Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase

Article abstract of DOI:10.3109/14756366.2015.1070846

Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened

Full text of DOI:10.3109/14756366.2015.1070846

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1070846
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RESEARCH ARTICLE
Bisphosphonic acids as effective inhibitors of Mycobacterium
tuberculosis glutamine synthetase
Paulina Kosikowska1, Marta Bochno1, Katarzyna Macegoniuk1, Giuseppe Forlani2, Paweł Kafarski1, and
Łukasz Berlicki1
1Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland and 2Department of Life Science
and Biotechnology, University of Ferrara, Ferrara, Italy
Abstract
Keywords
Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the
discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic
acids of various scaffolds, bearing aromatic substituents, were screened against recombinant
GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in
micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-
difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-
1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to
the reference compound – L-methionine-S-sulfoximine. Moreover, these inhibitors were found
to be much more effective against pathogen enzyme than against the human ortholog. Thus,
with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/
selectivity profile makes these compounds attractive agents for the treatment of bone
tuberculosis.
Bisphosphonates, enzyme inhibitors,
nitrogen metabolism, organophosphorus
compounds, tuberculosis
History
Received 12 May 2015
Revised 5 June 2015
Accepted 5 June 2015
Published online 3 August 2015
Introduction
recently characterised class (GS3) was first discovered in the
anaerobic bacterium Bacteroides fragilis and later found in other
anaerobes8. Its dodecameric structure is formed by association of
two hexameric rings, but on the opposite molecular surface than
found in GS1 and GS2.
Glutamine synthetase (GS, glutamate-ammonia ligase, E.C.
6.3.1.2), one of the key enzymes in nitrogen metabolism1,
catalyses the formation of glutamine from glutamate and ammo-
nia in the presence of adenosine triphosphate (ATP) (Figure 1).
This enzyme, together with glutamate synthase (GOGAT, E.C.
1.4.1.13), is a component of a metabolic cycle that enables the
incorporation of inorganic nitrogen (in the form of ammonium
ions) into the organic molecules present in living cells. Glutamate,
the product of the GS-GOGAT cycle, is the primary nitrogen
donor in the synthesis of purines, pyrimidines, amino acids,
glucosamine-6-phosphate, carbamoyl phosphate and a range of
other nitrogen-containing products.
The numerous implications of GS in various biological
processes make the enzyme indispensable for cell vitality in all
living organisms2. Bioinformatic studies using multiple sequence
alignment showed the gene encoding GS to be one of the oldest
yet found3. As the result of the long evolutionary process, three
distinct classes of GSs have been developed: GS1, GS2 and GS3.
GS1 is mainly expressed in prokaryotic cells and is constructed
from 12 identical subunits, forming two hexameric rings
connected face to face4,5. GS2 is primarily found in eukaryotes,
but its expression has also later been confirmed in some
actinomycetes and rhizobia6,7. This form of the enzyme is
composed of 10 subunits forming two pentameric rings. The most
The active site of GS is formed of two cones connected at their
narrow ends, in which two or three metal ions (Mg2+ or Mn2+
)
reside9. The glutamate and ammonium ion enter from one side,
while ATP arrives via the opposite end. In the first step of the
enzymatic reaction, glutamate is phosphorylated to yield
g-glutamyl phosphate. Ammonia then replaces the phosphate to
form the amide moiety of glutamine. The commonly held belief
that this catalytic mechanism is the same for all the existing forms
of GS is supported by the presence of highly conserved residues at
the glutamate and ammonium binding sites1,10
.
The most numerous group of GS inhibitors is composed of
glutamate analogues. The majority of these are organophosphorus
and organosulfur compounds, with L-methionine-S-sulfoximine
(MetSox, 1) and phosphinothricin (2) being the most represen-
tative examples (Figure 2)1,11. These structures are phosphory-
lated in the enzyme active site and subsequently act as transition
state analogues12,13
.
More recently, compounds that interact with the ATP binding
pocket have been developed. A number of aminomethylenebi-
sphosphonic acids (e.g. structure 3, Figure 2) were initially shown
to be effective inhibitors of plant GS14,15. Later, aromatic
derivatives of the imidazole functionality (e.g. compound 4)
were discovered as bacterial GS inhibitors16,17
.
Address for correspondence: Łukasz Berlicki, Faculty of Chemistry,
Department of Bioorganic Chemistry, Wrocław University of Technology,
Wrocław, Poland. E-mail: lukasz.berlicki@pwr.edu.pl
Currently, the most important application of GS inhibitors is in
the development of antibiotic strategies for use against the human

2
P. Kosikowska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
glutamine
synthetase
Figure 1. Reaction catalysed by glutamine
synthetase.
O
O
O
O
+ ATP + NH
+ ADP + Pi + H O
2
3
HO
OH
HO
NH
2
NH
NH
2
2
biochemical experiments. Escherichia coli GJ4547 (kindly
provided by Dr. Gowrishankar, Centre for DNA Fingerprinting
and Diagnostics, Hyderabad, India) and BL21(DE3) CodonPlus-
RIL (Stratagene, La Jolla, CA) strains were used for recombinant
M. tuberculosis and human GS expression, respectively. The
recombinant plasmids (Vector pTrc99c, Vector pNIC-BSA4) used
in GS studies were kindly provided by Prof. Sherry L. Mowbray
from Uppsala University. During the enzyme preparation, purity
of the obtained proteins was analyzed by means of SDS-PAGE
electrophoresis, while protein concentration was determined
according to Bradford protein assay with bovine serum albumin
used as standard.
O
O NH
S
HO
NH
2
1, methionine
sulfoxyimine (MetSox)
O
O OH
P
HO
NH
2
2, phosphinothricin (PPT)
Synthetic procedures
H
Cl
N
PO H
3 2
Compounds 5b, 5c, 5e–5w, 6m, 6k, 6r, 6t, 6w, 7c, 7h, 7w, 8c, 8g,
8h, 8k and 9a–w were described in preceding studies15,23–25
.
PO H
3
2
Aminomethylenebisphosphonic acids (5). The mixture of
amine (25 mmol), triethylortoformate (30 mmol) and diethyl
phosphite (100 mmol) was heated at 100–110 ꢀC for 6 h. The
volatiles were removed under reduced pressure and crystallized
compound was filtered and washed with hexane. Obtained crude
tetra-ester was dissolved in methylene chloride and TMSBr
(10 eq) was added. The mixture was stirred overnight.
Subsequently, methanol (10 mL) was added and stirring was
continued for 2 h. The reaction mixture was evaporated under
reduced pressure, and crude product was purified using flash
chromatography (C18 column, water/acetonitrile).
Cl
3
O
N
NH
N
H
2
N
4
N-phenylaminomethylenebisphosphonic acid (5a). Yield
56%. 1H NMR D2O d 3.97 (t, 1H, 2JPH ¼ 20.4, PCHP), 6.86–6.95
(m, 5H, Ph), 31P NMR D2O d 13.98, 13C NMR D2O d 53.92 (t,
1JPC ¼ 132.4, PCHP), 117.03, 122.37, 129.48, 142.87 (4 ꢁ s, Ph),
ESI-MS m/z: calc. 265.9984 [M–H+]ꢂ, found 265.9973 [M–H+]ꢂ.
N-(4-isopropylphenyl)aminomethylenebisphosphonic acid
Figure 2. Representative examples of glutamine synthetase inhibitors.
pathogen Mycobacterium tuberculosis. GS inhibitors were shown
to selectively block the formation of the poly(glutamine-glutam-
ate) cell walls of these bacteria18,19. The efficacy of MetSox was
proven in vivo20. M. tuberculosis infections are known to
frequently spread outside the lungs, reaching various parts of
the human body. One major site of extrapulmonary tuberculosis is
the bones21. Importantly in this respect, pharmacokinetic studies
of bisphosphonates indicate that, regardless their structure, the
overwhelming fraction of the absorbed active substance is
accumulated in the bones due to the presence of the bispho-
sphonate moiety22. Thus, the application of bone-targeting
bisphosphonate M. tuberculosis GS (MtGS) inhibitors may
prove an excellent strategy for treating osseous tuberculosis.
Herein, we present the results of an extensive screening of
compounds with various scaffolds containing the bisphosphonic
acid group, each designed to inhibit MtGS. Detailed kinetic
studies of the most active structures provided insight into their
molecular mode of action. Their selectivity towards MtGS was
estimated by comparison with the activities demonstrated against
recombinant human glutamine synthetase (HsGS).
1
(5d). Yield 63.5%. H NMR D2O d 1.07 and 1.02 (d each, 3H,
2
3JHH ¼ 6.9 and 7.0), 2.78 (m, 1H, CH), 3.84 (t, 1H, JHP ¼ 19.1,
3
CHP2), 7.06 (d, 2H, JHH ¼ 8.7, ArH), 7.20 (m, 2H, ArH), 31P
NMR D2O 11.97 d 13C NMR D2O d 23.54 (s, CH3), 32.46
1
(s, CH(CH3)2), 54.22 (t, JPC ¼ 130.0, PCHP), 112.92, 126.95,
136.05, 147.85 (4 ꢁ s, Ar), ESI-MS m/z: calc. 308.0453
[M–H+]ꢂ, found 308.0450 [M–H+]ꢂ.
N-(3-trifluoromethylphenyl)aminomethylenebisphosphonic
acid (5p). Yield 29.9%. 1H NMR D2O d 4.15 (t, 1H, 2JPH ¼ 21.3,
PCHP), 6.93 (d, 1H, J ¼ 8.3, Ar), 6.96 (d, 1H, J ¼ 7.6, Ar), 7.00 (s,
1H, Ar), 7.26 (t, 1H, J ¼ 8.0, Ar); 31P NMR D2O 16.64 d 13C
1
NMR D2O d 50.64 (t, JPC ¼ 138.1, PCHP), 109.65 (q, J ¼ 3.7,
Ar), 114.62 (q, J ¼ 3.8, Ar), 116.98 (s, Ar), 124.24 (q, J ¼ 271.4,
CF3), 129.94 (s, Ar), 130.87 (q, J ¼ 31.8, Ar),147.20 (s, Ar), ESI-
MS m/z: calc. 333.9857 [M–H+]ꢂ, found 333.9845 [M–H+]ꢂ.
Aminoethylidenebisphosphonic acids (6). Tetraethyl ethyli-
denebisphosphonate (1.0 g, 3.3 mmol) was dissolved in dry THF
and amine (3.3 mmol) was added. The reaction mixture was
stirred for 48 h at room temperature. Volatiles were evaporated
under reduced pressure to get crude tetraethyl aminoethylidenebi-
sphosphonate. The crude intermediate was dissolved in aceto-
nitrile and TMSBr (39 mmol) was added. The mixture was left
overnight at room temperature. Subsequently, methanol was
added (10 mL) and reaction was continued for 1 h. Mixture was
evaporated under reduced pressure, the residue crystallized from
water/ethanol.
Materials and methods
General
All used chemicals were obtained from commercial suppliers
(Sigma-Aldrich, Poznan, Poland; Merck, Warsaw, Poland;
Fermentas, Warsaw, Poland; POCh, Gliwice, Poland) as reagents
of analytical grade. Deionized ultra-pure water was used in all

DOI: 10.3109/14756366.2015.1070846
Bisphosphonate inhibitors of M. tuberculosis GS
3
N-phenylaminoethalidenebisphosphonic acid (6a). Yield addition of 100 mg/mL ampicillin at 37 ꢀC to OD600 of 1.0, when
2
3
58%. 1H NMR D2O d 2.34 (tt, 1H, JPH ¼ 21.1, JHH ¼ 7.6, expression was induced with 1 mM IPTG. Cells were incubated
PCHP), 3.59–3.71 (m, 2H, CH2), 7.35–7.45 (m, 5H, Ph), 31P overnight (18 h) at 25 ꢀC, then harvested by centrifugation,
1
NMR D2O d 15.92, 13C NMR D2O d 36.23 (t, JPC ¼ 117.3, washed with 50 mM Hepes buffer, pH 7.5 and stored at ꢂ20 ꢀC.
PCHP), 50.05 (s, CH2), 121.98, 129.08, 130.30, 135.49 (4 ꢁ s,
Recombinant HsGS was obtained as a product of GLUL gene
Ph), ESI-MS m/z: calc. 280.0140 [M–H+]ꢂ, found 280.0141 (encoding HsGS from human muscle) that was cloned into pNIC-
[M–H+]ꢂ.
N-(4-methylphenyl)aminoethalidenebisphosphonic
BSA4 vector and expressed in E. coli BL21 Codon Plus-RIL cells
acid (Stratagene). Cells were grown in a phosphate-buffered medium
1
(6b). Yield 15%. H NMR D2O d 2.26 (3H, s, CH3), 2.32 (tt, containing per liter 2.3 g KH2PO4, 3.78 g K2HPO4, 12 g tryptone,
2
3
3
1H, JHP ¼ 20.9, JHH ¼ 7.7, CH), 3.64 (td, 2H JHP ¼ 13.9, 24 yeast extract, 4 mL glycerol, with addition of 50 mg/mL
3JHH ¼ 7.6, CH2), 7.26–7.30 (m, 4H Ar); 31P NMR D2O d 19.29 kanamycin. When optical density of the culture (at 600 nm)
1
13C NMR D2O d 20.15 (s, CH3), 35.86 (t, JPC ¼ 118.6, PCHP), reached 1.0, expression was induced with 1 mM IPTG and
50.12 (s, CH2), 122.37, 130.80, 131.75, 140.65 (4 ꢁ s, Ar); ESI- continued for 18 h. After that, cells were harvested by centrifu-
MS m/z: calc. 294.0296 [M–H+]ꢂ, found 294.0294 [M–H+]ꢂ.
gation, washed with 50 mM phosphate buffer, pH 7.5 and stored
3,5-Difluorophenylaminoethylidenebisphosphonic
1
acid at ꢂ20 ꢀC until further use.
(6o). Yield 65%. 1H NMR D2O d 2.19 (tt, 1H, JHP ¼ 21.4,
3
3
3JHH ¼ 6.3, PCHP), 3.45 (td, 2H, JHP ¼ 14.1, JHH ¼ 6.8, CH2), Protein purification
6.20 (1H, tt, J ¼ 9.6, J ¼ 2.2, Ar); 6.29 (2H, m, Ar); 31P NMR D2O
Thawed bacterial cells were suspended in binding buffer supple-
d 19.00 13C NMR D2O d 38.74 (t, 1JPC ¼ 114.6, PCHP), 41.09 (s,
CH2), 92.75 (t, J ¼ 24.6, Ar), 96.67 (dd, J ¼ 22.5, J ¼ 6.2, Ar),
150.33 (t, J ¼ 13.1, Ar), 163.82 (dd, J ¼ 241.8, J ¼ 16.1, Ar); ESI-
MS m/z: calc. 315.9951 [M–H+]ꢂ, found 315.9947 [M–H+]ꢂ.
Hydroxybisphosphonic acids (7 and 8). Tris(trimethylsillyl)
phosphite (6 mmol) was added drop wise to acid chloride
(3 mmol). The reaction mixture was stirred for 1 h at room
temperature and volatiles were evaporated under reduced pres-
sure. Product was purified using flash chromatography with C18
column (water/acetonitrile).
mented with 1.0 mM EDTA and 2.0 mM b-mercaptoethanol and
were lysed by sonication. Crude extracts containing recombinant
protein were separated from cell debris by centrifugation (1 h,
˚
11 000 rpm) Supernatants were then loaded be means of AKTA
Prime Plus Liquid Chromatography system (GE Healthcare,
Warsaw, Poland) onto previously equilibrated pre-packed
HisTrapÔ FF column (3 ꢁ 1 mL, GE Healthcare). Unbound
proteins were removed by extensive column washing with
binding/wash buffer, the target protein was achieved with a
linear gradient of imidazole in elution buffer. Usually, single peak
of His-tagged protein with confirmed GS-activity appeared at
250–350 mM of imidazole.
In case of MtGS expressed in E. coli GJ4647, the wash/binding
buffer was composed of 50 mM HEPES, pH 7.5, 300 mM NaCl,
10% glycerol, 25 mM imidazole and elution buffer was 50 mM
HEPES, pH 7.5 with 300 mM NaCl, 10% glycerol and 450 mM
imidazole.
During purification of human GS expressed in E. coli BL21
Codon PLUS-RIL strain, the following buffers were used: the
wash/binding buffer consisted of 50 mM phosphate, 7.5, 500 mM
NaCl, 10% glycerol, 25 mM imidazole, while elution was done
with the same buffer containing 450 mM imidazole.
In the next step, obtained active fractions of the target proteins
(MtGS or HsGS) were pooled together, concentrated on Amicon
Ultra-15 Centrifugal Filter Units (NMWL 10 kDa, Merck,
Warsaw, Poland) and then loaded onto Sephacryl S-300 HR 26/
60 column. During gel-filtration step 20 mM HEPES, pH 7.5 with
the addition of 150 mM NaCl and 10% glycerol was used as the
working buffer (Figure S1 and S2). Identification of GS-
containing protein fraction was performed by means of biosyn-
thetic assay.
Phenylmethylidene-1-hydroxy-1,1-bisphosphonic acid (7a).
1
Yield 75%. H NMR D2O d 7.25, 7.59 (m, 5H, Ph), 31P NMR
1
D2O d 16.8, 13C NMR D2O d 75.63 (t, JPC ¼ 144.8, PCP),
125.79, 127.75, 128.11, 135.41 (4 ꢁ s, Ph), ESI-MS m/z: calc.
266.9824 [M–H+]ꢂ, found 266.9831 [M–H+]ꢂ.
Phenylethylidene-1-hydroxy-1,1-bisphosphonic acid (8a).
3
Yield 54%. 1H NMR D2O d 3.19 (t, 2H, JPH ¼ 13.4, CH2),
7.11–7.28 (m, 5H, Ph); 31P NMR D2O d 19.36; 13C NMR D2O d
38.19 (s, CH2), 73.62 (t, 1JCP ¼ 146, PCP), 126.87, 127.87, 131.08
3
(3 ꢁ s, Ph), 135.10 (3 ꢁ s, t, JCP ¼ 8.7, Ph), ESI-MS m/z: calc.
280.9980 [M–H+]ꢂ, found 280.9986 [M–H+]ꢂ.
4-Methylphenylethylidene-1-hydroxy-1,1-bisphosphonic
1
3
acid (8b). Yield 80%. H NMR D2O d 7.17 (d, 2H, JHH ¼ 7.8,
3
3
ArH), 7.03 (d, 2H, JHH ¼ 7.8, ArH), 3.16 (t, 2H, JHP ¼ 13.6,
CHP2), 2.16 (s, 3H, CH3); 31P NMR (d) 19.47. ESI-MS m/z: calc.
295.0136 [M–H+]ꢂ, found 295.0139 [M–H+]ꢂ.
3,4-Dichlorophenylethylidene-1-hydroxy-1,1-bisphosphonic
1
3
acid (8l). Yield 72%. H NMR D2O d 3.18 (t, 1H, JHP ¼ 13.1,
CH2), 7.18 (d, 1H, J ¼ 8.2, Ar), 7.31 (d, 1H, J ¼ 8.2, Ar), 7.43
(s, 1H Ar); 31P NMR D2O d 18.24; 13C NMR D2O d 37.57 (s,
CH2), 73.58 (t, J ¼ 145.4, PCP), 129.49, 129.99, 130.66, 130.87,
3
132.68 (5 ꢁ s, Ar), 136.12 (t, JCP ¼ 9.2, Ar); ESI-MS m/z: calc.
348.9201 [M–H+]ꢂ, found 348.9198 [M–H+]ꢂ.
Benzylethylidene-1-hydroxy-1,1-bisphosphonic acid (8x).
Biosynthetic assay
1
Yield 77%. H NMR D2O d 2.04–2.20 (m, 2H, CH2), 2.77–2.82 Biosynthetic activity of GS was measured by means of malachite
(m, 2H, CH2Ph), 7.10–7.26 (m, 5H, Ph) 31P NMR D2O d 19.86, green-acid molybdate assay enabling detection of inorganic
13C NMR D2O d 29.48 (t, 3JPC ¼ 6.7, CH2Ph), 35.74 (s, CH2CP), phosphate that is released during glutamine synthesis.
1
73.07 (t, JPC ¼ 146.8, PCP), 126.09, 128.30, 128.64, 142.04 Colorimetric reagent was composed of 6.3 mM malachite green
(4 ꢁ s, Ph), ESI-MS m/z: calc. 295.0136 [M–H+]ꢂ, found dye and 34 mM ammonium molybdate dissolved in 4M HCl. Both
295.0136 [M–H+]ꢂ.
of these components were mixed with each other in the 1:1 ratio
and finally diluted with two volumes of distilled water.
Colorimetric solution was stirred for 20 min, stabilized by
addition of CHAPS (2 mg/mL) and centrifuged for 3 min at
2500 g in order to remove undissolved particles.
Reaction mixture optimized for MtGS contained enzyme
solution, 100 mM HEPES buffer pH 7.5, 50 mM L-glutamate,
2.5 mM ATP, 10 mM MgCl2, 10 mM NH4Cl, yielding a final
volume of 100 mL. In case of HsGS, the highest enzymatic activity
Protein expression
In order to obtain unadenylated MtGS, pTrc99c plasmid5 with
glnA1 gene being under the control of trp/lac promoter, was
introduced to the competent cells prepared from genetically
modified E. coli GJ4547 strain that lacks GS adenyl transferase
activity. Transformed cells were grown in LB medium with

4
P. Kosikowska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
H
H
N
R
PO H
PO H
PO H
3
N
PO H
2 2
3
2
3
2
2
PO H
3
2
N
H
R
R
R
OH
PO H
OH
PO H
R
PO H
PO H
2 2
3
2
3
2
3
2
PO H
3
2
5
6
7
8
9
Cl
Cl
Cl
a
b
i
c
d
e
f
g
Cl
Cl
Cl
Cl
Cl
Br
Cl
Cl
Cl
F
Cl
Cl
2
Br
Cl
Cl
h
j
k
l
m
n
v
F C
3
NH
F C
3
O
CF
F
3
o
p
x
r
s
t
u
Ph
w
Figure 3. Scaffolds and range of substituents tested against Mycobacterium tuberculosis GS.
was observed when 100 mM HEPES, pH 8.0, 20 mM MgCl2,
Reversibility of inhibition was determined by measuring the
100 mM L-glutamate, 2.5 mM ATP and 10 mM NH4Cl were used. recovery of enzymatic activity after a rapid and large dilution of
In both cases, reaction was started by the addition of the the enzyme–inhibitor complex. Target enzyme at a concentration
enzyme. At the end of incubation, 1 mL of colorimetric reagent of 100-fold over the concentration used in activity assay was pre-
was added to the reaction mixture and after 1-min assay was incubated with inhibitor used at concentration equivalent to
accomplished by the addition of 100 mL of 34% citrate. Optical 10-fold the IC50. After 15-min equilibration time period, this
density of each probe was determined spectrophotometrically at mixture was diluted 100-fold into reaction buffer containing all
630 nm. Quantitative analysis of GS activity was done on the basis necessary substrates to initiate reaction. The progress curve for
of a standard curve obtained with proper dilutions of a 2 mM tested sample was measured and compared to that of control
solution of KH2PO4.
sample that was pre-incubated in the absence of inhibitor and
diluted the same way.
Inhibitory studies
Molecular modeling
IC50 determinations were conducted under the optimized condi-
tions of biosynthetic assay. GS inhibition was evaluated by
introduction of an appropriate dilution of freshly prepared 25 mM
stock solution of the analyzed compound (pH of dissolved
inhibitor was adjusted to 7.0–7.5) to the reaction mixture.
Reaction was started by the addition of the enzyme and had
been run for 30 min at 37 ꢀC in water bath. At least three
measurements were performed for each dose of tested inhibitor.
IC50 parameter was calculated using linear regression method by
means of Prism 5 software (GraphPad, La Jolla, CA). The activity
of enzyme was expressed as percentage of untreated control
plotted against logarithm of used inhibitor concentration.
Molecular modeling calculations were performed using Discover
Studio 3.1 program package (Accelrys, San Diego, CA). The
crystal structure of M. tuberculosis GS (PDB id 2BVC) was used
as starting point for calculations7. Initially, protons were added to
the protein molecule assuming pH 7. The model of bispho-
sphonate inhibitor was constructed and placed in ATP binding
site. Various positioning of bisphosphonate molecule was tested.
Partial charges were computed using Momany-Rone algorithm.
Minimizations were performed using CHARMm forcefield and
Smart Minimizer algorithm up to Energy change 0.0 or RMS
gradient 0.1. All residues that are not forming the active site cleft
were frozen.
For kinetic evaluations, MtGS was assayed in the presence of
increasing concentrations of tested compounds and substrate
concentrations ranging from 5 to 100 mM in case of glutamate
and from 0.25 to 5 mM for ATP. At least five inhibitor
concentrations ranging from 0.5 to 2-fold of IC50 value were
evaluated in triplicate for one concentration of substrate and fixed
concentration of the enzyme. The model of inhibition towards
ATP and glutamate was determined on the basis of Lineweaver–
Burk plots and curve-fitting method based on non-linear regres-
sion analysis using GraphPad Prism 5 software.
Results and discussion
We have previously shown aminomethylenebisphosphonic acids
to be effective inhibitors of plant GS15. The bisphosphonic group
is postulated to be an analogue of the terminal pyrophosphate
fragment of ATP, and is indispensable for potent inhibitory
activity. This mechanism was proven by the inactivity of a
structurally similar aminophosphonic acid bearing one phos-
phonic acid moiety. In the context of these studies, a broad

DOI: 10.3109/14756366.2015.1070846
Bisphosphonate inhibitors of M. tuberculosis GS
5
H
N
Table 1. Inhibitory activities of bisphosphonic and bis-H-phosphinic
acids against recombinant MtGS.
1. 6h, reflux
PO H
3
2
NH
2
HPO Et
3
HC(OEt)
3
+
+
R
2
R
2. TMSBr
3. MeOH
PO H
3
2
5
No
Compound
IC50[mM]
1. 48h, rt
R
PO H
3 2
PO Et
H
3
2
N
NH
2
+
H
PO H
3 2
N
R
PO H
2. TMSBr
3. MeOH
3
2
R
PO Et
3
2
6
PO H
3
2
PO H
3 2
OH
1. 1h, rt
O
5a
5b
5c
5d
5e
5f
5g
5i
5j
C6H5–
p-Me-C6H4–
3,5-diMe-C6H3–
p-iPr-C6H4–
o-Cl-C6H4–
246 11
525 31
227 15
+
P(OTMS)
3
R
PO H
3
2
R
Cl
2.
H O
2
7
8
or
231
8
358 30
240 22
375 19
447 21
331 16
317 28
228 13
NI
269 14
256 27
NI
273 28
278 34
286 20
250 40
O
P
1. 6h, reflux
H
m-Cl-C6H4–
p-Cl-C6H4–
N
PO H
2
2
R
NH
+
+
3
HC(OEt)
2
H
OEt
OEt
2. HBr/AcOH
R
PO H
2
2
2,4-diCl-C6H3–
2,5-diCl-C6H3–
3,4-diCl-C6H3–
3,5-diCl-C6H3–
3,5-diBr-C6H3–
3,5-diF-C6H3–
EtO
9
5l
Figure 4. Synthesis of bisphosphonic acids 5–9.
5m
5n
5o
5p
5r
5s
5u
5v
5w
m-CF3-C6H4–
3,5-diCF3-C6H3–
m-CONH2-C6H4–
5,6,7,8-tetrahydro-2-naphthyl
2,3-dihydro-1H-inden-5-yl
p-Bzl-C6H4–
screening of various structures bearing the bisphosphonic moiety
against MtGS has been envisaged (Figure 3).
In addition to the aminomethylenebisphosphonic acids (5)26,
related structures containing bisphosphonic fragments (6–8) were
examined. The position of hydrogen bond donor/acceptor (NH or
OH groups) and the linker length (zero to two atoms between the
methylenebisphosphonic group and the aromatic fragments) were
varied. Preliminary experiments (data not shown) showed
bisphosphonates with aliphatic substituents to be much less
effective than those bearing aromatic groups. As a consequence,
all of the proposed substitution patterns were based on modified
phenyl rings (fragments a–y). The aim of the screening was to
establish the influence of both the bisphosphonic scaffold and the
presence and positioning of substituents on the aromatic rings.
All bisphosphonic compounds were synthesized using standard
literature methods (Figure 4). Briefly, aminomethylenebispho-
sphonic acids were obtained using Kafarski’s method (tri-
component condensation of aromatic amine, diethyl phosphite
and triethyl orthoformate and subsequent transesterification
followed by hydrolysis)26. The analogous reaction done with
ethyl diethoxymethyl-H-phosphinate followed by HBr/AcOH
hydrolysis yielded aminomethylenebis-H-phosphinic acids 924.
The application of Hutchinson’s method (addition of aromatic
amine to tetraethyl ethylidenebisphosphonate and removal of
ethyl esters using TMSBr and MeOH) gave compounds 627.
Hydroxybisphosphonic acids 7 and 8 were synthesized by
Lecouvey’s method (the reaction of acid chlorides with
tris(trimethylsilyl)phosphite and subsequent hydrolysis)28.
R
PO H
3 2
N
H
PO H
3
2
6a
6b
6k
6m
6o
6r
C6H5–
174
8
p-Me-C6H4–
2,6-diCl-C6H3–
3,5-diCl-C6H3–
3,5-diF-C6H3–
249 13
180 19
82 16
82 25
3,5-diCF3-C6H3–
5,6,7,8-tetrahydro-1-naphthyl
p-Bzl-C6H4–
168 17
294 34
286 31
6t
6w
PO H
3
2
OH
R
PO H
3
2
7a
7c
7h
7w
C6H5-
348
6
3,5-diMe-C6H3–
2,3-diCl-C6H3–
p-Bzl-C6H4–
345 52
186 36
363 40
PO H
3
2
R
OH
PO H
3
2
8a
8b
8c
8g
8h
8k
8l
C6H5–
p-Me-C6H4–
3,5-diMe-C6H3–
p-Cl-C6H4–
2,3-diCl-C6H3–
2,6-diCl-C6H3–
3,4-diCl-C6H3–
C6H5CH2–
221
8
125 21
450 29
130 23
178 34
194 42
53 18
The inhibitory properties of 39 bisphosphonic (5–8) and four
bis-H-phosphinic (9) acids were analysed against the unadeny-
lated form of recombinant MtGS using a colorimetric assay based
on detection of phosphate released in physiologically relevant
biosynthetic reactions (Table 1). To obtain unadenylated MtGS,
the vector pTrc99c bearing the glnA1 gene of M. tuberculosis
(H37Rv strain) was used to transform an E. coli GJ4547 strain
lacking adenyl transferase activity, and thus unable to modify Tyr-
406 residues of MtGS subunits9. Enzymatic tests revealed that
almost all the bisphosphonates (with the exceptions of 5n and 5r)
exhibited inhibitory effects on MtGS with IC50 values in the
micromolar range, similar to the reference compound MetSox (1).
Comparison of the activities of the unsubstituted phenyl bispho-
sphonate compounds 5a, 6a, 7a and 8a indicated the aminoethy-
lidenebisphosphonic acid scaffold (compounds 6) to be the most
effective, although only slightly so. Moreover, the aminomethy-
lenebis-H-phosphinic acids (9) were found to be completely
8x
244 12
H
N
PO H
2
2
R
PO H
2
2
9a
9b
9m
9w
1
C6H5–
p-Me-C6H4–
3,5-diCl-C6H3–
p-Bzl-C6H4–
L-MetSox
NI
NI
NI
NI
104 21
NI, no inhibition.

6
P. Kosikowska et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
mM
0
mM
0
Figure 5. Kinetic analysis of MtGS inhibition
by compound 6m at varying concentration of
glutamine (panel A) and ATP (panel B).
(A)
(B)
0.075
0.085
0.100
0.125
0.150
0.025
0.0375
0.05
0.075
0.125
20
10
0
10
5
0
0.0
0.1
1/C (Glu) [mM]
0.2
0
1
2
3
4
−1
−1
1/C (ATP) [mM]
Table 2. Inhibition constants (Ki) against MtGS and IC50 values against HsGS for selected compounds.
MtGS
HsGS
IC50 [mM]
No
Structure
Ki (Glu) [mM]
Ki (ATP) [mM]
6m
25.0 2.7
40.8 7.1
780 30
6o
35.0 7.9
49.6 10.0
2430 120
8l
1
21.0 8.2
25.7 2.7
17.0 5.1
400 40
ND
6400 780
O
O
S
HO
NH
NH
2
ND, not determined.
inactive, indicating that all three oxygen atoms of the phosphonate were undertaken (Figure 5). Due to the complexity of the
groups on 5–8 are required for successful inhibition of the possible interactions between the enzyme, the inhibitor and the
enzyme.
substrates of the reaction, a clear interpretation of the results is
In general, substitution of the phenyl ring did not significantly not obvious. The most consistent mechanism that can be
influence the inhibitory activity. Halogen atoms, alkyl groups or proposed based on linear and non-linear regression analyses is
even larger substituents like benzyl groups placed in various that bisphosphonates act like a mixed-type inhibitor with respect
positions on the aromatic ring yielded similar IC50 values in the to glutamate, and as uncompetitive inhibitors with respect to
majority of cases. However, it was noted that scaffolds with ATP. This characteristic denotes the ability of these inhibitors
shorter linkers (5 and 7) are slightly more sensitive to the presence to interact with both the free enzyme and the enzyme–glutamate
of substituents than the more flexible compounds (6 and 8). For complex at a site other than the glutamate-binding site.
example, compound 5r showed no inhibitory activity, while its Moreover, the inhibitors interact with the enzyme after ATP
homologue 6r (IC50 ¼ 168 mM) was equipotent to the unsubsti- binding.
tuted derivative 6a. Among all of the tested bisphosphonates,
When the tested inhibitors were applied at concentrations
three compounds (6m, 6o and 8l) exhibited IC50 values lower than higher than 150 mM, GS activity failed to display typical enzyme
the reference compound 1. kinetics, most likely because of non-specific interactions between
To gain insight into the mechanism of inhibition, detailed the inhibitor and metal ions present in the reaction mixture,
kinetics studies on the most active compounds (6m, 6o and 8l) which reduce the availability of the metal ions for the enzyme.

DOI: 10.3109/14756366.2015.1070846
Bisphosphonate inhibitors of M. tuberculosis GS
Molecular modelling
7
To elucidate the molecular mode of binding of the most active
bisphosphonic MtGS inhibitors, the structure of 8l-MtGS was
modeled (Figure 6). The bisphosphonate fragment of the inhibitor
is tightly bound by two magnesium ions and a net of hydrogen
bonds formed by the side chains of Lys215, Asn229, His278,
Arg347 and Arg352. Importantly, all of the phosphonate oxygen
atoms are engaged in interactions with the enzyme, a fact that
is consistent with the lack of inhibitory activity of the bis-H-
phosphinates with two oxygen atoms on phosphorus atom. The
aromatic fragment of the inhibitor is located in a cleft formed by
Arg364, Ala362, Thr356, Ile355, Pro98, Leu70 and Ala52. The
space available in this site does not significantly restrict the sizes
and positions of the phenyl ring substituents. This phenomenon
also correlates well with the experimental observations showing
no direct dependence of inhibitory activity on the phenyl
substitution pattern.
Conclusions
In summation, extensive screening of bisphosphonic acids
representing varied, albeit similar, structural scaffolds resulted
in the discovery of effective MtGS inhibitors with potencies
similar to the reference compound L-methionine-S-sulfoximine
(1). Importantly, the binding mode of the bisphosphonic inhibitors
differs significantly from that of compound 1. While L-methio-
nine-S-sulfoximine is a typical transition state analogue of
glutamate, bisphosphonates bind to the active site from the
opposite side, partially overlapping the ATP binding site. Because
of the low degree of conservation in this portion of enzyme
among species, different effects against bacterial and human
orthologs were observed. The combination of high efficiencies,
selectivities and bone-targeting properties makes these bispho-
sphonate GS inhibitors an attractive class of compounds in the
development of treatments for osseous tuberculosis.
Acknowledgements
The authors would like to thank Dr. Jayaraman Gowrishankar, Centre for
DNA Fingerprinting and Diagnostics, Hyderabad, India, for the kind gift
of GJ4547 E. coli strain and Prof. Sherry L. Mowbray from Uppsala
University for recombinant plasmids encoding MtGS and HsGS. The
Discovery Studio package was used under a Polish country-wide license.
The use of software resources (Discovery Studio program package) of the
Wrocław Centre for Networking and Supercomputing is also kindly
acknowledged.
Figure 6. Modelled complex of MtGS and hydroxybisphosphonate 8l
(front view – panel A and top view – panel B). The inhibitor molecule is
shown in ball-and-stick representation, enzyme residues with stick
representation, and metal ions in CPK representation. Hydrogen bonds
between the ligand and the enzyme are marked by thin green lines. The
enzyme surface is colored according to interpolated charge (blue –
positive, gray – neutral, red – negative) (color figure can be viewed in the
online issue).
Declaration of interest
Ł. B. acknowledges the financial support from the Polish Ministry of
Science and Higher Education (grant N N302 159937). The project was
supported by Wroclaw Centre of Biotechnology, programme The Leading
National Research Centre (KNOW) for years 2014–2018. The authors
report no declarations of interest.
These deviations from the linear models hindered further analysis
of the mode of inhibition (Figure S3).
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Supplementary material available online
Supplementary Figures S1–S3