Bioorganic & Medicinal Chemistry Letters 24 (2014) 1432–1436

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Inhibition of glutamate racemase by substrate–product analogues Mohan Pal a, Stephen L. Bearne a,b,⇑ a b

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada

a r t i c l e

i n f o

Article history: Received 6 December 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: D-Glutamate Glutamate racemase Fusobacterium nucleatum Inhibition Substrate–product analogues Cyclic glutamate analogue

a b s t r a c t 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 (Ki = 3.1 ± 0.6 mM, cf. Km = 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 (Ki = 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. 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 catalyzes the reversible conversion of L-glutamate to D-glutamate.10–12 D-Glutamate is a key component of the peptidoglycan layer, which encapsulates the bacterial cell wall in a number of pathogenic organisms and protects them against osmotic lysis.13– 15 Previously, we described the overexpression, kinetic properties, 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 facilitating the co-aggregation of different bacterial species in oral biofilms, leading to the permanent establishment of pathogenic strains within dental plaque and periodontal disease.18,19 F. nucleatum is also associated with extraoral disease such as intrauterine infections associated with pregnancy complications20–22 and colorectal 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 therapeutic target for development of drugs directed against periodontal disease and colorectal cancer. ⇑ Corresponding author. E-mail address: [email protected] (S.L. Bearne). http://dx.doi.org/10.1016/j.bmcl.2013.12.114 0960-894X/Ó 2013 Elsevier Ltd. All rights reserved.

GR catalyzes the stereoinversion of L-glutamate and D-glutamate via a two-base mechanism wherein one enantiospecific Brønsted base abstracts the proton from L-glutamate and the conjugate acid of a second enantiospecific Brønsted base protonates the intermediate to form D-glutamate, and vice versa (Scheme 1A). For GRs, two cysteine residues serve as the Brønsted acid/base catalysts within the active site.26–29 The development of inhibitors for GRs has been particularly challenging.6,9,30 Amino acid derivatives including L-serine-O-sulfate,31 D-N-hydroxyglutamate,32 aziridino glutamate,33 (2R,4S)-4substituted D-glutamate analogues,4 and boron- and imide-containing 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 (mechanistically 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

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

Scheme 1. (A) Two-base racemization mechanism of cofactor-independent race mases. For glutamate racemase, R1 ¼ NHþ 3 and R 2 ¼ CH2 CH2 CO2 ; for mandelate racemase, R1 = OH and R2 = Ph; and for aspartate racemase, R1 ¼ NHþ and 3 R2 ¼ CH2 CO 2 . 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 et al.45 showed that the substrate–product analogues a-(hydroxymethyl)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 bound D- and L-glutamate.12 Unlike mandelate racemase and aspartate racemase, wherein one of the groups attached to the stereogenic center moves during catalysis (i.e., the phenyl ring of 44 mandelate46 and apparently the CH2 CO 2 side chain of aspartate,   þ respectively), GR holds the a-CO2 , c-CO2 , and a-NH3 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

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suggested by the crystal structure (Fig. 1). Hence, the alternative binding positions of the methylene groups of D- and L-glutamate might be mimicked by cyclic structures (substrate–product analogues) 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 analogues 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-phosphorinane (1) was accomplished in a 6-step sequence (Scheme 2) starting from H3PO2. The intermediate 1-methoxy-1-oxo-4-phosphorinanone (10) was synthesized using a route similar to that described by Wróblewski and Verkade.51 The Michael-type addition of the methyl hypophosphite P–H unit, generated in situ by reaction of concd hypophosphorous acid with trismethoxy(propyl)-silane, 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 solubility issues in subsequent steps. Using the Strecker synthesis,53 10 was converted to a 1:2 mixture of diastereomers of 11, as determined 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 preparation of the target racemic cyclic phosphorinane 1 on a scale sufficient for enzyme assays. The cyclic analogue, 4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (2), was prepared by deprotection of the commercially available 4-N-Boc-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran using TFA/CH2Cl2.54 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 significantly 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

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 L-glu (green carbons) and D-glu (blue carbons), respectively, are superposed. Oxygen, nitrogen, and sulfur are colored  red, blue, and yellow, respectively. H-bonds securing the a-COO, a-NHþ 3 , and c-COO groups are indicated by the blue dashed lines. The L- and D-enantiospecfic Brønsted acid/base catalysts are Cys 185 and Cys 74, respectively. This figure was prepared using MacPyMOL.50

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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.

Table 1 Inhibition of FnGR by cyclic and linear chain substrate–product analogues Entry

Inhibitor

IC50 (mM)

Ki (mM)

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 — — — — 18.4 ± 1.2

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. b At the highest concentration of 4 (64 mM) compatible with the CD-based assay, 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 concentrations 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 active ESI complex (Scheme 3). Examination of the inhibition kinetics at [1] 1).55

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 text using 0 mM (s), 1.15 mM (4), 2.30 mM (h), 4.60 mM (}), and 6.90 mM (r) of 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 (Scheme 3) are 1, 0.60 ± 0.01, 1, 147 ± 4 lM min1, 1.41 ± 0.06 mM, and 3.1 ± 0.6 mM, respectively with [FnGR] = 6.25 lg/mL. See Supporting Information for experimental details.

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 c-COO of 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-phosphonobutyric 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 particularly 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, respectively, 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 affinity 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 nonhydrated 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 phosphate buffer (pD 8.0).

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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 revealed 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 inhibitor57 (Fig. 3) wherein the 1/x-intercepts of the Lineweaver–Burk plot (i.e., apparent Km) varied exponentially with the inhibitor concentration (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 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 exhibited for the substrate (i.e., Km = 1.41 ± 0.06). The origin of the complex 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 surprisingly, were similar to the substrate binding affinity. The same

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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 affinities that exceed that of the substrate, it has rationally afforded potential 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 carbanion 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 observed with the present substrate–product analogues could also arise from interactions at an allosteric site. 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. References and notes 1. 2. 3. 4.

5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Figure 3. Inhibition of FnGR by 6. A representative Michaelis–Menten plot (A) with fixed concentrations of 6 at 0 (s), 10.0 (4), 20.0 (h), 25.0 (}), and 30.0 mM (r), and the corresponding Lineweaver–Burk plot (B) are shown. A representative Dixon plot (C) with fixed substrate concentrations of 0.5 (s), 1.0 (4), 2.0 (h), 3.0 (}), 5.0 (r), 7.5 (.), and 15.0 mM (/) reveals the nonlinear behaviour of the inhibition. The apparent Km increases markedly with increasing concentrations of 6 (D). The curve shown in panel D was obtained by fitting the dependence of the apparent Km values on [6] using Eq. 5S. The values of b, n, Vmax, Km, Ki, and aKi (Scheme 3) are 0, 4.4 ± 1.2, 150 ± 3 lM min1, 1.41 ± 0.06, 18.4 ± 1.2 mM, and 28.0 ± 5.1 mM, respectively with [FnGR] = 6.25 lg/mL. See Supporting Information for experimental details.

21. 22. 23.

24.

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