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-

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

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

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

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

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

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-

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

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-

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).

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

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-

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
) 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

(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

(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

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

26
244–246 °C, lit. 158–
195 °C (free base) [42].
1
H NMR (dimethylsulfoxide-d
6
) d:
9.33 (s, 1H, 2-H), 7.75 (d, J ¼ 9 Hz, 1H, 7-H), 7.32 (s, 1H,
6-H), 7.17 (d, J ¼ 9 Hz, 1H, 7-H), 4.8–4.2 (br, 3H, NH,
NH
2
), 3.98 (s, 3H, NCH
3
). MS (EI): 185 (M
+
). Found: C,
43.19; H, 5.00; N, 18.31. C
8
H
9
N
3
Æ2HClÆO.2H
2
O requires:
C, 42.95; H, 5.14; N, 18.78%.
Enzyme purification
MetS was purified from rat liver, as previously described
[14]. Briefly, rat liver homogenate was prepared in ice-cold
50 mm potassium phosphate buffer, pH 7.0, containing

active fractions were pooled, desalted and concentrated by
ultrafiltration, as described above. The final enzyme pre-
paration 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 phos-
phate buffer (pH 7.4), 227 lm
14
C-5-methyltetrahydrofolate
[2077 disintegrations per min (dpm)
30
Ænmol
)1
], 23 mm dio-
thiothreitol, 40 lm S-AdoMet, 60 lm hydroxycobalamin,
the enzyme source and (when applicable) dimethylsulfoxide
solutions of the inhibitors (maximum volume 5 lL) in a
total volume of 300 lL. Incubations were performed in
light-excluding sealed serum vials under nitrogen. The reac-
tion mixture was pre-incubated for 5 min, the reaction was
initiated by the addition of 500 lm (dl)-homocysteine and
incubated at 37 °C for a further 30 min, unless otherwise
stated. The reaction was terminated by the addition of ice-
cold water (400 lL). The reaction mixture was passed
through a 0.5 · 5 cm AG1-X8 resin column, [
14
C] methion-
ine was eluted with 2 mL of water and quantified using

Protein content was determined using the Protein Assay kit
based on the method of Bradford [44]. Standards and sam-
ples were assayed in triplicate, according to the manufac-
turer’s instructions. Sample absorbances were read against
a BSA standard curve to determine protein content.
Molecular model construction
Protein sequences were identified using the Human Gen-
ome Mapping Project Centre and the SWISSPROT data-
bases (). Sequence alignment
was performed using clustalw dynamic programming.
The model was constructed using a Silicon Graphics
workstation with sybyl software (Tripos Inc.) for model
building, charmm minimization and molecular dynamics
E. C. Banks et al. Methionine synthase inhibitors
FEBS Journal 274 (2007) 287–299 ª 2006 The Authors Journal compilation ª 2006 FEBS 297
[31] for structure optimization. Ligands were parameter-
ized using partial atomic charges and other values
obtained from quantum mechanic modelling (Hartree-
Fock 6–31G*) of the ligand structure using pc spartan
pro (Wavefunction Inc.).
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