Tài liệu Báo cáo khoa học: Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles pptx

Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles Elizabeth C. Banks 1 , Stephen W. Doughty 2, *, Steven M. Toms 1 , Richard T. Wheelhouse 1 and Anna Nicolaou 1 1 School of Pharmacy, University of Bradford, UK 2 School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, UK Methionine synthase (MetS) (5-methyltetrahydrofolate- homocysteine transmethylase) 1 (EC.2.1.1.13) is one of two established mammalian enzymes that utilize a bio- logically active cobalamin derivative [methylcobalamin (CH 3 -Cbl)] as a cofactor [1]. MetS catalyses the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine via the CH 3 -Cbl cofactor, with cycling of cobalamin between the +1 [Cbl(I)] and +3 [Cbl(III)] valency states (Fig. 1). Studies on the Escherichia coli and Homo sapiens cobalamin-dependent MetS have revealed that it is a large, conformationally flexible pro- tein, consisting of four functional domains arranged in a linear manner. Each one of these domains binds a different substrate or cofactor. In detail, the N-terminal module is the homocysteine (Hcy)-binding domain; the second domain binds 5-methyltetrahydrofolate, the third domain binds CH 3 -Cbl; and the fourth domain (C-term- inal module) binds S-adenosyl-methionine (S-AdoMet), an allosteric cofactor required for reductive reactivation [2]. X-ray crystal structures of the cobalamin-, S-AdoMet- and 5-methyltetrahydrofolate-binding sites have only been reported for the bacterial enzyme [3–5]. The reaction products methionine and tetrahydro- folate are further metabolized through the one-carbon methionine transmethylation and folate cycles. MetS is therefore intimately linked to important biochemical Keywords benzimidazole; benzothiadiazole; inhibition; methionine synthase; molecular modelling Correspondence A. Nicolaou, School of Pharmacy, University of Bradford, Richmond Road, Bradford BD7 1DP, UK Fax: +44 1274 235600 Tel: +44 1274 234717 E-mail: a.nicolaou@bradford.ac.uk *Present address Faculty of Health and Biological Sciences, School of Pharmacy, University of Notting- ham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia (Received 14 July 2006, revised 7 November 2006, accepted 9 November 2006) doi:10.1111/j.1742-4658.2006.05583.x The cobalamin–dependent cytosolic enzyme, methionine synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to methionine using 5-methyltetrahydrofolate as the methyl donor. The products of this remethylation – methionine and tetrahydrofolate – participate in the active methionine and folate pathways. Impaired methionine synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders. Although the need for potent and specific inhibitors of methion- ine synthase has been recognized, there is a lack of such agents. In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles. Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver methionine synthase, whilst the most active benzothiadiazole (IC 50 ¼ 80 lm) exhibited characteristics of uncompetitive inhibition. A model of the methyltetrahydrofolate-binding site of the rat liver methionine synthase was constructed; docking experiments were designed to elucidate, in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of methionine synthase. Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence of a nitro functional group and two or three probable hydrogen-bonding interactions. Abbreviations CH 3 -Cbl, methylcobalamin; DHPS, dihydropteroate synthase; Hcy, homocysteine; IC 50 , half-inhibitory concentration; MeTr, methyltransferase protein; MetS, methionine synthase; S-AdoMet, S-adenosylmethionine. FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 287 pathways. These include the reactions of trans-sulfura- tion through the production of homocysteine, biologi- cal methylations of DNA, lipids and proteins, and polyamine biosynthesis through the production of methionine and S-AdoMet [6,7]. Furthermore, MetS is the only human enzyme that metabolizes methyltetra- hydrofolate to tetrahydrofolate, thereby facilitating the recycling of this major form of folates to other bio- active folates that provide one-carbon units for purine and pyrimidine synthesis. Impaired function of MetS has been linked to megaloblastic anaemias and neuro- logical disorders [8], atherosclerosis [7] and carcinogen- esis [9,10]. Although the need to develop inhibitors of MetS as drug candidates has long been recognized [11], there are a limited number of reports on agents inhibiting this enzyme. The anaesthetic gas N 2 O is possibly the only selective inhibitor of MetS reported to date, its action mediated through the oxidation of the cobala- min cofactor [12]. Other compounds that have been shown to inhibit this enzyme are the cell-signalling molecule nitric oxide [13,14], chloroform and carbon tetrachloride [15], methylmercury [16], ethanol and acetaldehyde [17], hydrazine [18], S-AdoMet deriva- tives [19] and a series of cobalamin analogues [20]. Polyamines have been shown to stimulate MetS activity [21], whilst methotrexate has been shown to indirectly inhibit the enzyme in vivo through depletion of its substrate, 5-methyltetrahydrofolate [22]. In a new strategy for discovering specific inhibitors of MetS, drug-like, benzo-fused heterocycles that mimic substructures of 5-methyltetrahydrofolate have been evaluated in a cell-free system. The inhibitory activity and mechanism of action have been probed by kinetic studies using purified rat liver enzyme, whilst a structure–activity relationship study has been discerned using a model of the 5-methyltetrahydrofolate-binding site constructed by homology modelling. Results and Discussion The test compounds 1a–k and 2a–c (Table 1) were designed to mimic the pteridine substructure of 5-methyltetrahydrofolate, one of the two substrates of MetS 2 (Fig. 1), and carry functionalities that may facili- tate molecular recognition. The synthesis of compounds 1c–k followed adapta- tions of known methodologies. Substituted phenylene- diamines were prepared by selective reduction of nitroanilines [23] and cyclized with formic acid [24] to give the desired substituted benzimidazoles. The benz- imidazoles 1a–b and the benzothiadiazoles 2a–c were commercially available. All the compounds were tested against highly puri- fied rat liver MetS [14] and the half-inhibitory con- centrations (IC 50 ) are presented in Table 1. The benzimidazoles 1c, 1h and 1k, and the nitrobenzothiad- iazole 2b, gave IC 50 values close to or below 100 lm, with 2b being the most potent inhibitor (IC 50 ¼ 80 lm). From these results it was apparent that the presence of a nitro group at the 5-position was associ- ated with stronger inhibition (1c compared with 1d; 1h with 1i) and this was positively associated with the presence of the 3-methoxy group (1c compared with 1h). However, the aminobenzimidazole 1k showed a marginally stronger inhibition than the corresponding nitrobenzimidazole 1j. This result may indicate that there is more than one mode of interaction with the active site that affects the activity of more-highly sub- stituted molecules such as 1j and 1k. Furthermore, the presence of an N-methyl group on the benzimidazole ring (position similar to the one in the substrate 5-methyltetrahydrofolate; Fig. 1) was detrimental to the IC 50 (comparing 1c with 1f and 1h with 1j). Finally, the inhibitory activity of the benzothiadiazoles was improved by the nitro substitution ( 2b compared with 2a or 2c). To explore further the molecular mechanism of action of those two classes of substituted benzohetero- cycles, the kinetic parameters of inhibition were meas- ured. Compounds 1c and 2b were chosen as being representative of each class as a result of their good inhibitory activity and availability. Figure 2 shows the Lineweaver–Burk, Dixon and Cornish–Bowden plots for the uninhibited and inhibited reactions. The K m values for the uninhibited reaction were calculated to be 25 lm for 5-methyltetrahydrofolate and 0.6 lm for homocysteine, both results being in fair agreement Fig. 1. The cobalamin-dependent methionine synthase catalysed reaction. Cbl(I), cob(I)alamin; CH 3 -Cbl, methylcobalamin; R, ptero- glutamate. Methionine synthase inhibitors E. C. Banks et al. 288 FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS with previously published data for the pig liver enzyme (16.8 and 2.16 lm, respectively) [25]. The Lineweaver– Burk plots for the inhibited reactions showed that the nitrobenzimidazole 1c exhibits the characteristics of mixed inhibition (Fig. 2A) (K i ¼ 26 lm), whilst the nitrobenzothiadiazole 2b is an uncompetitive inhibitor of MetS with respect to 5-methyltetrahydrofolate (K i ¼ 17 lm) (Fig. 2B). These findings were confirmed by the Dixon (Fig. 2C,D) [26] and Cornish–Bowden (Fig. 2E,F) [27] plots for the two compounds. When 1c and 2b were assessed with homocysteine as the vari- able substrate, the Lineweaver–Burk, Dixon and Corn- ish–Bowden plots indicated that both compounds exhibited characteristics of mixed inhibition (Fig. 3), with the nitrobenzothiadiazole 2b presenting a strong component of uncompetitive inhibition (Fig. 3D). It must also be noted that both compounds were very weak inhibitors when assessed with respect to homo- cysteine, with detectable inhibition noted mainly at high concentrations of the inhibitors (0.5 and 1 mm; Fig. 3A). The results of these studies suggest that the two clas- ses of substituted benzo-fused heterocycles may act by two distinct mechanisms. The mixed inhibition exhib- ited by the nitrobenzimidazole 1c is a pattern usually observed in multisubstrate enzyme-catalysed reactions such as MetS. However, the uncompetitive inhibition, shown by the nitrobenzothiadiazole 2b, indicates that there is no reversible link between the inhibitor and the variable substrates. Plausible rationalization includes the possibility that the relatively small nitro- benzothiadiazole may displace the dimethylbenzimidaz- ole side chain of the cobalamin-cofactor or that it may act on the binding site of the MetS allosteric cofactor, S-AdoMet, both effects which could explain the observed uncompetitive inhibition. Furthermore, clo- sely related 1,2,3-benzothiadiazoles and 1,2,4-thiazoles act as potent electron acceptors in biological systems. Thus, 1,2,4-thiadiazoles can be used to trap cysteine residues by mixed disulfide formulation [28]. Alternat- ively, 1,2,3-benzothiazoles have been shown to inhibit cytochrome P450 metabolites by interference with elec- tron transport within the catalytic cycle of cytochrome P450 [29]. Details of the potential binding and electron transfer events that may account for the uncompetitive inhibition of MetS by nitrobenzothiadiazole 2b are the subject of continuing investigation in this laboratory. To elucidate further the mechanism of action of the substituted benzo-fused heterocycles, to explore the interactions occurring at the binding site, and to develop a tool that could assist further optimization of inhibitors, a molecular model of the methyltetra- hydrofolate-binding domain of the rat liver MetS was constructed. In the absence of a high-resolution struc- ture of the methyltetrahydrofolate-binding site for the mammalian enzyme, a model based on the X-ray crys- tal structure of the methyltetrahydrofolate corrinoid iron-sulfur methyltransferase protein (MeTr) from Clostridium thermoaceticum, as determined by Doukov et al. [30], was constructed. It has been suggested that Table 1. Structures and half inhibitory concentrations (IC 50 ) of the series 1 and 2 substituted benzo-fused heterocycles. IC 50 values were determined using highly purified rat liver methionine synthase. Series Compound IC 50 (lM) Series Compound IC 50 (lM) 1a R¼HX¼HY¼H > 150 2a Z¼H > 150 1b R¼CH 3 X¼HY¼H > 150 2b Z¼NO 2 80 ± 6 1c R¼HX¼HY¼NO 2 120 ± 8 2c Z¼NH 2 > 150 1d R¼HX¼HY¼NH 2 > 150 1e R¼HX¼HY¼OCH 3 > 150 1f R¼CH 3 X¼HY¼NO 2 > 150 1g R¼CH 3 X¼HY¼NH 2 > 150 1h R¼HX¼OCH 3 Y¼NO 2 100 ± 13 1i R¼HX¼OCH 3 Y¼NH 2 > 150 1j R¼CH 3 X¼OCH 3 Y¼NO 2 150 ± 9 1k R¼CH 3 X¼OCH 3 Y¼NH 2 95 ± 17 N N R X Y 1 5 7 N S N Z E. C. Banks et al. Methionine synthase inhibitors FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 289 the methyltetrahydrofolate-binding domain of MetS, and indeed of other methyltransferases, share architec- tural similarities [30]. We therefore constructed the rat liver MetS methyltetrahydrofolate-binding site (resi- dues 359–639) model based on the homology of this protein with that of MeTr (residues 1–262). Using information from the Brookhaven protein data bank, sequence homologues of the two proteins were obtained (Fig. 4). This led to the deduction of a back- bone structure that was modelled to the conserved TIM barrel fold (triose phosphate isomerase type structure), a common feature for globular proteins that B A D C F E Fig. 2. Lineweaver–Burk plots for nitrobenzimidazole 1c (A) and nitrothiadiazole 2b (B), with respect to methyltetrahydrofolate (MTHF), at inhibitor concentrations of 1000 l M (·), 500 lM (m), 100 lM (d) and 0 lM (j ). Dixon plots for nitrobenzimidazole 1c (C) and nitrothiadiazole 2b (D), and Cornish–Bowden plots for nitrobenzimidazole 1c (E) and nitrothiadiazole 2b (F), with respect to the inhibitor, at methyltetrahydro- folate (MTHF) concentrations of 11 l M (·), 22 lM (m), 67 lM (d) and 224 lM (j). Methionine synthase inhibitors E. C. Banks et al. 290 FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS has been predicted to occur in the pterin-binding site of related methyltransferases [30]. The nonconserved sequences were altered and any insertions or deletions were applied using the molecular modelling program, sybyl 3 , to construct and refine the model. A gradual refinement of the resulting structure was performed using minimization through application of the charmm 4 program and force field [31]. A C E B D F Fig. 3. Lineweaver–Burk plots for nitrobenzimidazole 1c (A) and nitrothiadiazole 2b (B), with respect to homocysteine (Hcy), at inhibitor con- centrations of 1000 l M (·), 500 lM (m), 100 lM (d) and 0 lM (j). Dixon plots for nitrobenzimidazole 1c (C) and nitrothiadiazole 2b (D), and Cornish–Bowden plots for nitrobenzimidazole 1c (E) and nitrothiadiazole 2b (F), with respect to the inhibitor, at homocysteine (Hcy) concen- trations of 1.1 l M (·), 2.2 lM (m), 6.5 lM (d) and 11 lM (j). E. C. Banks et al. Methionine synthase inhibitors FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 291 Docking of the substrate 5-methyltetrahydrofolate in the model of the active site was based on the approach followed by Doukov et al. [30] to identify the inter- actions between the pterin cofactor and MeTr. This approach was based on the assumption that the strong structural homology of MeTr and the dihydropteroate synthases (DHPS) allows the prediction of interactions between the pterin ring and MeTr based on the binding of hydroxymethylpterin pyrophosphate to DHPS. Following the same approach, and in order to identify the orientation of the substrate when bound to the active site, we superimposed the rat liver MetS model on the experimentally determined structure of hydroxymethlypterin pyrophosphate bound to DHPS [32]. The result of this approach indicated the orienta- tion of 5-methyltetrahydrofolate in the MetS active site. Figure 5 shows the superimposed structures of DHPS and 5-methyltetrahydrofolate. The model of the methyltetrahydrofolate-binding site of MetS with one molecule of the substrate included was further opti- mized using charmm, and the ligand was parameter- ized using partial atomic charges and other parameters obtained from quantum mechanic modelling (Hartree- Fock 6–31G* within the Spartan PCPro package) 5 of the ligand structure. Electrostatic surfaces of the meth- yltetrahydrofolate-binding site domain were generated to show the size of the active site (Fig. 6). The negat- ively charged areas may indicate the need of the inhib- itor to have positively charged regions for favourable interactions to take place. Figure 7 highlights the amino acyl residues that are proposed to interact with 5-methyltetrahydrofolate, according to this model. Calculations using interaction potentials produced predicted values for the percentage inhibition of each of the tested compounds. These data were then compared with the experimentally determined data (percentage inhibition at 100 lm), and the results are presented in Fig. 8. The predicted activities of seven heterocycles (1j, 1k, 1f, 1g, 1a, 2a , 2c) were found to have good cor- relation with the experimentally determined inhibition Fig. 4. Sequence homology of the template sequence of MeTr and the methyltetrahydrofolate-binding domain of rat liver MetS. The residues shown in bold indicate conserved homology between the MeTr and MetS proteins. Marked with a cross (+) are the residues that have a high degree of similarity so that although the sequence is not identical, the function of the residues is expected to remain the same. Fig. 5. The orientation of hydroxymethylpterin pyrophosphate (HMPP) when bound to dihydropteroate synthase, and the super- imposed structure of methyltetrahydrofolate showing the proposed orientation in the methionine synthase active site. HMPP is repre- sented as a stick structure; methyltetrahydrofolate is represented as a wire structure. Methionine synthase inhibitors E. C. Banks et al. 292 FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS (percentage inhibition ± 10, Fig. 8), including two of the five most active compounds (i.e. the benzimidazoles 1j and 1k). Interestingly, 2b, the most active compound of this series, was not predicted to have strong inhibitory activity. This finding is consistent with the kinetic evalu- ation for this inhibitor that suggests a different mode of action (i.e. through noncompetitive inhibition, as shown in Fig. 2) unrelated to direct binding to the methyltetra- hydrofolate site. Overall, the biological evaluation and molecular modelling studies indicate two routes for the develop- ment of the next generation of inhibitors. Specifically, the molecules need to be relatively small or not carry bulky substituents in order to enter the active site. They require a planar region that can be positioned in the centre of the active site, a nitro functional group and two or three possible hydrogen-bonding groups. Further refinement of this model could assist the dis- covery of the next generation of inhibitors for MetS. Moreover, the observed noncompetitive inhibition pat- tern, with respect to the methyltetrahydrofolate-bind- ing site of MetS, implies the existence of other binding sites that may also be investigated for the development of inhibitors, whilst the potential reactivity of the benzothiadiazole ring opens the possibility for design- ing mechanism-based inhibitors. Overall, this approach may lead to the identification of compounds with potential therapeutic value, in particular as chemo- therapeutic agents for methionine-dependent cancers in combination with methionine-depleted treatments [33]. As the enzyme and its related metabolites have been involved in many disorders, including cardiovascular disease, neurodegenerative diseases and cancer, potent and specific inhibitors will also be valuable tools for defining the exact role of MetS in the pathophysiology of these diseases. Experimental procedures dl-homocysteine, S-AdoMet (iodide salt), 5-methyl- tetrahydrofolic acid (barium salt), diothiothreitol, hydroxycobalamin, dimethylsulfoxide, ascorbic acid, phenyl- methanesulfonyl fluoride, Na-p-tosyl-l-lysylchloromethyl ketone, trypsin inhibitor, aprotinin, DEAE-cellulose, and phosphate buffers were purchased from Sigma (Poole, UK). 5-[ 14 C]-methyl]methyltetrahydrofolic acid (barium salt) (56 mCiÆmmol )1 ) was purchased from Amersham (Little Chalfont, UK). AG1-X8 resin (200–400 mesh chloride form) 6 and the Protein Assay kit were from Bio-Rad (Hemel Hemp- stead, UK). Q-Sepharose Fast Flow and Hydroxyapatite were from Pharmacia 7 (Chalfont St Giles, UK). Optiphase HiSafe 3 scintillation cocktail was from Fisher Scientific (Leicester, UK). Amicon ultrafiltration membranes, of 30 kDa, were purchased from 8 Millipore (Watford, UK). Benzimidazole (1a), 1-methylbenzimidazole (1b), 2,1,3-ben- zothiadiazole (2a), 4-nitro-2,1,3-benzothiadiazole (2b), 4-amino-2,1,3-benzothiadiazole (2c) and 4-methoxyaniline were obtained from Aldrich (Poole, UK); and 1,3-dinitroben- zene was from Avocado (Hewsham, UK). Solvents were of the highest purity commercially available and were A B C Fig. 6. Snapshot pictures showing the electrostatic surfaces of the methyltetrahydrofolate-binding domain of rat liver MetS (A) with 5-methyltetrahydrofolate bound showing the open cleft binding side from a side angle, (B) from a reverse angle, and (C) from above. Red indicates negatively charged surfaces, and blue indicates posi- tively charged surfaces. E. C. Banks et al. Methionine synthase inhibitors FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 293 purchased from Sigma or BDH (Poole, UK). TLC plates (sil- ica gel 60 F 254 ) and silica gel (particle size 40–63 lm) for chromatography were from Merck (Beeston, UK). Deuterat- ed solvents were from Goss (Glossop, UK). Melting points were determined using an Electrothermal IA9200 digital melt- ing point apparatus. IR spectra were recorded on a Perkin Elmer 9 (Paragon 1000) FT-IR Spectrophotometer (Perkin Elmer, Seer Green, UK). 1 H and 13 C NMR spectra were acquired at 270.05 and 67.80 MHz, respectively, on a JEOL GX270 spectrometer (JEOL UK, Welwyn, UK) 10 ; 13 C assign- ments were made using the DEPT135 experiment. Mass spectra were obtained from the EPSRC National Mass Spectrometry Service Centre, University of Wales (Swansea, UK). Synthesis of the substituted benzimidazoles 1c–k 1,3,5-Trinitrobenzene [34] 1,3-Dinitrobenzene 50 g (0.297 mol) was dissolved in fum- ing nitric acid (130.5 mL) and fuming sulphuric acid (243.5 mL), then heated under reflux at 150 °C for 7 days. The reaction was cooled slowly to room temperature. On addition to ice-cold distilled water, a solid precipitated which was collected by filtration and recrystallized from glacial acetic acid to give 1,3,5-trinitrobenzene (61.01 g, 97%), melting point (m.p.) 118–119 °C, literature 11 122 °C [34]. 1 H NMR (CDCl 3 ) d: 9.41 (s 12 , 2-H, 4-H, 6-H). 13 C NMR (CDCl 3 ) d: 149.6 (C-NO 2 ), 124.4 (CH-Ar). MS (EI): 213 (M + ). IR v max Æcm )1 3104s 13;14 (C-H aromatic), 1624s (C¼C aromatic), 1475m 13;14 (C¼C aromatic), 1544s (N¼O, asym- metric), 1345s (N¼O, symmetric), 900s (C-H bend). Fig. 7. Detailed view into the 5-methyltetrahydrofolate-binding pocket of rat liver MetS. Atoms within 7 A ˚ of the docked ligand are shown: hydrophobic amino acid residues are coloured bronze, and hydrophilic acid residues are coloured blue. Blue lines indicate the putative hydro- gen bonding interactions with ASN100. H-bond lengths are shown on the drawing, deviations from linearity are < 15° for both. Fig. 8. Correlation of experimentally determined inhibition (percent- age inhibition at 100 l M) with computer-predicted inhibition, based on the calculation of interaction potentials. Methionine synthase inhibitors E. C. Banks et al. 294 FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 3,5-Dinitroanisole [35] 1,3,5-Trinitrobenzene (5 g, 0.023 mol) was dissolved in methanol (75 mL) with gentle heating. To this hot solution, a hot solution of potassium bicarbonate (0.5 mol, 7.5 g) in water (30 mL) and methanol (20 mL) was added. The mix- ture was heated at reflux for 2.5 h, cooled to room tem- perature and the methanol evaporated under reduced pressure. The aqueous residue was extracted with chloro- form (3 · 40 mL), the chloroform extracts combined, dried over MgSO 4 and the solvent evaporated. The product was recrystallized from ethanol to give 3,5-dinitroanisole (3.36 g, 74%), m.p. 15 98–100 °C, lit. 104–106 °C [35]. 1 H NMR (CDCl 3 ) d: 8.65 (d 16 , J ¼ 2 Hz, 1H, 4-H), 8.06 (d, J ¼ 2 Hz, 2H, 2-H, 6-H), 4.01 (s, 3H, OCH 3 ). 13 C NMR (dimethylsulfoxide) d: 164.3 (C-1), 152.7 (C-3,5), 118.9 (C-2,6), 114.3 (C-4), 61.07 (CH 3 ). MS (EI): 198 (M + ). IR, v max Æcm )1 : 3098s (C-H aromatic), 2862w 17 (C-H sp 3 ), 1600m (C¼C aromatic), 1544s (N¼O, asymmetric), 1345s (N¼O, symmetric), 1080s (C-O). 2,3,5-Trinitroanisole [36] 3,5-Dinitroanisole (1.5 g, 0.007 mol) was dissolved in con- centrated sulfuric acid (20 mL) with gentle heating. The solution was placed in an ice bath and fuming nitric acid (4.2 mL) was added dropwise over a period of 10 min. The mixture was kept on ice for 20 min, with constant stirring, and monitored by TLC. The reaction was stopped by the addition of distilled water (50–100 mL) and the product was extracted into ether (3 · 40 mL). The ether extracts were combined, dried over MgSO 4 and the solvent evaporated. The product was recrystallized from ethanol to give 2,3,5-trinitroanisole (1.6 g, 96%), m.p. 100–104 °C, lit. 104 °C [36]. 1 H NMR (CDCl 3 ) d: 8.59 (d, J ¼ 2 Hz, 1H, 4-H), 7.99 (d, J ¼ 2 Hz, 1H, 6-H), 4.06 (s, 3H, OCH 3 ). 13 C NMR (dimethylsulfoxide- d 6 ) d: 155.1 (C-1), 152.5 (C-5), 143.9 (C-3), 140.0 (C-2), 116.5 (C-6), 107.4 (C-4), 60.2 (CH 3 ). MS (EI): 243 (M + ). IR v max Æcm )1 : 3117m (C-H aromatic), 2992w (C-H sp 3 ), 1600m (C¼C aromatic), 1469m (C¼C aromatic), 1046s (C-O symmetric). 2-Amino-3,5-dinitroanisole [37] 2,3,5-Trinitroanisole (1 g, 0.004 mol) was dissolved in abso- lute ethanol (54 mL), cooled to 2 °C, and concentrated NH 3 (6 mL) was added. The mixture was heated under reflux for 3 h, cooled to room temperature and the solvent evaporated under reduced pressure. After isolation by flash chromatography [chloroform ⁄ petroleum ether (70 : 30; v ⁄ v)], the product was recrystallised from ethanol to give 2-amino-3,5-dinitroanisole (0.86 g, 80%), m.p. 182–184 °C, lit. 180 °C [37]. 1 H NMR (CDCl 3 ) d: 8.82 (d, J ¼ 2 Hz, 1H, 4-H), 9.0–5.0 (br 18 , 2H, NH 2 ), 7.72 (d, J ¼ 2 Hz, 1H, 6-H), 4.05 (s, 3H, OCH 3 ). MS (EI): 214(M + ). IR v max ⁄ cm )1 : 3465s (NH asymmetric), 3322s (NH symmetric), 3098w (C-H aromatic), 2992w (C-H sp 3 ), 1600m (C¼C aro- matic), 1456m (C¼C aromatic), 1550s (N¼O asymmetric), 145s (N¼O symmetric), 1059s (C-O). 2-N-Methylamino-3,5-dinitroanisole [37] 2,3,5-Trinitroanisole (1 g, 0.004 mol) was dissolved in tetrahydrofuran (THF) (5 mL) and methylamine in THF 19 (10 mL, 2 m), then the solution was heated in a Young’s tube for 4 h, cooled and the solvent evaporated under reduced pressure. The product was isolated by flash chro- matography [diethyl ether ⁄ hexane (60 : 40, v ⁄ v)] to give 2-aminomethyl-3,5-dinitroanisole (0.8 g, 89%), 220–222 °C, lit. 230 °C [37]. 1 H NMR (CDCl 3 ) d: 8.76 (d, J ¼ 2 Hz, 1H, 4-H), 8.49 (br, 1H, NH), 7.65 (d, J ¼ 2 Hz, 1H, 6-H), 3.94 (s, 3H, OCH 3 ), 3.37 (d, J ¼ 6 Hz, 3H, NCH 3 ). 13 C NMR (CDCl 3 ) d: 149.6 (C-1), 148.4 (C-5), 138.8 (C-3), 137.2 (C-2), 124.3 (C-6), 118.9 (C-4), 57.2 (OCH 3 ), 33.6 (NCH 3 ). MS (EI): 198 (M + ). 2,3-Diamino-5-nitroanisole, 3-amino-2-methylamino- 5-nitroanisole [37] The same method was 20 applied to both 2-amino-3,5-dinitro- anisole and 2-N-methylamino-3,5-dinitroanisole. The appro- priate compound (1.0 mmol) was dissolved in methanol (30 mL) and water (2 mL). Ammonium chloride (573 mg, 10 mmol) and ammonium carbonate (350 mg, 3.64 mmol) were added to the solution. A hot solution of sodium sulfide (110 mg, 1.41 mmol in 1.5 mL water) was added dropwise over a time-period of 5 min and the solution heated at reflux for 2 h. The reaction mixture was allowed to cool slowly to room temperature, the volatile solvent evaporated under reduced pressure and the product was isolated by flash chromatography, eluted with methanol: c. NH 3 : chloroform (1 : 1 : 98). 2,3-Diamino-5-nitroanisole: (0.150 mg, 77%), m.p. 173–175 °C, lit.165–167 °C [37]. 1 H NMR (CDCl 3 ) d: 7.41 (d, J ¼ 2 Hz, 1H, 4-H), 7.39 (d, J ¼ 2 Hz, 1H, 6-H), 4.05 (s, 2H, NH 2 ), 3.93 (s, 3H, OCH 3 ), 3.79 (s, 2H, NH 2 ). 3-Amino-2-methylamino- 5-nitroanisole: (0.181 mg, 80%), m.p. 158–160 °C. 1 H NMR (CDCl 3 ) d: 7.32 (d, J ¼ 2 Hz, 1H, 4-H), 7.24 (d, J ¼ 2 Hz, 1H, 6-H), 4.1 (br, s, 3H, NH), 3.87 (s, 3H, OCH 3 ), 2.82 (s, 3H, NCH 3 ). 5-Nitro-7-methoxybenzimidazole (1h) and 1-methyl- 5-nitro-7-methoxybenzimidazole (1j) [37] The same method was 21 applied to both 2,3-diamino-5-nit- roanisole and 3-amino-2-methylamino-5-nitroanisole. The compound (1.0 mmol) was dissolved in formic acid (5 mL) and heated at reflux for 2 h. The reaction was removed E. C. Banks et al. Methionine synthase inhibitors FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 295 from the heat, cooled to room temperature, and toluene (20 mL) and water (1 mL) were added. The volatile sol- vents were evaporated under reduced pressure and the resi- due poured into water (30 mL) and extracted with ethyl acetate (3 · 30 mL). The combined organic extracts were washed with water (20 mL), dried over MgSO 4 and evapor- ated under reduced pressure. The product was recrystallized from ethyl acetate. 1h: (216 mg, 96%), m.p. 254–256 °C, lit. 258–260 °C [37]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 13.25 (br, 1H, NH), 8.44 (s, 1H, 2-H), 8.13 (d, J ¼ 2 Hz, 1H, 4-H), 7.57 (d, J ¼ 2 Hz, 1H, 6-H), 4.04 (s, 3H, OCH 3 ). MS (EI): 193 (M + ). 1j: (202 mg, 98%), m.p. 146–148 °C. 1 H NMR (dimethylsulfoxide-d 6 ) d: 8.35 (s, 1H, 2-H), 8.18 (d, J ¼ 2 Hz, 1H, 4-H), 7.06 (d, J ¼ 2 Hz, 1H, 6-H), 4.03 (s, 3H, OCH 3 ), 3.29 (s, 3H, NCH 3 ). HRMS (ES) (M + H) 208.0717, C 9 H 10 N 3 O 3 requires 208.0717. 5-Amino-7-methoxybenzimidazole (1i) [37] and 5-amino-7-methoxy-N1-methylbenzimidazole (1k) [38] The same method was 22 applied to both 5-nitro-7-methoxy- benzimidazole (1h) and 1-methyl-5-nitro-7-methoxybenzimi- dazole (1j). The compound (0.6 mmol) was dissolved in ethanol (30 mL) with 2 drops of concentrated HCl, and 10% weight of palladium on a carbon catalyst was added. The system was evacuated and the mixture stirred vigo- rously under a hydrogen atmosphere until the reaction was complete (approximately 2–3 h by TLC). The catalyst was removed by filtration through celite and washed with copi- ous amounts of ethanol. The solvent was evaporated under reduced pressure and the product was recrystallized from ethanol and ethyl acetate. An alternative method involved using eight equivalents of ammonium formate as the hydro- gen source and reacting the mixture, as above, for 2 h in an evacuated system. 1i: (0.08 mg, 82%), m.p. 220–223 °C, lit. 216–218 °C [37]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 8.72 (s, 1H, 2-H), 8.20 (d, J ¼ 2 Hz, 1H, 4-H), 7.65 (d, J ¼ 2 Hz, 1H, 6-H), 5.81–6.12 (br, 3H, NH, NH 2 ) 4.07 (s, 3H, OCH 3 ). 1k: (0.081 mg, 76%), m.p. 180–182, lit. 178 °C [38]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 8.64 (s, 1H, 2-H), 8.26 (d, J ¼ 2 Hz, 1H, 4-H), 7.12 (d, J ¼ 2 Hz, 1H, 6-H), 5.23–5.65 (br, 2H, NH 2 ), 4.01 (s, 3H, OCH 3 ), 3.26 (s, 3H, NCH 3 ). 5-Nitrobenzimidazole (1c) and 5-aminobenzimidazole (1d) [39] These compounds were synthesized, according to the meth- ods described above, from 2,4 dinitroaniline. 1c: (2.5 g, 88%), m.p. 203–204 °C, lit. 204–205 °C [39]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 8.54 (s, 1H, 2-H), 8.51 (d, J ¼ 2 Hz, 1H, 4-H), 8.44 (s, 1H, NH), 8.13 (dd 23 , J ¼ 2 Hz, J ¼ 8 Hz, 1H, 6-H) 7.09 (d, J ¼ 8 Hz, 1H, 7-H). MS (EI): 164(M + ). 1dÆ2HClÆ0.2H 2 O: (3.1 g, 90%), m.p. decomposition 24 > 230 °C, lit. 165–166 °C (free base) [39]. 1 H NMR (dimethyl- sulfoxide-d 6 ) d: 9.46 (s, 1H, 2-H), 7.80 (d, J ¼ 8 Hz, 1H, 7- H), 7.58 (br, s, 1H, 4-H), 7.33 (dd, J ¼ 2 Hz, J ¼ 8 Hz, 1H, 6-H), 5.6–3.4 (br, s, 3H, NH + NH 2 ). MS (EI): 134(M + ). Found: C, 40.53; H, 4.44; N, 19.55. C 7 H 7 N 3 Æ2HClÆO.2H 2 O requires: C, 40.10; H, 4.52; N, 20.04%. 5-Methoxybenzimidazole (1e) 2-Amino-4-methoxy-2-aniline hydrochoride (0.8 g, 4.60 mmol) was dissolved in formic acid (50 mL) and heated at 120 °C for 12 h. The mixture was evaporated to dryness and partitioned between ethyl acetate (100 mL) and concen- trated NH 3 (20 mL). The layers were separated and the aqueous layer further extracted with ethyl acetate (2 · 30 mL). The combined organic extracts were dried over MgSO 4 and evaporated under reduced pressure. The residue was dissolved in isopropanol, treated with 5 mL of concentrated HCl, evaporated twice from isopropanol then recrystallized from isopropanol-ether to yield a grey solid, 1eÆHCl: (0.50 g, 59%), m.p. 202–206 °C, lit. 199–202 °C [40]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 15.01 (br, 2H, 2 · NH), 9.43 (d, J ¼ 5 Hz, 1H, 2-H), 7.71 (d, J ¼ 9 Hz, 7-H), 7.23 (d, J ¼ 2 Hz, 1H, 4-H), 7.14 (dd, J ¼ 9Hz, J ¼ 2 Hz, 1H, 6-H), 3.83 (s, 3H, OCH 3 ). MS (EI) (free base): 148 (M + ). 1-Methyl-5-nitro-benzimidazole (1f) Chlorodinitrobenzene (0.81 g, 4.0 mmol), dissolved in THF (5 mL) and methylamine (10 mL · 2 m THF) 25 , was heated for 12 h at 90 °C in a Young’s tube. The reaction was monitored using TLC with diethyl ether as the eluant. The mixture was cooled to room temperature and the solvent evaporated under reduced pressure. The resulting diamino compound was then cyclized in formic acid (5 mL) heated at reflux for 2 h, after which the reaction was cooled to room temperature and toluene (20 mL) and water (1 mL) were added. The volatile solvent was evaporated under reduced pressure and the residue was poured into water (30 mL) and extracted with ethyl acetate (3 · 30 mL). The combined organic extracts were washed with water (20 mL), dried over MgSO 4 and evaporated under reduced pressure. The product was recrystallized from ethyl acetate (0.23 g, 32%), m.p. 213–215 °C, lit. 209–211 °C [41]. 1 H NMR (dimethylsulfoxide-d 6 ) d: 8.73 (d, J ¼ 2 Hz, 1H, 4-H), 8.25 (d, J ¼ 9 Hz, 2H, 6-H), 8.04 (s, 1H, 2-H), 7.44 (d, J ¼ 9 Hz, 1H, 7-H), 3.92 (s, 3H, NCH 3 ). MS (EI): 178 (M + ). 1-Methyl-5-amino-benzimidazoleÆ2HClÆ0.2H 2 O (1g) 1-Methyl-5-nitro-benzimidazole (1f) (0.097 g, 0.548 mmol) was dissolved in ethanol (20 mL), 10% palladium on carbon Methionine synthase inhibitors E. C. Banks et al. 296 FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... structure of B12-binding domains of methionine synthase Science 266, 1669–1674 5 Dixon MM, Huang S, Matthews RG & Ludwig M (1996) The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12 Structure 4, 1263–1275 6 Finkelstein JD (1990) Methionine metabolism in mammals J Nutr Biochem 1, 228–237 7 Fowler B (2005) Homocysteine: overview of. .. acid derivatives as inhibitors of vitamin B12 dependent methionine synthase Biochem Soc Trans 24, 265S Stabler SP, Brass EP, Marcell PD & Allen RH (1991) Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats J Clin Invest 87, 1422–1430 Kenyon SH, Nicolaou A, Ast T & Gibbons WA (1996) Stimulation in vitro of vitamin B-12-dependent methionine synthase by polyamines Biochem J 316, 661–665... inactivation of methionine synthase by nitrous oxide J Biol Chem 261, 15823–15826 13 Nicolaou A, Waterfield CJ, Kenyon SH & Gibbons WA (1997) The inactivation of methionine synthase in isolated rat hepatocytes by sodium nitroprusside Eur J Biochem 244, 876–882 14 Nicolaou A, Kenyon SH, Gibbons JM, Ast T & Gibbons WA (1996) In vitro inactivation of mammalian 298 17 18 19 20 21 22 23 32 24 25 26 27 28 29 methionine. .. mammalian 298 17 18 19 20 21 22 23 32 24 25 26 27 28 29 methionine synthase by nitric oxide Eur J Clin Invest 26, 167–170 Alston TA (1991) Inhibition of vitamin B12-dependent methionine biosynthesis by chloroform and carbon tetrachloride Biochem Pharmacol 42, R25–R28 Smith JR & Smith JG (1990) Effects of methylmercury in vitro on methionine synthase activity in various rat tissues Bull Environ Contam Toxicol... MR & Wei Q (2005) Polymorphisms of methionine synthase and methionine synthase reductase and risk of lung cancer: a case-control analysis Pharmacogenet Genomics 15, 547–555 10 Mason JB & Choi SW (2005) Effects of alcohol on folate metabolism: implications for carcinogenesis Alcohol 35, 235–241 11 Matthews RG, Drummond JT & Webb HK (1998) Cobalamin-dependent methionine synthase and serine hydroxymethyltransferase:... RV & Matthews RG (1990) Cobalamin-dependent methionine synthase FASEB J 4, 1450–1459 2 Ludwig ML & Matthews RG (1997) Structure-based perspectives on B12-dependent enzymes Annu Rev Biochem 66, 269–313 3 Evans JC, Huddler DP, Hilgers MT, Romanchuk G, Matthews RG & Ludwig ML (2004) Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase Proc Natl Acad Sci... concentrated by ultrafiltration, as described above The final enzyme preparation was stored at )20 °C, in 20% (v ⁄ v) glycerol Handling of enzyme solutions was performed at low temperature, out of direct light Methionine synthase assay MetS activity was determined using the assay described by Kenyon et al [43] Briefly, reactions contained 50 mm phosphate buffer (pH 7.4), 227 lm 14C-5-methyltetrahydrofolate... Toxicol 45, 649–654 Kenyon SH, Nicolaou A & Gibbons WA (1998) The effect of ethanol and its metabolites upon methionine synthase activity in vitro Alcohol 15, 305–309 Kenyon SH, Waterfield CJ, Asker DS, Kudo M, Moss DW, Bates TE, Nicolaou A, Gibbons WA & Timbrell JA (1999) Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes... 14C-5-methyltetrahydrofolate Determination of Km and Vmax for 5-methyltetrahydrofolate Assays were incubated for 10 min at a fixed concentration of Hcy (500 lm) and varying concentrations of 5-methyltetrahydrofolate (i.e 0, 50, 100, 500 and 1000 lm) During preliminary experiments, the enzyme concentration and the incubation time were varied in order to establish conditions for linear kinetics The concentration of protein used... Z, Crippen K, Gulati S & Banerjee R (1994) Purification and kinetic mechanism of a mammalian methionine synthase from pig liver J Biol Chem 269, 27193– 27197 Dixon M (1953) The determination of inhibitor constants J Biochem 55, 170–172 Cornish–Bowden A (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors Biochem J 137, 143–144 . Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles Elizabeth C. Banks 1 ,. inactivation of mammalian methionine synthase by nitric oxide. Eur J Clin Invest 26, 167–170. 15 Alston TA (1991) Inhibition of vitamin B12-dependent methionine