Inhibition of serine and proline racemases by substrate-product analogues

Article abstract of DOI:10.1016/j.bmcl.2013.10.061

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (K _i = 167 ± 21 mM, K_i′ = 661 ± 81 mM, cf. K_m = 19 ± 2 mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5 mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (K_i = 111 ± 15 mM, cf. K_m = 5.7 ± 0.5 mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.

Full text of DOI:10.1016/j.bmcl.2013.10.061

Bioorganic & Medicinal Chemistry Letters 24 (2014) 390–393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of serine and proline racemases by substrate-product
analogues
a
a
a
b
b,
⇑
Matthew Harty , Mitesh Nagar , Logan Atkinson , Christina M. LeGay , Darren J. Derksen ,
a,c,
⇑
Stephen L. Bearne
a
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
Department of Chemistry, St. Francis Xavier University, Antigonish, Nova Scotia B2G 2W5, Canada
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
D
-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or
act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of -amino
acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of -serine (a
modulator of neurotransmission) from -serine, while proline racemase (an essential enzymatic and
mitogenic protein in trypanosomes) catalyzes the reversible conversion of -proline to -proline. We
Received 22 September 2013
Revised 28 October 2013
Accepted 28 October 2013
Available online 4 November 2013
D
D
L
L
D
show the substrate-product analogue
of serine racemase from Schizosaccharomyces pombe (K
a
-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor
Keywords:
Racemases
0
i
= 167 ± 21 mM,
K
i
= 661 ± 81 mM, cf.
K
m
= 19 ± 2 mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-
D
-Proline
-Serine
1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29%
D
inhibition at 142.5 mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-
Inhibition
Substrate-product analogues
1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii
i m
(K = 111 ± 15 mM, cf. K = 5.7 ± 0.5 mM). These results suggest that substrate-product analogue
inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when
the inhibitor is sufficiently flexible.
Ó 2013 Elsevier Ltd. All rights reserved.
1
5
Although
may be employed as specific metabolites, regulators, or used as
specific biosynthetic building blocks required by organisms.
Amino acids may be generated or degraded through the action of
racemases, which catalyze the inversion of stereochemistry at
L
-amino acids predominate in nature,
D
-amino acids
(Scheme 1A). SR is activated by ATP and divalent metal ions such
2
+
2+
as Mg and Ca , and utilizes a two-base mechanism wherein one
enantiospecific Brønsted base abstracts the proton from the -Ser-
PLP aldimine and the conjugate acid of a second enantiospecific
Brønsted base protonates the intermediate to form the -Ser-PLP
aldimine, and vice versa (Scheme 1A). SR also catalyzes the
,b-elimination of - and -serine to yield pyruvate and ammonia.
SR activity in the brain serves as the only endogenous mecha-
nism for generating -serine, a co-agonist of the N-methyl- -aspar-
tate receptor (NMDAR) which mediates glutamatergic
neurotransmission. Inhibition of SR has been suggested as a
means of controlling -serine levels to limit the extent of
NMDAR-mediated neurotoxicity in Alzheimer’s disease
amyotrophic lateral sclerosis, to ameliorate neuropathic pain,
and to protect against cerebral ischemia. Indeed, a number of
SR racemase inhibitors have been developed including dicarboxylic
small peptides containing a 3-phenylpropanoic acid
moiety, and hydroxamic acids. However, the development of
potent and selective SR inhibitors remains a challenge.
The X-ray crystal structures of human, rat, and Schizosaccha-
romyces pombe (fission yeast)
D
-
L
D
the
a-carbon. That some of these molecules of unusual stereo-
chemistry play unique roles in organisms or lead to particular
a
L
D
pathologies if under- or overproduced, has led to the recognition
1
–
of racemases (and epimerases) as potential therapeutic targets.
Our studies on mandelate racemase
D
D
8
9–12
revealed that mimicking
1
6
the structure of both enantiomeric substrates (substrate-product
analogues) may serve as a useful design strategy for developing
inhibitors of racemases. For example, benzilate competitively
D
1
7
18–20
and
1
1
1
3
21
22
inhibits mandelate racemase with K
i
= 0.7 mM (cf. K
S
= 0.7 mM).
23
Inspection of the X-ray crystal structures of serine racemase
1
4
and proline racemase suggested that these two enzymes might
be susceptible to inhibition by substrate-product analogues.
2
4
acids,
0
25
26
Serine racemase (SR, EC 5.1.1.18) is a pyridoxal-5 -phosphate
7
(
PLP)-dependant enzyme that catalyzes both the racemization
1
6
16
and ,b-elimination reaction of the amino acid serine
a
1
3,27
SRs have been solved. The amino
acid sequence of SR from S. pombe (SpSR) shares 35% identity with
human SR, and the overall structure appears to be conserved for
⇑
Corresponding authors.
E-mail addresses: dderksen@stfx.ca (D.J. Derksen), sbearne@dal.ca (S.L. Bearne).
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.061

M. Harty et al. / Bioorg. Med. Chem. Lett. 24 (2014) 390–393
391
mammalian SRs.13 To delineate the binding of serine within the
1
3
active site of SpSR, Goto et al. generated a model using the struc-
ture of lysino- -alanyl-modified SpSR and rat serine dehydratase
with bound O-methyl- -serine. This model suggested that the
D
L
hydroxymethyl side chain of the substrate could occupy one of
two binding pockets depending on which enantiomer was bound
(Fig. 1A). Consequently, we anticipated that the substrate-product
analogue -(hydroxymethyl)serine (1) might be an inhibitor of SR.
a
We sub-cloned the open reading frame encoding SpSR from S.
pombe and expressed and purified the enzyme as a fusion protein
6
bearing an N-terminal hexahistidine (His ) tag (see Supplementary
data). Inhibition studies with 1 (prepared by the copper-catalyzed
2
8
condensation of formaldehyde with glycine) were conducted by
following the production of pyruvate (dehydratase activity) using
2
7
a coupled assay with
L-lactic dehydrogenase.
As shown in
Fig. 2, 1 is a modest, linear mixed-type inhibitor of SpSR. (We also
verified the mode of inhibition by plotting the initial velocity data
2
9
in the form of Cornish–Bowden plots.) The inhibition constants
for binding to the free enzyme (K
i
) and the enzyme-substrate com-
0
plex (K
i
) are equal to 167 ± 21 mM and 661 ± 81 mM, respectively.
Thus, 1 binds to the free enzyme with an ꢀ8-fold weaker affinity
relative to that of the substrate (i.e., K = 19 ± 2 mM). Although
m
the model presented by Goto et al. (Fig. 1A) suggests that SpSR
should be able to accommodate the hydroxymethyl side chains
of L- and D-serine simultaneously, as would be the case for binding
of 1, our results suggest that such a binding scenario is unfavor-
able. It is conceivable that the polar b-OH of serine is bound at only
a single site and therefore an inhibitor bearing two hydroxyl
functions is not tolerated. Alternatively, the packing arrangement
within the active site is not flexible enough to accommodate the
added steric bulk of 1, or a conformational change occurs in the en-
Scheme 1. Two-base racemization mechanisms of serine racemase (A) and proline
racemase (B); and substrate-product analogues (C). For SpSR, B
1
2
and B are Lys 57
and Ser 82, respectively.13 For TcPR,
1 2
B and B are Cys 130 and Cys 300,
zyme as it converts between the substrate-bound
L- and D-forms
respectively.14
such that only a single side chain can be bound at the active site.
Figure 1. Stereoviews (wall-eyed) showing putative motion of amino acid side chains during racemization (red arrows). Panel A shows a model of SpSR with bound PLP-L-
13
serine aldimine (green) and PLP-
serine dehydratase complexed with O-methyl-
W61) with space available on either side of the planar ring to accommodate the side chains of
Cys residues is shaded yellow, while surfaces corresponding to Phe 102 and Phe 290 are shaded magenta. This figure was prepared using MacPyMOL.
D-serine aldimine (blue) superimposed at the active site. This model was constructed using the X-ray structures of SpSR (PDB 2ZR8) and rat
3
0
L
-serine (PDB 1PWH). Panel B shows the active site structure of TcPR with the transition state analogue P2C bound (PDB
1
D- and
L
-proline.14 The van der Waals surface corresponding to the active site
31

3
92
M. Harty et al. / Bioorg. Med. Chem. Lett. 24 (2014) 390–393
Scheme 2. Synthetic route to 2. Reagents and conditions: Key: (a) Boc
dioxane, water; (b) (i) MsCl, NEt , DCM, (ii) t-BuOK, THF; (c) s-BuLi, TMEDA, DMF;
d) 2-methyl-2-butene, NaClO , NaH PO , t-BuOH, H O; (e) TFA, DCM.
2
O, NaOH,
3
(
2
2
4
2
two-step procedure incorporating
a
formylation-oxidation
sequence was employed. The formyl substituent was introduced
by quenching the lithiated carbanion, generated in situ, with
DMF. After prompt but careful purification of the formylated inter-
mediate 7, the final oxidation to the carboxylic acid 8 was com-
pleted in high yield using buffered sodium chlorite conditions
(
Pinnick oxidation). By optimization of this key carboxylation se-
quence, we were able to prepare the target bicyclic analogue 2
on a sufficient scale required for the enzyme assays. Compound 3
was obtained from a commercial source.
To test the inhibition properties of the bicyclic substrate-prod-
uct analogues 2 and 3, we sub-cloned the open reading frame
encoding PR from Clostridium sticklandii (CsPR) and expressed and
purified the enzyme as a fusion protein bearing an N-terminal
Figure 2. Inhibition of SpSR by 1. A representative double-reciprocal plot is shown
in panel A. Assays were conducted as described in the Supplementary data with
concentrations of 1 equal to 0 mM (s), 100 mM (4), 200 mM (h), and 300 mM (}).
Representative replots of the apparent 1/Vmax (panel B) and apparent K
m
/Vmax
(
panel C) values obtained from fitting the initial velocity curves as a function of L-
serine concentration to eqn. 1S are shown and the x-intercepts yield the values of
0
K
i
and K
i
according to eqn. 3S, respectively.
hexahistidine (His
cantly alter the kinetic properties of the enzyme (see Supplemen-
tary data) and, therefore, the His -tagged enzyme was used for
the inhibition studies. At the highest concentration of 2 compatible
with the circular dichroism-based assay (142.5 mM), only 29 (±2)%
inhibition was observed (see Supplementary data). Assuming 2
6 6
) tag. Removal of the His -tag did not signifi-
Proline racemase (PR, EC 5.1.1.4) catalyzes the reversible
interconversion of - and -proline. Like SR, PR is a two-base
racemase (Scheme 1B); however, the enzyme is not PLP-depen-
L
D
6
3
2–34
dent.
PR from the eukaryotic parasite Trypanosoma cruzi
(
TcPR) plays a role in immune evasion and in the regulation of
4,35
intracellular metabolism.
Consequently, the enzyme has been
acts as a noncompetitive inhibitor (cf. 3, vide infra), the K
i
value
identified as a target for the development of drugs directed against
can be estimated to be 349 ± 22 mM (with K = 5.7 mM, [L-
m
3
6
Chagas’ disease. Development of inhibitors of PR has been partic-
Pro] = 5.0 mM), or 187 ± 12 mM if one assumes competitive inhibi-
tion. The bicyclic analogue 2 is therefore only a weak inhibitor of
CsPR, binding with an apparent affinity ꢀ61-fold weaker than that
exhibited for the substrate. Since it was possible that the weak
binding could have arisen because of the rigid nature of the bicyclic
system, we tested 3 as a potential inhibitor. We anticipated that
the two 6-membered rings in 3 might afford greater conforma-
tional flexibility relative to the two 5-membered rings in 2. Indeed,
the IC50 value for 3 was 67 ± 5 mM (Fig. 2S, Supplementary data).
8
ularly challenging. Recent attempts to develop more soluble
analogues of the transition state analogue inhibitor pyrrole-2-car-
boxylate (P2C) proved unsuccessful; however, in silico screening
revealed two lead compounds, (E)-4-oxopent-2-enoic acid and its
derivative (E)-5-bromo-4-oxopent-2-enoic acid, that were shown
to be irreversible competitive inhibitors of TcPR.36
The X-ray crystal structure of TcPR with bound P2C revealed
that the carboxylate is held firmly in place by 5 H-bonds and the
ligand is completely buried in a predominately hydrophobic active
site with no solvent channel connecting the active site to the bulk
solvent.1 In addition, the quaternary aminium group of the
4
substrate is held in place by 2 H-bonds.8
,14,37
Crystallographic evi-
dence suggests that the enzyme undergoes a significant conforma-
tional transition upon binding ligands, with the loop containing
Cys 300 being highly mobile.14 Considering the conformational
flexibility of PR and that the
a
-carboxylate and
a-aminium groups
are held in place via H-bonds, we anticipated that the b-,
c
-, and d-
carbons of the proline ring of the substrate may move during catal-
ysis and that the hydrophobic pocket surrounding the proline ring
site might be flexible enough to accommodate substrate-product
analogues such as the bicyclic compounds 2 and 3 (Scheme 1C).
The synthesis of 2 was accomplished using a modification of the
route described by Xiong et al. (Scheme 2; see Supplementary
data). In our hands, the direct carboxylation of the deprotonated
bridgehead carbon with carbon dioxide proved problematic in
terms of yield and purification. To overcome these challenges, a
Figure 3. Inhibition of CsPR by 3.
shown in panel A. Assays were conducted as described in the Supplementary
A representative double-reciprocal plot is
3
8
data with concentrations of 3 equal to 0 mM (s), 25 mM (4), 50 mM (h), and
7
5 mM (}). A representative replot of the apparent 1/Vmax values obtained from
fitting the initial velocity curves as a function of
1S is shown in panel B.
L-proline concentration to eqn.

M. Harty et al. / Bioorg. Med. Chem. Lett. 24 (2014) 390–393
393
Although Lineweaver–Burk plots could be fit quite well to either
noncompetitive inhibition or linear mixed-type inhibition, Cor-
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