Inhibition of glutamate racemase by substrate-product analogues

Article abstract of DOI:10.1016/j.bmcl.2013.12.114

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (K_i = 3.1 ± 0.6 mM, cf. K _m = 1.41 ± 0.06 mM). The cyclic substrate-product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ～30% inhibition at a concentration of 40 mM. The related cyclic substrate-product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (K_i = 18.4 ± 1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate-product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.

Full text of DOI:10.1016/j.bmcl.2013.12.114

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1432–1436
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of glutamate racemase by substrate–product analogues
Mohan Pal a, Stephen L. Bearne a,b,
⇑
a Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
b Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
D
-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bac-
Received 6 December 2013
Accepted 27 December 2013
Available online 8 January 2014
terial cell wall. Glutamate racemase catalyzes the reversible formation of -glutamate from -glutamate
D
L
and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate–product
analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive
inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part,
for periodontal disease and colorectal cancer (Ki = 3.1 0.6 mM, cf. Km = 1.41 0.06 mM). The cyclic sub-
strate–product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor,
giving only ꢀ30% inhibition at a concentration of 40 mM. The related cyclic substrate–product analogue
1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (Ki = 18.4 1.2
mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimick-
ing the structure of both enantiomeric substrates (substrate–product analogues) serves as a useful design
strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve
as potential lead compounds for further development.
Keywords:
D-Glutamate
Glutamate racemase
Fusobacterium nucleatum
Inhibition
Substrate–product analogues
Cyclic glutamate analogue
Ó 2013 Elsevier Ltd. All rights reserved.
The rise of antibiotic resistance in pathogenic organisms has led
to an increasing need to develop antibacterial agents and to
identify new drug targets.1–3 One such bacteria-specific target is
glutamate racemase (GR).4–9 This cofactor-independent enzyme
GR catalyzes the stereoinversion of
mate via a two-base mechanism wherein one enantiospecific
Brønsted base abstracts the proton from -glutamate and the con-
jugate acid of a second enantiospecific Brønsted base protonates
the intermediate to form -glutamate, and vice versa
L-glutamate and D-gluta-
L
catalyzes the reversible conversion of
L
-glutamate to
D
-gluta-
D
mate.10–12
D-Glutamate is a key component of the peptidoglycan
(Scheme 1A). For GRs, two cysteine residues serve as the Brønsted
layer, which encapsulates the bacterial cell wall in a number of
pathogenic organisms and protects them against osmotic lysis.13–
acid/base catalysts within the active site.26–29
The development of inhibitors for GRs has been particularly
15
Previously, we described the overexpression, kinetic properties,
challenging.6,9,30 Amino acid derivatives including
L
-serine-O-sul-
-N-hydroxyglutamate,32 aziridino glutamate,33 (2R,4S)-4-
substituted
-glutamate analogues,4 and boron- and imide-con-
and quaternary structure of GR from the opportunistic pathogen
Fusobacterium nucleatum (FnGR).16 This gram-negative, obligate
anaerobe17 promotes the onset of periodontal disease by facilitat-
ing the co-aggregation of different bacterial species in oral bio-
films, leading to the permanent establishment of pathogenic
strains within dental plaque and periodontal disease.18,19 F. nucle-
atum is also associated with extraoral disease such as intrauterine
infections associated with pregnancy complications20–22 and colo-
rectal cancer.23,24 Recently, it was established that F. nucleatum
promotes colorectal cancer by adhering to, invading, and inducing
oncogenic and inflammatory responses to stimulate the growth of
colorectal cancer cells.25 Consequently, FnGR is a potential thera-
peutic target for development of drugs directed against periodontal
disease and colorectal cancer.
fate,31
D
D
taining glutamate analogues34 have been reported as GR inhibitors.
A number of non-amino acid inhibitors have also been reported
including 9-benzyl purines,35 8-benzyl pteridinediones,5
pyrazolopyrimidinediones,12,36,37 and benzodiazepine amines.7
However, many of these compounds suffered from poor water
solubility and low bioavailability.9 Some large-molecule38,39 and
peptide-based inhibitors40 have also been reported. More recently,
Spies and co-workers8,41,42 employed in silico screening against a
‘transition state conformation’ of GR to identify several cyclic
inhibitors bearing anionic groups (vide infra).
Previously, we reported that the substrate–product analogue
benzilate is a competitive inhibitor of mandelate racemase (mech-
anistically similar to GRs), binding with an affinity slightly better
than that observed for the substrate mandelate (Ki = 0.67 mM vs
Km = 1.0 mM).43 Similarly, Ohtaki et al.44 reported that citric acid
⇑
Corresponding author.
E-mail address: sbearne@dal.ca (S.L. Bearne).
http://dx.doi.org/10.1016/j.bmcl.2013.12.114
0960-894X/Ó 2013 Elsevier Ltd. All rights reserved.

M. Pal, S. L. Bearne / Bioorg. Med. Chem. Lett. 24 (2014) 1432–1436
1433
suggested by the crystal structure (Fig. 1). Hence, the alternative
binding positions of the methylene groups of - and -glutamate
D
L
might be mimicked by cyclic structures (substrate–product ana-
logues) such as 1 and 2 (Scheme 1B). Indeed, crystallographic
and computational studies have suggested that GR is quite flexible
and therefore likely to accommodate cyclic substrate–product ana-
logues within its active site.8,26,47–49 Herein, we report that these
compounds and related cyclic structures (Scheme 1C) are modest
inhibitors of FnGR, binding with affinities between 2- and 13-fold
less than that of the substrate.
The synthesis of 1-hydroxy-1-oxo-4-amino-4-carboxy-phos-
phorinane (1) was accomplished in a 6-step sequence (Scheme 2)
starting from H3PO2. The intermediate 1-methoxy-1-oxo-4-phos-
phorinanone (10) was synthesized using a route similar to that de-
scribed by Wróblewski and Verkade.51 The Michael-type addition
of the methyl hypophosphite P–H unit, generated in situ by reac-
tion of concd hypophosphorous acid with trismethoxy(propyl)-si-
lane, to methyl acrylate gave
7
in 62% yield.52 Subsequent
Michael-type addition of the other P–H unit in 7 to methyl acrylate
followed by Dieckmann cyclization of 8 gave enol 9, which after
decarboxylation with 1 M HCl afforded 10 in 35% yield. We did
not hydrolyze the methyl ester in 10 at this stage to avoid solubil-
ity issues in subsequent steps. Using the Strecker synthesis,53 10
was converted to a 1:2 mixture of diastereomers of 11, as deter-
mined using NMR spectroscopy (Figs. 13S–15S). Treatment of 11
with 6 M HCl gave a yellow crude mixture containing 1 and NH4Cl.
The NH4Cl was removed by precipitation from methanol/CHCl3
(10:90) and filtration to yield white crystals of 1. Optimization of
the NH4Cl removal and crystallization conditions permitted prepa-
ration of the target racemic cyclic phosphorinane 1 on a scale suf-
ficient for enzyme assays.
Scheme 1. (A) Two-base racemization mechanism of cofactor-independent race-
mases. For glutamate racemase, R1 ¼ NHþ3 and R2 ¼ CH2CH2CO2ꢁ; for mandelate
racemase,
R
1 = OH and R2 = Ph; and for aspartate racemase, R1 ¼ NHþ3 and
R2 ¼ CH2COꢁ2 . The (S)- and (R)-enantiospecific Brønsted acid/base catalysts are
labeled generically as B1 and B2, respectively. The proposed cyclic substrate–
product analogues (1, 2) are shown in (B); and the related linear chain molecules (3,
4) and cyclic (5, 6) analogues are shown in (C).
was a substrate–product analogue for aspartate racemase, acting
as a competitive inhibitor (Ki = 7.4 mM vs Km = 0.7 mM). Harty
The cyclic analogue, 4-amino-4-carboxy-1,1-dioxo-tetrahydro-
thiopyran (2), was prepared by deprotection of the commercially
available 4-N-Boc-amino-4-carboxy-1,1-dioxo-tetrahydrothiopy-
ran using TFA/CH2Cl2.54
et al.45 showed that the substrate–product analogues
a-(hydroxy-
methyl)serine and tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate
were modest inhibitors of serine racemase and proline racemase,
respectively. Figure 1 shows the superposition of the X-ray crystal
structures of the active sites of GR from Enterococcus faecalis with
To test the inhibitory properties of the cyclic substrate–product
analogues 1 and 2, we purified FnGR as a fusion protein bearing an
N-terminal hexahistidine (His6) tag as described previously.16 The
(His)6-tagged enzyme was used for all inhibition studies since it
had previously been shown that removal of the tag did not signif-
icantly alter the kinetic properties of the enzyme.16 Compound 1
inhibited FnGR with an IC50 value of 24.1 3.8 mM (Table 1;
Fig. 44S). Interestingly, we could not achieve 100% inhibition, even
with inhibitor concentrations up to 37 mM—the highest inhibitor
concentration compatible with the circular dichroism (CD)-based
bound
D- and L
-glutamate.12 Unlike mandelate racemase and
aspartate racemase, wherein one of the groups attached to the ste-
reogenic center moves during catalysiꢁs (i.e., the phenyl ring of
mandelate46 and apparently the CH2CO2 side chain of aspartate,44
respectively), GR holds the
a c a
-COꢁ2 , -COꢁ2 , and -NHþ3 groups of
glutamate firmly in place by multiple H-bonds. Therefore, we
anticipated that only the two methylene groups of the side chain
might move within the active site during racemization as
Figure 1. Stereoview (wall-eyed) showing putative motion of the glutamate side chains during racemization. Active site residues of GR from Enterococcus faecalis (PDB
2JFO)12 corresponding to the A and B chains with bound
-glu (green carbons) and -glu (blue carbons), respectively, are superposed. Oxygen, nitrogen, and sulfur are colored
red, blue, and yellow, respectively. H-bonds securing the - and -enantiospecfic Brønsted
-COOꢁ, -NHþ3 , and -COOꢁ groups are indicated by the blue dashed lines. The
acid/base catalysts are Cys 185 and Cys 74, respectively. This figure was prepared using MacPyMOL.50
L
D
a
a
c
L
D

1434
M. Pal, S. L. Bearne / Bioorg. Med. Chem. Lett. 24 (2014) 1432–1436
Scheme 2. Synthetic route to 1. Reagents and conditions: (a) n-PrSi(OMe)3, CH3CN;
(b) H2C@CHCO2Me, i-Pr2NEt; (c) H2C@CHCO2Me, MeONa; (d) t-AmONa, benzene;
(e) 1 M HCl; (f) NH4Cl, 30% NH4OH, NaCN; (g) 6 M HCl; (h) 2.5 M HCl.
Figure 2. Inhibition of FnGR by 1. Representative Michealis–Menten (A) and
Lineweaver–Burk (B) plots are shown. Assays were conducted as described in the
Table 1
text using 0 mM (s), 1.15 mM (4), 2.30 mM (h), 4.60 mM (}), and 6.90 mM (
r
) of
Inhibition of FnGR by cyclic and linear chain substrate–product analogues
1. (C) A representative replot of the apparent Vmax values from the Michaelis–
Menten plot versus inhibitor concentration. The values of
a
, b, n, Vmax, Km, and Ki
1.41 0.06 mM, and
Entry
Inhibitor
IC50 (mM)
Ki (mM)
(Scheme 3) are 1, 0.60 0.01, 1, 147
3.1 0.6 mM, respectively with [FnGR] = 6.25
for experimental details.
4
l
l
M minꢁ1
,
1
2
3
4
5
6
(R,S)-1
(R,S)-2
3
(R,S)-4
5
6
24.1 3.8
215 70a
60.4 1.3
105 39b
47.7 5.6
21.2 1.5
3.1 0.6
—
—
—
g/mL. See Supporting Information
—
18.4 1.2
concentration compatible with CD-based assay (40 mM). The
greater affinity of FnGR for 1, relative to 2, likely arises because
the negatively charged phosphinate in 1 mimics the
a
At the highest concentration of 2 (40 mM) compatible with the CD-based assay,
only ꢀ30% inhibition was observed. IC50 is estimated using Eq. 2S.
c
-COOꢁ of
b
At the highest concentration of 4 (64 mM) compatible with the CD-based assay,
the substrate, while such negative charge is absent in sulfone 2.
This suggested that the negative charge is required for better
binding to FnGR. The ability of two linear analogues of 1, that is,
bis(2-carboxyethyl)-phosphinic acid (3) and ( )-2-amino-4-phos-
phonobutyric acid (4), to inhibit FnGR was also examined. At the
highest concentration of 3 (50 mM) and 4 (64 mM), only 40% and
15% inhibition were observed, respectively, which suggests that a
cyclic structure is preferred and that the active site of FnGR is par-
only ꢀ15% inhibition was observed. IC50 is estimated using Eq. 2S.
assay (Fig. 44S). The IC50 curve exhibited a 40–45% drop in activity
for concentrations of 1 equal to ꢀ10–15 mM, but at higher concen-
trations of 1, the activity leveled off at a constant value. In accord
with this observation, inhibition studies with concentrations of 1
up to 23 mM gave Michaelis–Menten plots (Fig. 48S) in which
the apparent Vmax values were reduced with increasing inhibitor
concentrations up to ꢀ7 mM, but showed little change at higher
concentrations of 1. These results suggested the formation of an ac-
tive ESI complex (Scheme 3). Examination of the inhibition kinetics
at [1] <7 mM (Fig. 2) revealed that 1 behaved as a partial noncom-
petitive inhibitor of FnGR with b = 0.61 0.01 and Ki = 3.1 0.6
mM.55 The cyclic analogue 1 is therefore a good inhibitor of FnGR,
binding with an apparent affinity only 2-fold weaker than that
exhibited for the substrate (i.e., Km = 1.41 0.06 mM). In contrast
to 1, compound 2 showed only ꢀ30% inhibition at the highest
ticularly sensitive to the distance between the
a-carbon and the
anionic group on the side chain.8,49
Bearing in mind the preference exhibited for cyclic analogues,
we examined the ability of 5 and 6, precursors for 1 and 2, respec-
tively, to inhibit FnGR. The synthesis of 5 was accomplished in 65%
yield by hydrolysis of 10 with 2.5 M HCl, while 6 was commercially
available. The IC50 values for 5 and 6 were 47.7 5.6 mM and
21.2 1.5 mM, respectively (Table 1). Since the negatively charged
phosphinate group led to better inhibition of FnGR by 1, relative to
2, we were surprised that FnGR bound 6 with a 2-fold greater affin-
ity than it exhibited for 5. Since the ketone group may exist in an
equilibrium with its hydrated form in water,56 we examined the
hydration equilibria for 5 and 6 using NMR spectroscopy in
DMSO-d and D2O. Using the 1H, 31P, and 13C NMR spectra of 5 in
DMSO-d (25 mM) to assign signals corresponding to the non-
hydrated form (5), we were able to assign signals in the spectra
obtained in D2O to both the non-hydrated (5) and hydrated (50)
species. At pD = 1.5–1.6, the ratio of 5:50 was 1:1.8 (Figs. 24S–
29S). Using a similar approach, the ratio of the non-hydrated form
(6) to the hydrated form (60) was found to be 1:14.8 at pD = 7.6–7.7
(Figs. 33S–36S). In order to estimate the extent of hydration under
assay conditions (10 mM potassium phosphate buffer, pH 8.0), we
conducted NMR studies in deuterated, 10 mM potassium phos-
phate buffer (pD 8.0).
Scheme 3. Kinetic mechanism for the inhibition of FnGR by 1 and 6. Compound 1 is
a partial noncompetitive inhibitor (a = 1, 0 < b < 1, n = 1), while 6 is a cooperative,
mixed-type inhibitor (1 <
a
< 1, b = 0, n >1).55

M. Pal, S. L. Bearne / Bioorg. Med. Chem. Lett. 24 (2014) 1432–1436
1435
The ratio of 5:50 was 2:1 (Figs. 30S–32S) and 6:60 was 1:15
(Figs. 37S–38S). Thus, under the assay conditions, both 5 and 6
are present as a mixture of their hydrated and non-hydrated forms.
Since 6 is present mostly as the hydrated species (93.7%), while 5
exists ꢀ33% as the hydrate, it appears that the hydrated species
may be the better inhibitor. Unfortunately, NMR experiments re-
vealed little change in the ratio of hydrated to non-hydrated forms
of both 5 and 6 between pD 6.0 and 8.0 where the enzyme is active,
so we were not able to assess the effect of altering the ratio of 6:60
on inhibition by altering the assay pD. Additional inhibition studies
with 6 revealed that 6 behaved as a cooperative mixed-type inhib-
itor57 (Fig. 3) wherein the ꢁ1/x-intercepts of the Lineweaver–Burk
plot (i.e., apparent Km) varied exponentially with the inhibitor con-
centration (Fig. 3D). The cooperative effect of the inhibitor is also
evident in the curvature present in the Dixon plot (Fig. 3C). Fitting
the apparent Km data to Eq. 4S (see Supporting Information),
based on the kinetic mechanism shown in Scheme 3, gives
is true for the cyclic analogues of glutamate (i.e., 1 and 6) reported
in the present study. The lack of potency of the present substrate–
product analogues may arise because of a reduction of the volume
(ꢀ29 Å3) of the active site in the enzyme reactive conformation
versus the conformation present in crystal structures.8 Although
this design strategy has not furnished inhibitors with binding affin-
ities that exceed that of the substrate, it has rationally afforded po-
tential lead compounds for further development. In fact, this
rational approach suggested cyclic structures that are remarkably
similar to dipicolinic acid (DPA),49 3-sulfo-benzoic acid,49 croconic
acid,8 and 4-hydroxy-1,3-benzenedisulfonic acid8 identified by
Spies and co-workers through high throughput, in silico screening
approaches based on interactions of GR with the glutamate carban-
ion intermediate. Interestingly, although DPA was a weak inhibitor
of GR from Bacillus subtilis (Ki = 2.7 mM),49 it proved to be a potent
noncompetitive inhibitor of GR from Bacillus anthracis (Ki = 92 lM),
binding at an allosteric site.41 The complex inhibition kinetics ob-
served with the present substrate–product analogues could also
arise from interactions at an allosteric site.
Ki = 18.4 1.2 mM,
aKi = 28.0 5.1 mM, and n = 4.4 1.2. Thus,
inhibition by 6 is highly cooperative and free enzyme binds the
inhibitor with an affinity that is ꢀ13-fold weaker than that exhib-
ited for the substrate (i.e., Km = 1.41 0.06). The origin of the com-
plex inhibition kinetics is unclear, but may arise due to effects on
the monomer–dimer equilibrium,12,16,58 or binding at an allosteric
site (vide infra).12,41
Previously, we demonstrated that substrate–product analogues
were good to modest inhibitors of mandelate racemase,43 serine
racemase,45 and proline racemase.45 In addition, citrate has been
reported as a substrate–product analogue inhibitor of aspartate
racemase.44 For some of these amino acid racemases, substrate–
product analogues that incorporate structural features of both
enantiomeric substrates were bound with affinities that, not sur-
prisingly, were similar to the substrate binding affinity. The same
Acknowledgments
This work was supported by a Discovery Grant from the Natural
Sciences and Engineering Research Council (NSERC) of Canada
(SLB).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.114.
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),
r
), and
(C) with fixed substrate concentrations of 0.5 (s), 1.0 (4), 2.0 (h), 3.0 (}), 5.0 (
r
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3
l
M minꢁ1, 1.41 0.06, 18.4 1.2 mM, and 28.0 5.1 mM, respec-
tively with [FnGR] = 6.25 lg/mL. See Supporting Information for experimental
details.

1436
M. Pal, S. L. Bearne / Bioorg. Med. Chem. Lett. 24 (2014) 1432–1436
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