Photon Factory Activity Report 2018 #36 (2019)

BL-5A, AR-NW12A, AR-NE3A/2013G738, 2014G190, 2016G199

Structure-based discovery of inhibitors of human DJ-1,a Parkinson’s disease associated protein.
1Jose M.M. Caaveiro1,2,*, Shinya TASHIRO1, and Kouhei TSUMOTO1,3,*
Dept. of Bioengineering, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
2
Global Healthcare, Graduate School of Pharmacy, Kyushu University, Fukuoka 812-8582, Japan
3
Institute of Medical Science, The University of Tokyo, Tokyo 108-8639

DJ-1 is a Parkinson’s disease associated protein but its exact role in the progression of the disease remains uncertain. We
have employed X-ray crystallography to identify the binding mode of low-affinity inhibitors. Based on the structuraldata, optimized inhibitors displaying high-affinity were obtained.
1 Introduction
Parkinson’s disease is a devastating neurodegenerative
disorder [1]. The brains in patients with advanced disease
are profoundly damaged, and show insufficient levels of
the neurotransmitter dopamine. Mutation in the protein DJ1 produces a condition termed early onset Parkinson's
disease [2]. Unfortunately, the molecular mechanism
leading to this condition is unknown
Among the reasons hampering the characterization ofDJ-1 is the absence of a well-defined inhibitor. The
evidence reported so far suggested that an inhibitor binding
to the Cys106 may inhibit the biological function of DJ-1.
Herein, we have identified and optimized compounds witha well-defined inhibition mechanism against DJ-1. These
compounds belong to the isatin family and showed strong
inhibitory properties in vitro and in cell. We hope thesecompounds will contribute to understand the function ofDJ-1 and its connection to Parkinsonism.

1). This is the first evidence of a small molecule directly
bound to the key Cys106 of DJ-1 suggesting that isatin(and isatin-like compounds) may be a promising family ofcompounds inhibiting the activity of DJ-1.

2 Experimental
Human DJ-1 was expressed in high-levels inEscherichia coli, and purified to homogeneity. Crystals ofDJ-1 in complex with inhibitors were prepared by thesoaking method. Crystals of reduced DJ-1 were obtainedby the hanging-drop method by mixing the protein (20 mgmL-1) with a solution containing 100 mM Tris-HCl (pH8.5), 200 mM sodium citrate, 30% (v/v) PEG-400, and 5
mM DTT. Crystals of DJ-1 were subsequently soaked withcompounds 1 ~ 40 mM for 1 ~ 24 hr. The selected crystals
were frozen in liquid N2 until data collection.
Data were collected at BL-5A, AR-NE3A and ARNW12A of the Photon Factory (Tsukuba, Japan) undercryogenic conditions (100 K). Data were processed with
MOSFLM and SCALA. Structures of DJ-1 in complex
with various compounds were determined by the methodof molecular replacement with PHASER and refined withREFMAC5 and COOT.

Fig. 1: Structure of isatin (1) bound to DJ-1. (a) Position ofisatin in the dimer. (b) Close-up view of the active site. (c)Environment of isatin when bound to DJ-1. Adapted from [3].

3 Results and Discussion
A screening of DJ-1 with a small library of fragmentcompounds found that the molecule of isatin binds to DJ-1
with significant affinity. This observation wascorroborated by other techniques [3]. The crystal structureof DJ-1 in complex with isatin at 1.5 Å resolution revealeda covalent bond between the Cys106 residue and DJ-1 (Fig.

The analysis of the interactions between isatin and DJ-1
revealed some critical elements necessary for binding, butalso regions suitable for optimization. The structural
information guided the optimization of isatin to increase
the affinity. In particular the addition of a phenyl group anda halogen at specific positions increased the affinity of thecompounds for the protein (Fig. 2). These compounds bindin the same conformation as isatin, and also engage in acovalent bond with Cys106.

Fig. 2: Optimization of isatins. Affinity expressed as KD.

Photon Factory Activity Report 2018 #36 (2019)

Importantly, these compounds do not only bind, but also
inhibit the enzymatic activity of DJ-1 (Fig. 3). Because
these compounds make a covalent bond with Cys106, it
was hypothesized these compounds could behave asinhibitors. Among several biological functions, DJ-1 is an
enzyme with glyoxalase and deglycation activity [4]. The
enzymatic assays in the presence of several compounds
indeed showed robust inhibitory properties, thefluoride/phenyl derivative having the strongest effect(compound 15) (Fig. 3).

Fig. 3: Enzymatic inhibition by isatins. Compound 2
corresponds to an isatin derivative not binding to DJ-1 because itlacks one of the carbonyl oxygens. Adapted from [3].

In summary, we have determined the crystal structure ofisatin bound to human DJ-1. Based on this structure wehave increased the affinity of the hit-compound anddemonstrated that these compounds also inhibit theenzymatic activity of the protein. We hope thesecompounds will help to clarify the function of DJ-1 inParkinsonism and other biological processes.
Acknowledgement
We thank the staff of the Photon Factory for excellent
technical support. This work was supported by thePlatform for Drug Discovery, Informatics and Structural
Life Science from the Ministry of Education, Culture,Sports, Science and Technology of Japan (to KT), by JSPS
KAKENHI-A grants 25249115 and 16H02420 (to KT), bya JSPS KAKENHI-C grant 15K06962 (to JMMC), and by
a Grant-in-Aid for JSPS Fellows (to ST).
References[1] P.M.A. Antony et al., FEBS J. 280, 5981 (2013).
[2] V. Bonifati et al., Science 299, 256 (2003).
[3] S. Tashiro et al., ACS Chem. Biol. 13, 2783 (2018).
[4] G. Richarme et al., Science 357, 208 (2017).
*Corresponding
authors:
jose@phar.kyushu-u.ac.jp(JMMC), and tsumoto@bioeng.t.u-tokyo.ac.jp (KT)