Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase

Article abstract of DOI:10.1016/j.bmcl.2017.12.021

Most of the endogenous free D-serine (about 90%) in the brain is produced by serine racemase (SR). D-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR. Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.

Full text of DOI:10.1016/j.bmcl.2017.12.021

Accepted Manuscript
Design, synthesis, and evaluation of novelinhibitors for wild-type human serine
racemase
Satoyuki Takahara, Kiyomi Nakagawa, Tsugumi Uchiyama, Tomoyuki
Yoshida, Kazunori Matsumoto, Yasuo Kawasumi, Mineyuki Mizuguchi,
Takayuki Obita, Yurie Watanabe, Daichi Hayakawa, Hiroaki Gouda, Hisashi
Mori, Naoki Toyooka
PII:
S0960-894X(17)31180-0
https://doi.org/10.1016/j.bmcl.2017.12.021
BMCL 25477
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 September 2017
1 December 2017
10 December 2017
Please cite this article as: Takahara, S., Nakagawa, K., Uchiyama, T., Yoshida, T., Matsumoto, K., Kawasumi, Y.,
Mizuguchi, M., Obita, T., Watanabe, Y., Hayakawa, D., Gouda, H., Mori, H., Toyooka, N., Design, synthesis, and
evaluation of novelinhibitors for wild-type human serine racemase, Bioorganic & Medicinal Chemistry Letters
(2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.021
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Bioorganic & Medicinal Chemistry Letters
jour nal homepage: w ww.elsevier. com
Design, synthesis, and evaluation of novel inhibitors for wild-type human serine
racemase
Satoyuki Takaharaa, Kiyomi Nakagawab, Tsugumi Uchiyamac, Tomoyuki Yoshidaa,d, Kazunori
Matsumotod, Yasuo Kawasumid, Mineyuki Mizuguchi*,a,d, Takayuki Obitad, Yurie Watanabee, Daichi
Hayakawae, Hiroaki Gouda*,e, Hisashi Mori*,a,d and Naoki Toyooka*,a,b,c
a
Graduate School of Innovative Life Science, University of Toyama
b
Graduate School of Science and Engineering, University of Toyama
c Faculty of Engineering, University of Toyama
d Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama
e School of Pharmacy, Showa University
ARTICLE INFO
ABSTRACT
Article history:
Received
Most of the endogenous free D-serine (about 90%) in the brain is produced by serine racemase
(SR).
D-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an
Revised
Accepted
Available online
endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected
to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved
the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico
screening using the structural information. As a result, we identified two hit compounds by their
in vitro evaluations using wild-type SR.
Keywords:
Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and
evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C
showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent
inhibitor than compound 24, which was synthesized by our group based upon the structural
information of the mutant-type SR.
Serine racemase inhibitor
D-serine
In silico
In vitro
Serine racemase (SR) is a pyridoxal 5’-phosphate (PLP)-
previously reported crystal structure of malonate-bound form of
human SR with C2D and C6D mutations and the homology-
modeled structure of ligand-free form of human SR with C2D
and C6D mutations, which was derived from the X-ray crystal
structure of ligand-free rat SR16. However, enzymatic activity of
the mutant-type SR and the effect of inhibitors on mutant-type
SR are significantly different from those of wild-type SR. Thus,
we expressed and purified recombinant wild-type SR and solved
the crystal structure of wild-type SR. Based on the obtained
structural information of the crystal structure of wild-type SR, we
tried to identify new compounds by in silico screening, the
procedure of which is described in supplementary material. We
purchased 9 candidates of possible SR inhibitors, and identified 2
hit compounds (Fig, 1) by their in vitro evaluations using wild-
type SR as shown in Table 1.
dependent enzyme that catalyzes both
L
-serine to
from
generating pyruvate and ammonia 1. A significant amount of free
-serine is found in forebrain regions2. Most of the endogenous
-serine (about 90%) in the brain is produced by SR as revealed
in Srr-knockout (Srr-KO) mouse studies3, 4, 5
-Serine is a co-
agonist of the N-methyl -aspartate-type glutamate receptors
(NMDARs). Full activation of the NMDARs requires
endogenous agonist -glutamate and co-agonist -serine or
D
-serine and
also
the
elimination
of
water
L-serine,
D
D
.
D
D
L
D
glycine. NMDARs are involved in many physiological functions
such as neurotransmission in the central nervous system, higher
brain functions of learning and memory, synaptic plasticity, and
neuronal network formation6, 7. Over activation of the NMDARs
is involved in neuronal cell damage observed in many
neurodegenerative disorders and epileptic states8. This neuronal
cell damage is attenuated in Srr-KO mice3, 9. Thus, SR inhibitors
are expected to be novel therapeutic candidates for the treatment
of these disorders.
Several efforts to discover novel SR inhibitors based on the
structure of mutant-type mouse10, 13, 14A or rat14B SR have been
reported. The investigations of the activities and properties of
14D,
wild-type human SR14C,
14E, and a few efforts to discover
Many efforts, including those of our group, to develop SR
inhibitors have resulted in the generation of some relatively low-
affinity SR inhibitors10-15, thus, further development of novel SR
inhibitors is desired. In our previous in silico screening of the
candidate compounds of human SR inhibitor, we used the
novel SR inhibitors with the wild-type human SR12, 14F have also
been reported. However, this approach with the wild-type human
SR still has remained as difficult target. In this study, we report
the X-ray crystal structure of ligand-free (apo) form of wild-type
human SR, and development of novel inhibitors of human SR

based on the X-ray structure of the apo human wild-type SR for
the first time.
Figure 1. Structure of two hit compounds for wild-type SR
Scheme 1. Synthesis of derivatives 5A, B
Table1. Results of in vitro assay using wild-type SR. The
activity of compounds (1 mM) was evaluated with the percentage
of the D-serine production and compared with vehicle (DMSO).
Next, we examined the effect of the substituent on the right
benzene ring. Condensation of aniline 6 with 7 provided amide 8,
which was converted to eight derivatives 9A-H. Some of the des-
methyl derivatives 9A-H showed good inhibitory effects on wild-
type SR. In particular, the inhibitory effect of derivative 9C was
superior to that of hit compound 1. (Table 2)
The X-ray crystal structure of human wild-type SR was
determined at 1.8 Å resolution. The wild-type structure is an
open form: helix 5, helix 6, stand 3, and strand 4 in the small
subdomain are moved away from the large subdomain (PDB
code 5X2L). The positions of the strand 4, the helix 6, and the
loop between them in the human wild-type SR structure are
different from those in the homology-modeled structure of
ligand-free form of human SR with C2D and C6D mutations in
our previous study (PDB code 3L6B)14A. It was suggested that
both R- and S- isomers of 1 could bind to SR in almost the same
fashion, but the binding affinity of the R-isomer would be
somewhat stronger than that of the S-isomer, because the binding
free energy (∆Gbind) of the R-isomer was estimated to be about
0.9 kcal/mol more stable than that of the S-isomer. Figure 2
shows the interaction model of the R-isomer of 1 with wild-type
SR.
The interaction model of 9C with wild-type SR is presented in
the supplementary material. The estimated ∆Gbind of 9C was
suggested to be about 1.2 kcal/mol more stable than that of the R-
isomer, which is consistent with the trend of inhibitory activity.
Although the interaction of 9C with SR was very similar to that
of 1, the linker region between the methoxybenzene and ester
groups of 9C was suggested to be more deeply located in the
pocket of SR than that of 1, as shown in the supplementary
material. This seems to lead to a greater interaction energy of 9C.
Scheme 2. Synthesis of derivatives 9A-H
Two bis-amide derivatives such as derivative 12A having the
same substituent (o-Cl) in the right benzene ring to 1 and 12B
having the same substituent (o-MeO) to 9C were synthesized
from amide 11 as shown in Scheme 3. Derivative 12A showed
almost the same inhibitory effects for hit compound 1, however
they were less potent than those of 9C. (Table 2)
Figure 2. Interaction model of 1 with wild-type SR. (A)
Interaction Diagram. (B) Compound 1 is shown in stick display.
(C) Compound 1 is shown in space-filling display.
According to the interaction model, the acetoamide group in
the left benzene ring offers the hydrogen donor to Glu283, and
the central two carbonyl groups provide hydrogen acceptors to
His87 and Ser242, respectively. To verify the importance of
acetoamide substituent on the left benzene ring, we first prepared
two derivatives (5A, B) as shown in Scheme 1. After acetylation
of 3, the resulting carboxylic acid was converted to amides 4A,
B, which was transformed into 5A, B in two steps. Both 5A and
5B showed no inhibitory effects on in vitro evaluations. (Table
2)
Scheme 3. Synthesis of derivatives 12A, B

Because derivative 9C showed potent inhibitory effects, we
tried to synthesize and assay des-methyl derivatives 15A, B from
amide 14. However, no inhibitory activity was shown. (Table 2)
silico screening of candidates of SR inhibitors, and in
vitro evaluation of them. As the previously reported structure and
activity of mutant-type SR are different from those of wild-type
SR, we will open new rational approach to discover the inhibitors
of SR for the treatment of pathological states involving over-
activation of NMDAR.
Human serine racemase (SR) cDNA clone (Clone ID,
NT2RP3002501) was obtained from the National Institute of
Technology and Evaluation (NITE) Biological Resource Center
(NBRC). A His-tag sequence for affinity purification was
introduced at the carboxyl-terminal of SR. The SR cDNA was
PCR-amplified
CATATGTGTGCTCAGTATTGCATCTCC-3’
with
primers
5’-
5’-
and
CTCGAGAACAGAAACAGACTGATAAGAAGCT-3’ and cut
with NdeI and XhoI. The modified SR cDNA fragment was
cloned into Escherichia coli expression vector pET21a (Novagen)
digested with NdeI and XhoI to yield the plasmid pETSR. The
pETSR was introduced into BL21 (DE3) E coli. cells (Novagen)
for protein expression. The DNA sequences of PCR fragments
were confirmed with a DNA sequencer (ABI, Genetic analyzer
3100). The amino acid sequence of the SR is the same as full
length human serine racemase (NCBI accession No. CAG33581)
except for the addition of EDHHHHHH (one letter amino acid
sequence) at the carboxyl –terminus.
Scheme 4. Synthesis of derivatives 15A, B
The des-methyl derivative 9C was intriguing as a new
inhibitor for wild-type SR, and we next prepared ketone 21 and
C-derivative 23 as shown in Scheme 5. However, both
derivatives showed no or only weak inhibitory effects. (Table 2)
Figure 3. Dose-response curves of compounds inhibition
against the SR activity. Curves of 24 (◇) and 9C (〇) are
indicated. Data are presented as mean SD (n = 5-16 in each
concentration). The IC50 values of 24 and 9C are 1.603 mM, and
0.836 mM, respectively. The Inhibitory effect of two compounds
is significantly different (p = 1.80 x 10-28, Two-way ANOVA).
Scheme 5. Synthesis of derivatives 21 and 23
Protein expression was performed at 37 °C in LB medium
supplemented with 50 µg/mL ampicillin and 0.01% pyridoxine.
When the optical density at 600 nm was from 0.8 to 0.9, protein
expression
was
induced
with
isopropyl
β-D-
thiogalactopyranoside at a final concentration of 0.3 mM. After
cultivation for 19 hours at 37 °C, cells were collected by
centrifugation. The cells were resuspended in buffer A (20 mM
Tris at pH 8.0, 100 mM NaCl and 20 mM imidazole) and
sonicated on ice. The protein was detected in a soluble fraction
and purified with a His-trap column (GE Healthcare, Bio-
Sciences, Pittsburgh, PA, USA) equilibrated with buffer A. The
protein was finally purified by gel-filtration on a Superdex 26/60
column (GE Healthcare, Bio-Sciences, Pittsburgh, PA, USA) in
buffer B (20 mM Tris at pH 7.4, 100 mM NaCl, 1 mM
Table 2. Results of in vitro assay using wild-type SR.
In a previous paper14, we reported compound 24 as a novel
inhibitor of mutant-type SR (IC50 value to mutant-type SR is 0.14
mM). Finally, we compared the IC50 values of 9C and 24 using
wild-type SR. As shown in Fig. 3, the IC50 value of new
derivative 9C (0.836 mM) was about two times lower than that of
24 (1.603 mM). Here we report for the first time the
identification of novel inhibitors against wild-type SR using the
newly solved crystal structure of wild-type SR, in

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