soaking the SET7/9–SAM
crystals into a crystallization drop containing 3 mM of 1for overnight. The crystals of the ternary complex ofSET7/9–sinefungin–2 were also obtained by the soaking
method, where sinefungin was used as a SAM analogue.
The crystals of the SET7/9–sinefungin complex, grownunder similar conditions to those of the SET7/9–SAMcomplex,
were soaked into a crystallization dropcontaining 3 mM of 2 for ten days. The diffraction datafrom the SET7/9–SAM-1 and SET7/9–sinefungin–2
crystals were collected at AR-NW12A and BL-17A of
Photon Factory, respectively. Data processing, molecularreplacement, and structure modelling and refinement werecarried out similarly for the both complexes, using
HKL2000, CCP4 programs, Phaser, Coot and Phenix. The
apo form structure of SET7/9 (PDB: 1N6A) was used forthe search model in the molecular replacement for
SET7/9–SAM–1, while the refined SET7/9–SAM–1
structure was used as the starting model for SET7/9–sinefungin–2. The initial coordinates and parameters forthe compounds were generated using eLBOW in thePhenix suite. The final coordinates and structure factors
have been deposited in PDB with the accession codes
5AYF for SET7/9–SAM–1 and 5YLT for SET7/9–sinefungin–2.
3 Results and Discussion
The crystal structure of SET7/9 in complex with SAM
and 1 was determined at 2.01 Å resolution. The bound 1
molecule was unambiguously identified in the electron
density map. Compound 1 binds to the peptide-binding site
in the SET domain of the protein, which is on the opposite
side of the domain from the SAM-binding site (Fig. 2). The
methylpiperidine ring of 1 is accommodated in the lysine
access channel. The methylpiperidine nitrogen atom forms
a 2.7 Å hydrogen bond with the carbonyl oxygen of Thr266.
The methylpiperidine ring makes hydrophobic contacts
with Tyr335 by stacking its ring to that of Tyr335 (Fig. 3A).
The dibenzocycloheptene ring of 1 occupies the positionof the Tyr337 aromatic ring in a substrate-lysine boundSET7/9 structure. The electron density of Tyr337 is absent
in the compound 1-bound structure, suggesting theconformation of the Tyr337 side chain is not fixed. Astretch of weak electron density was observed above thedibenzocycloheptene rings, to which the main chain of six

Photon Factory Activity Report 2018 #36 (2019)

Fig. 2: Overall structure of SET7/9 with bound 1 (cyan)
and SAM (orange).
residues were modelled as a part of the flexible loop before
the C-terminal α helix (Fig. 3A).
The crystal structure of SET7/9 in complex withsinefungin and 2 was determined at 1.69 Å resolution. The
electron density map showed clearly the molecule of 2 witha bulge that corresponds to the 2-hydroxy group (Fig. 3B).
Compound 2 binds to SET7/9 at the same position and withthe same orientation as 1. As in the SET7/9–SAM–1
complex, the methylpiperidine nitrogen atom forms a 2.6
Å hydrogen bond with the carbonyl oxygen atom ofThr266, and the methylpiperidine ring makes hydrophobic
contacts with the side chain of Tyr335 (Fig. 3B).
Importantly, the hydroxy group of 2 forms 2.6 and 3.0 Åhydrogen bonds with the side-chain oxygen atom andmain-chain nitrogen atom of Asp338, respectively, which
contribute to the increased affinity for SET7/9. The side

chain of Tyr337 was found completely flipped outward.
The dibenzosuberene moiety is surrounded byhydrophobic residues Pro341, Pro342, Pro350, Trp352,and Tyr353. The residues from Tyr337 to Ser345 werevisible in the electron density map of SET7/9–sinefungin–2, with only the residues 346–348 just before the Cterminal helix lacking enough electron density formodelling. The interactions between 2 and Asp338 seem torestrain the conformation of the flexible loop, andaccordingly Tyr337 and the subsequent residues, which
was not clearly visible in the SET7/9–SAM–1 structure,
became visible in the electron density map (Fig. 3B).
In summary, we determined the crystal structure of
SET7/9–SAM–1 and elucidated how compound 1 binds tothe substrate-binding site of SET7/9. Based on thestructure, we developed a more potent 2 and determinedthe crystal structure of SET7/9–sinefungin–2. The hydroxygroup of 2 forms hydrogen bonds with Asp338 and restrain
the conformation of the flexible loop before the C-terminalhelix.
Acknowledgement
We thank the staff at AR-NW12A and BL-17A ofPhoton Factory for their help with X-ray diffraction datacollection. We are grateful to Prof. Minoru Yoshida (theUniversity of Tokyo and RIKEN), Prof. Akihiro Ito (Tokyo
University of Pharmacy and Life Sciences and RIKEN),
and Prof. Tomoya Hirano (Tokyo Medical and Dental
University) for collaboration.
References[1] Y. Takemoto et al., J. Med. Chem. 59, 3650 (2016).
[2] T. Hirano et al., ChemMedChem 13, 1530 (2018).
* takashi.umehara@riken.jp

Fig. 3: The binding modes of the inhibitors. (A) Compound 1 is shown in cyan, SET7/9 residues are in blue, and the mainchain only residues in the weak electron density are in green. The position of missing Tyr337 is indicated with the label inparentheses. (B) Compound 2 is shown in magenta and SET7/9 residues in orange. In the both figures, electron density forthe inhibitors are shown in gray meshes.