Inhibitory effects of decavanadate on several enzymes and Leishmania tarentolae in Vitro

Article abstract of DOI:10.1016/j.jinorgbio.2011.09.009

Multiple studies report apparent effects of vanadium on various systems in vivo and in vitro. Vanadium species may be possible deterrents for the growth of the Leishmania parasite, which causes the sometimes deadly diseases known as leishmaniasis. The cur

Full text of DOI:10.1016/j.jinorgbio.2011.09.009

Journal of Inorganic Biochemistry 108 (2012) 96–104
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Inhibitory effects of decavanadate on several enzymes and Leishmania tarentolae
In Vitro
Timothy L. Turner, Victoria H. Nguyen, Craig C. McLauchlan ⁎, Zaneta Dymon, Benjamin M. Dorsey,
Jaqueline D. Hooker, Marjorie A. Jones ⁎
Illinois State University, Department of Chemistry, Campus Box 4160, Normal, IL 61790–4160, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2011
Received in revised form 12 August 2011
Accepted 6 September 2011
Available online 14 September 2011
Multiple studies report apparent effects of vanadium on various systems in vivo and in vitro. Vanadium spe-
cies may be possible deterrents for the growth of the Leishmania parasite, which causes the sometimes deadly
6-
diseases known as leishmaniasis. The current studies focus specifically on decavanadate V10
O
28 (V10), which
has a potential to be a potent effector for disease treatment. The X-ray structure of a new solvate salt of V10,
namely (NH 28·5H O, is also reported. Other vanadium complexes with imidazole carboxylate, an-
thranilate, or picolinate were also evaluated. The yellow-orange oxoanion, used as the (NH 28·6H
)
4 6
10
V O
2
) V
4 6 10
O
2
O
Keywords:
salt, was tested (at 1–100 μM) directly with two strains of Leishmania tarentolae promastigotes in culture
to evaluate the effect on cell viability. Vanadium coordination complexes are known effective inhibitors of
phosphatases. Using the artificial phosphatase substrate para-nitrophenylphosphate in the presence of a bo-
vine calf intestine alkaline phosphatase enzyme, V10 (from 5 to 100 μM) was shown to be a mixed inhibitor
for this enzyme and decreased the activity of the other two phosphatases tested. The effect of V10 and the
other vanadium complexes on the activity of phosphoglycerate mutase B (PGAM), an important enzyme in
glycolysis and gluconeogenesis, was also evaluated. At 10 μM, V10 was the most potent inhibitor of PGAM,
with an apparent reduction of about 50%. Taken together, we speculate that V10 could have a role in treating
Leishmania diseases.
Decavanadate
Vanadium complexes
Phosphatase inhibition
Leishmania
Phosphoglycerate mutase
©
2011 Elsevier Inc. All rights reserved.
1
. Introduction
leishmaniasis thus chemotherapy is most commonly used for treat-
ment. The first line of treatment is usually an organic salt of Sb(V)
[7] but an increase in resistance to these pentavalent antimonial ther-
apies has been reported [8-12]. Some species only show resistance in
either the promastigote form or the amastigote form, but not both [8].
Because most forms of leishmaniasis are not fatal, development of
new leishmaniasis treatments has not been considered a priority
and, therefore, leishmaniasis is considered a neglected disease by
the World Health Organization [2,13]. Therefore, a need exists for de-
velopment of new directions of leishmaniasis treatment.
These studies focus on potential new directions for treatment of
leishmaniasis using vanadium complexes and evaluating inhibition
of growth of the model system Leishmania tarentolae, in culture.
This species was selected owing to the ease and safety of culturing,
with optimum growth at 26 °C and no known capability to infect
humans, and its physiological similarity to species which can infect
humans [14,15].
1
.1. Leishmania and leishmaniasis
Leishmaniasis is a potentially deadly group of diseases caused by
infection from the parasitic protozoan Leishmania [1], with 1.5 to 2
million new cases reported annually [2]. These eukaryotic parasites
are endemic to at least 82 countries, but are most often found in
warmer climates such as Central and South America, Africa, India
and the Middle East [3]. There are at least twenty Leishmania species
capable of infecting humans and some domesticated animals leading
to leishmaniasis [4], each of which are hosted and transmitted
through small female phlebotomine sandflies [2]. When the sandflies
ingest their blood meal, Leishmania are transmitted to the alternative
host. Once inside their new host, Leishmania undergo a morphological
change from their promastigote form to their amastigote form. In
their amastigote form, they reside in the host macrophage cells
resulting in diseases, in three general forms (cutaneous, mucocutane-
ous, and visceral) for which the current therapies are costly and have
severe side effects [5,6]. There are no current vaccines available for
1.2. Vanadium and phosphatases
There are numerous studies on the actual mechanism of action of
some vanadium complexes in the body [16-20], and it is reported that
the vanadium compounds directly inhibit protein tyrosine phospha-
tase 1B (PTP1B) [21], a regulatory enzyme in the insulin receptor
⁎
Corresponding authors. Fax: +1 309 438 5538.
E-mail addresses: McLauchlan@IllinoisState.edu (C.C. McLauchlan),
majone3@IllinoisState.edu (M.A. Jones).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.09.009

T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
97
response pathway. Orthovanadate (“vanadate,” VO3-
) has been
4
6
-
O
O
shown to be a potent competitive inhibitor of phosphatases [22] in-
cluding PTPs [23-25]. An X-ray crystal structure of Yersinia PTP (pro-
tein tyrosine phosphatase) complexed with vanadate suggests that
the vanadate occupies the active site of the phosphatase enzyme.
Within the active site, the vanadium compound forms a thiol-vanadyl
ester linkage with a catalytic cysteine residue, similar to the covalent
thiol-phosphate linkage formed during normal enzymatic catalysis
O
O
O
O
V3
V3
O
O
O
O
O
O
V2
V2
V1
O
O
O
O
V2
[26]. Similar structures are known for acid phosphatases [27] and al-
O
V2
V1
O
O
O
kaline phosphatases [28] which are also inhibited by vanadium com-
pounds [29,30]. We have previously reported in vitro inhibition of
acid, alkaline, and tyrosine phosphatases with several novel vanadi-
um complexes [31] and here we extend this to report that decavana-
O
O
O
O
O
V3
V
3
6–
O
V O
28
1
0
6
-
10
date (V10
O
28, V10) affects these same enzymes. In addition, the effect
O
O
on another enzyme catalyzing reactions involving phosphate groups,
phosphoglycerate mutase B (PGAM), is investigated.
6
-
Fig. 1. Decavanadate structure, V10O28, V10.
1
.3. Phosphatases and phosphomutases
Here, we report effects of V10 and other vanadium complexes
when added to L. tarentolae in culture as well as its effects on four dif-
ferent enzymes, alkaline phosphatase (EC # 3.1.3.1, ALP), acid phos-
phatase (EC # 3.1.3.2, AP), protein tyrosine phosphatase 1B (EC #
3.1.3.48, PTP1B), and phosphoglycerate mutase B (EC # 2.7.5.3), in
vitro.
Phosphatases are enzymes found throughout nature that catalyze
the removal of phosphate groups from a variety of substrates. This de-
phosphorylation is especially important in the regulation of carbohy-
drate metabolism in metabolic pathways such as glycolysis and
gluconeogenesis.
Phosphoglycerate mutase is an enzyme that is critical in both gly-
colysis and gluconeogenesis, catalyzing the inter-conversion of 3-
phosphoglycerate (3PG) to 2-phosphoglycerate. It has been reported
that Leishmania are highly dependent on gluconeogenesis [32-34],
thus without adequate PGAM activity, gluconeogenesis is restricted,
eliminating one method of energy production within Leishmania.
Therefore, inhibition of this enzyme in Leishmania could prove an ef-
fective deterrent to their growth and virulence.
2
. Materials and methods
2.1. General considerations and instrumentation
The 13 complexes or compounds under investigation are shown in
Figs. 1 and 2. The complexes V(pic)
2·H O) [58], and NH [VO (pic) ]∙H
thesized with slight modification to the reported procedures as de-
3
∙H
2
2
O (1·H
2
O) [57], VO(pic)
2
2
∙H O
(
2
4
2
2
O ([NH
4
]3·H
2
O) [58] were syn-
tailed in [31]. V(imc)
3
(4), VO(imc)
(7), VO(anc)
2
∙H
∙H
2
O
(5·H
2
O), [NH
4
]
]
1
.4. Vanadium and Leishmania
[VO
VO
[NH
ing the methods of Johnson and Murmann [59]. Sodium orthovanadate
(Na VO , Na 11) was purchased from Acros Organics, imidazole (1,3-
2
(imc)
(anc)
[V10
2
] ([NH
4
]6), V(anc)
3
2
2
O (8·H O), and [NH
2
4
[
2
2
] ([NH
4
]9) were synthesized as previously reported [31].
O (V10, [NH 10·6H O) was synthesized follow-
Despite the challenges of using metals in medicine, a number of
4
]
6
O28]·6H
2
]
4 6
2
vanadium-containing species have been examined as potential agents
against trypanosomes [35-37], including Leishmania [25,38]. Toxicity
with metals in medicine, including vanadium, is often cited as a con-
cern for undertaking these studies [39-42], but these concerns are less
warranted when trying to replace the current treatment for leish-
maniasis. Some recent studies on Leishmania donovani with diperox-
ovanadates in conjunction with sodium antimony gluconate have
demonstrated possible cooperative effects of mixed treatments [43].
There have been no previous reports that focus on V10 as an anti-
Leishmania drug. Vanadium has known effects on several metabolic
pathways [44,45] and the decameric V10 may have a relatively ampli-
fied effect when incubated with these same systems, particularly
those involving phosphatases.
3
4
3
diaza-2,4-cyclopentadiene, 12) was purchased from Sigma, and 1H-
imidazole-4-carboxylic acid (Himc, H13) was purchased from May-
1
13
51
bridge. NMR spectra ( H, C, V) were collected using a 500 MHz
Bruker spectrometer and were referenced to residual solvent [60] or ex-
51
ternal, neat VOCl
3
(
V). Magnetic susceptibility measurements were
calculated via a Johnson-Matthey magnetic susceptibility balance. UV-
visible (UV-Vis) data were acquired with an Agilent 8453 UV–vis spec-
troscopy system or with a Bio-Rad Benchmark microplate reader. Infra-
red spectra were collected as neat solids using a Perkin-Elmer FTIR with
ATR attachment.
Cell density, motility, and shape were evaluated through a Jenco
inverted compound microscope (model CP-2A1). Confocal microsco-
py was carried out with a Leica TCS SP2 confocal microscope, with
an Ar laser excitation at 514 nm and emissions evaluated between
1
.5. Decavanadate
5
50–600 nm. The MitoSox™ Red (Invitrogen) superoxide detecting
A polymeric oxocluster complex of vanadium that occurs in aque-
ous solutions, i.e. decavanadate, V10O28 (Fig. 1) was first described by
6-
probe was utilized to detect the presence of superoxide production
within cells [61]. Cell viability was evaluated at 595 nm using the
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Berzelius [46], and clusters of this type have long been investigated
for, inter alia, their interaction with proteins, inhibition of ATPases,
and interaction with cation channels [47-51]. A number of peptide
species have been bound to decavanadate to study the possible inter-
action of decavanadate with proteins [48,49,52]. Decavanadate effects
in biological systems have been recently reviewed [48,53,54] and dif-
ferences in the effects of decavanadate and simpler clusters have been
highlighted. For instance, in vivo studies have shown that mitochon-
drial uptake is more pronounced with decavanadate than with ortho-
vanadate [48], depending on the vanadium concentration and mode
of administration [55,56].
(
Sigma)) spectrophotometric assay developed by Mossman [62] fol-
lowing the procedure of Morgenthaler et al. [63] using Falcon 96-well
microtiter plates (with flat-bottomed wells) from Becton Dickinson.
2.2. Preparation of [NH
4
]
6
V
10 2
O28·5H O
The decameric species [NH
acidification of an aqueous solution of 0.133 g (1.14 mmol) of
NH VO and 15 mL of deionized H O with 2.28 mmol of anthranilic
4
]
6
[V10O28]·5H O was prepared by
2
4
3
2

98
T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
Fig. 2. Vanadium coordination complexes and compounds under investigation in this study.
acid. The solution was gravity filtered and reduced to dryness in vacuo
to afford a yellow-orange solid. Anal. Calc (found) for H32 10: C,
O, VOCl
514, -497, -422. Single crystals of this compound were additionally
analyzed by X-ray diffraction.
tarentolae promastigotes. It should be noted that throughout this
work these concentrations indicated are for decavanadate concentra-
tion, not total vanadium concentration. This distinction is critical be-
cause the effects of a given concentration of decavanadate often differ
(and often are superior [56]) from those for a ten-fold-more-concen-
trated solution of a monomeric species, e.g. orthovanadate. After ad-
dition of V10, cultures were divided into two groups, one that
received two hours of exposure to a fluorescent lamp (350–750 nm,
6 33
N O V
51
0
-
.0 (0.6); H, 3.0 (2.7); N, 7.3 (6.8). NMR δ V(400 MHz; D
2
3
)
2
.3. Leishmania tarentolae growth studies
7
50 LUX) and one that received no additional light exposure. The cul-
Studies were conducted with two L. tarentolae strains, #30143
from the American Type Culture Collection) and LEM-125 (provided
tures were evaluated at selected times (0, 2, 12, and 24 hours) after
V10 addition to determine L. tarentolae viability using the MTT assay.
Background controls consisted of medium with V10, and the mean
absorbance value (n=6) was subtracted from the mean experimen-
tal values (n=6 for each V10 concentration used) and reported as
corrected values. In some cases data are reported as corrected percent
of the control cell cultures (no added V10). All other complexes tested
(Fig. 2) with Leishmania were done using the #30143 promastigotes
in BHI and evaluated as above.
(
by Dr. Larry Simpson of the University of California, Los Angeles).
Cells were incubated in polystyrene 25 cm2 flasks (Corning) or 24-
well plates (Becton Dickinson) at room temperature. Two media
were used for cell culturing and viability; brain-heart infusion (BHI) pre-
pared per the manufacturer's (Sigma-Aldrich) protocol and Schneider's
Insect Medium (SIM) [64]. To the BHI, 20 μM final concentration
hemin was added whereas 10% (v/v) Fetal Bovine Serum (Invitro-
gen) and 2 mM L-glutamine were added to the SIM. All media and
cells were handled under sterile conditions. Penicillin and strepto-
mycin (10,000 units/mL and 10 mg/mL, respectively) were added
to the BHI medium.
2.4. Phosphatase enzyme kinetic studies
Various final concentrations (0, 1, 10, 50, and 100 μM) of V10
freshly prepared in BHI or SIM) were added to replicate cell cultures
in one of the growth media as well as one of the two strains of L.
Kinetic studies were carried out using bovine calf intestine alkaline
phosphatase enzyme (P7923, Sigma-Aldrich, 2KU diluted with 300 μL
of pH 7.8 buffer as stock stored at 4 °C), para-nitrophenylphosphate
(

T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
99
(
pNPP) substrate (freshly prepared following recommendations of the
Table 1
Details of crystallographic results.
supplier, Sigma), and several concentrations of V10. A pH 7.8 buffer con-
sisting of 25 mM TES, 25 mM Trizma® base, and 1.5 mM MgCl
2
was
[NH ] [V10O28]·5H O
4
6
2
used. Reactions were conducted at room temperature in 1.5 mL poly-
propylene tubes in a final volume of 1 mL. Sixteen substrate concentra-
tions and five V10 concentrations (n=3) were utilized under initial
velocity conditions (20 min). The enzyme (stock diluted to 2.2×10-4
units of activity per assay) was preincubated for 6 minutes± V10 before
addition of pNPP. The reactions were stopped by addition of 60 μL of
Formula
FW
Temperature (K)
λ (Å)
Crystal System
Space Group
a (Å)
H
34
N
6
O
33
V
10
1155.73
173 (2)
0.71073
Monoclinic
C2/c
11.5743(16)
17.119(2)
16.980(3)
108.369(2)
3193.1(8)
4
1
0 M NaOH. The solutions were then heated in an 80 °C water bath
b (Å)
c (Å)
for 10 minutes to allow for more accurate product determination (see
Section 3.3 and Supplementary Information), allowed to cool to rt in a
water bath, and evaluated at 405 nm. Blanks consisted of buffer with
enzyme± V10 to which 60 μL of 10 M NaOH was added so that the en-
zyme was denatured for 5 minutes before adding pNPP. Data are
reported as μM product formed per minute.
Acid phosphatase (AP) and protein tyrosine phosphatase (PTP1B)
were also assayed± preincubation (6 min) with V10, following the
methods reported by McLauchlan et al. [31].
β (°)
3
V (Å )
Z
-3
D
χ
, Mg m
2.404
0.0307
0.0710
a
R
1
(F)
2
)b
wR2(F
o
a
b
2
2
2
2
4
1/2
2
2
R (F)=Σ(|F | - |F |)/ Σ|F |. wR2(F )=[Σ[w(F – F ) ]/ ΣwF ] ; w=1/[\σ (F )+
1
o
c
o
o
o
c
o
o
2
2
2
(0.0339P) +5.2273P] where P=(F
o
c
+2F )/3.
3
. Results and discussion
2
.5. Phosphoglycerate mutase B (PGAM) sensitivity to various vanadium
compounds
3.1. Synthesis and characterization of [NH ] V10O28·5H O
4
6
2
Effects of 11 vanadium complexes (Figs. 1 and 2: V(pic)
1·H O), VO(pic) ∙H O (2·H O), [NH ][VO (pic) ]∙H O ([NH ]3·H
V(imc) (4), VO(imc) ∙H O (5·H O), [NH ] [VO (imc) ] ([NH
V(anc) (7), VO(anc) ∙H (8·H O), [NH ][VO (anc)
NH [V10 28]·6H O ([NH 10·6H O), and Na VO
3
∙H
2
2
O
O),
It has long been known that a series of polyoxovanadate clusters
6-
(
2
2
2
2
4
2
2
2
4
including the decavanadate V10
28
O can readily be formed from the
3
2
2
2
4
2
2
4
]6),
]9),
acidification of a solution of aqueous vanadium(V) salts [59,68-70]
and crystal structures with counterions ranging from simple metals
[70] to protonated amines have been previously reported [49,52,71,72].
Here we isolated the decameric cluster when we serendipitously acidi-
fied our ammonium vanadate solution with anthranilic acid in the
attempted preparation of [NH ][VO (anc) ] [31]. The complex is easily
3
2
2
O
2
4
2
2
]([NH
4
[
]
4 6
O
2
4
]
6
2
3
4
(Na
3
11)) on
PGAM activity were measured by use of a reaction coupled to the oxida-
+
tion of NADH to NAD , following the methods of Takeda et al. [65]. Each
reaction (final volume of 100 μL) was evaluated at 340 nm under initial
4
2
2
velocity conditions. A reaction cocktail in a buffer of 25 mM TES,
isolated as a yellow-orange solid. Spectroscopic analysis of the complex
2
2
5 mM Tris, 1.5 mM MgCl
,3-bisphosphoglycerate (2,3-BPG), 6 units / mL lactate dehydrogen-
2
, pH 7.9 containing 100 mM KCl, 0.1 mM
confirms that this is the well-studied decameric vanadium complex
6-
28
V10O (10, V10, Figs. 1 and S3 and Supplementary Information), which
ase/pyruvate kinase (LDH/PK), 1 unit / mL enolase, 5 units / mL PGAM,
0 μM adenosine diphosphate (ADP) and 2 mM NADH was used. The re-
action was started by addition of 2 mM substrate (3PG). 3PG, PGAM, 2,3-
BPG, and LDH/PK were purchased from USB Corporation (#26118). Eno-
lase, NADH, ADP, and KCl were purchased from Sigma. All reagents were
used as received without further purification.
had crystallized as an unusual solvate (Fig. S1). Formulation of the spe-
cies as written is described below in Section 3.2. Preliminary enzyme in-
hibition studies were conducted with this polymorph of V10 in aqueous
2
6-
28
solutions, but all subsequent studies involving V10O were conducted
using the well-known hexahydrate salt described by Johnson and Mur-
mann, i.e. [NH ] V10O28·6H O [59].
4
6
2
Using the coupled reaction, ± preincubation with selected vanadium
complexes, the background rate (rate A over time of 1 min) prior to ad-
dition of the 3PG substrate was determined for each reaction. A second
rate was determined upon addition of the substrate (rate B over time of
3.2. X-ray structure of [NH
4
]
6 2
[V10O28]•5H O
1
min). The mean difference (rate B – rate A) for each added vanadium
Single crystals of [NH
4
]
6 2
[V10O28]•5H O suitable for X-ray diffrac-
complex was compared with the mean difference of the control reac-
tion (without vanadium complex) to determine the effect of the vana-
dium complexes on PGAM activity. Data are reported as percent of
control reactions. The individual enzymes in this coupled reaction
tion studies were isolated by slow re-crystallization from an aqueous
solution. Selected crystallographic information is shown in Table 1.
Bond distances and angles are contained in the Supplementary Infor-
mation, Table S1. The decameric polyvanadate contains a typical core
V
(
PK, enolase, and LDH) were also tested with V10 pre-incubation
arrangement of ten V O
6
edge-shared octahedra, Fig. S1 [49,52,70-
which in all cases exhibited only modest (about 10%) effects on cat-
alytic activity.
72]. There is evidence for extensive hydrogen bonding in the solid
state (Fig. S1, Table S2). Based solely upon X-ray diffraction analysis,
the formulation of this cluster is ambiguous. The protonation states
of such species are often unclear and are determined based on the
+
2
.6. X-ray crystallography
number of counter ions present [73]. With the use of NH
4
as a coun-
ter ion and the presence of isoelectronic waters of crystallization, this
method for determination of protons is unhelpful. Elemental analyses
results of the bulk materials, which is admittedly not necessarily the
same as the single crystal used for structure determination, afford in-
termediate values for the bulk assignment. The best match with the
experimental elemental analysis results are achieved for the formula-
Intensity data were collected at −93 °C using a Bruker SMART Apex
diffractometer equipped with a CCD area detector using graphite mono-
chromated Mo Kα radiation. Data were reduced and corrected for ab-
sorption using the SAINT+Software Suite [66]. Structure solutions
2
were obtained by direct methods and were refined on F with the use
of full-matrix least squares techniques [67]. All non-hydrogen atoms
were refined anisotropically and hydrogen atoms were refined with a
riding model. Pertinent crystallographic details are shown in Table 1
and in the Supplementary Information.
tion [NH
4 5 2
] [HV10O28]•2H O, which does not match with the electron
density map derived from the X-ray diffraction study, which shows
evidence for 11 NH
molecule, O3W, sits on an inversion center in this monoclinic cell.
+
4 2
/H O units per decavanadate unit. One water

100
T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
5
1
V NMR studies show that the species has identical shifts to those of
a
6
-
known V10
to have strong impacts on
have been previously reported for [NH
the crystal systems differ from this species and the V MAS SSNMR
spectra for the isolated pentahydrate polymorph versus that known
complex are quite different (O. Goncharov-Zapata, M. Li, J. Yehl, K.
Ooms, S. Bolte, P. Chatterjee, C. McLauchlan, D. Crans, T. Polenova,
manuscript in preparation).
O
28 species and differences in protonation are well-known
1.8
.6
1.4
51
V NMR shifts [74]. Crystal structures
[V10 28]•6H O [49,73], but
1
4
]
6
O
2
0
5
µM V10
µM V10
51
1.2
10 µM V10
50 µM V10
1
1
00 µM V10
0
0
0
0
.8
.6
.4
.2
0
3
.3. Decavanadate stability in BHI and various buffers
There is a known propensity for V10 to degrade into its tetra-, di-,
and ortho- vanadate forms (V4, V2, and V1, respectively) in neutral or
alkaline environments [48]. Vanadate and its oligomers have a well-
known equilibrium (Fig. S2) [75-78] and interactions with buffer/
solution species have been well-studied [79]. Thus, tests were con-
ducted to determine V10 stability for the studies conducted here. Stabil-
ity of V10 in solution was assessed by 51 V NMR (Fig. S3a,b) and UV–vis
studies (Fig. S3c), including in buffer, ± enzyme, ± substrate, ± NaOH,
with varying temperatures (room temperature, 37 °C and 80 °C), and±
ambient room light. The stability of the substrate, pNPP, and product
were also tested± light, at varying temperatures, and over varying
lengths of time. In BHI, which has a pH of ca. 7.2, we observed that the
V10 species decomposes quickly with measurable decay in as little as
one hour. In our hands, the dominant species after 19 hours is V4. Al-
most no V10 was detected by 42 h while V4 still dominates at 42 h
and 66 h. Trace amounts of V1, V2, and the pentameric V5 were
detected at all times, with 0.26, 0.14, 0.19 molar equivalents to 1 for
V4 based on NMR integrations at 44 and 66 h. A UV–vis kinetics study
of the decay in the alkaline phosphatase buffer, pH 7.8 carried out by fol-
lowing disappearance of peak at 430 nm (Fig. S3c) indicated that V10
0
50
100
pNPP (µM)
150
200
250
b
14
1
2
0 µM V10
5 µM V10
1
5
1
0 µM V10
0 µM V10
00 µM V10
10
8
1
/V (min/mcM)
6
4
2
0
-
0.02 -0.01
0
0.01 0.02 0.03 0.04 0.05
/[S] (mcM–1)
1
Fig. 3. a) ALP product formation per minute with varying substrate concentrations
± V10 preincubation with enzyme. b) Lineweaver-Burk transformation of V10 inhibi-
tion of ALP data shown in a).
½
degrades approximately 15% per hour at rt (t =6 h under first order
kinetics). Under alkaline conditions at 80 °C, about 95% of the V10
decomposed in 10 minutes. In Schneider's Insect Medium, which has
a pH of 5.4, the decomposition of V10 is substantially slower and after
2
hours, only 0.06 molar equivalent of V4 was detectable and the rest
previously to be a competitive inhibitor, may both be competitive of
these two enzymes while having a different type of inhibition on
the ALP activity as evaluated here. This is of interest since V10 has
been shown to degrade, as a function of pH, to other vanadium com-
pounds in addition to orthovanadate.
remained as V10. Our data correlate with reports that vanadates can in-
teract with buffer systems around them as a function of pH [54,79,80].
Decavanadate stability thus is an issue especially since it has been
reported that each V species may have different effects on enzymes
[
81,82], proteins [81,83-88], and membranes [83,89]. However, with
PTP has been previously shown to have a role in Leishmania. For
instance, the known PTP inhibitor potassium bisperoxo(1,10-phenan-
throline)oxovanadate has been tested against L. major-induced infec-
tions in mice and L. donovani in culture, and shows a dramatic effect
on controlling progression/growth of the parasites [25]. There are now
many reports of PTP1B inhibition by V species [24,29,31,38,91-95].
our short enzyme incubation times in this work, we anticipated a
minimal effect of the V10 degradation and thus observed effects
may be attributed to V10. Longer incubation times may lead to effects
due to other vanadium species.
3
.4. Enzyme kinetic studies of ALP, AP, and PTP1B
As shown in Fig. 3a, V10 was shown to be an effective inhibitor of
3.5. Decavanadate phosphoglycerate mutase B studies
the bovine calf intestine ALP enzyme. In the presence of 5 μM V10,
product formation was reduced by nearly 50%. When incubated
with 100 μM V10, product formation was reduced by almost 80%.
The Lineweaver-Burk plot of the data (Fig. 3b) is consistent with
For PGAM, the various vanadium complexes tested (Figs. 1 and 2)
in general had only modest effects at 100 μM on this enzyme, except
–
V10, 10, and VO
2
imc
2
, 6, which appeared to inhibit the enzyme 100%.
V10 acting as a mixed inhibitor, [90] with an increased K
m
and a de-
At 10 μM, V10 and VO
2
imc-
2
inhibited the enzyme activity about 50%
creased Vmax. In addition to its effects on ALP, V10 also is a potent in-
hibitor of both AP and PTP1B. Following the methods reported by
McLauchlan et al. [31] using a variety of vanadium compounds,
Table 2
Comparison of Kinetic Constants of three phosphatases with and without V10
preincubation.
2
m
30 μM V10 preincubated with AP increased the K value approxi-
mately 40 fold (from 0.16 to 6.9 μM) while having only a modest ef-
fect on the Vmax value and thus appears to be a competitive
inhibitor of the type 1 wheat germ acid phosphatase enzyme. With
max (μM min-1
)
Addition
None
Km (μM)
V
Alkaline phosphatase
Acid phosphatase
PTP1B
10
1.7
0.5
5.9
18
16
8.8
+
V10 (100 μM)
120
0.16
6.9
0.73
17.6
PTP1B, 23 μM V10 increased the K
m
value approximately 24 fold
None
+ V10 (230 μM)
None
(
from 0.73 μM to 17.6 μM) with only a modest effect on Vmax thus be-
having as a competitive inhibitor of this enzyme also. Kinetic data are
summarized in Table 2. Thus, V10 and orthovanadate, reported
+
V10 (23 μM)

T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
101
and 30%, respectively (Fig. 4). Orthovanadate, 11, was a modest inhib-
itor (about 25% inhibition at 100 μM) of the enzyme.
after 2–12 hours of incubation; in all cases, this effect is reduced to
control values by 24 hours. The imidazole carboxylate (10 μM, 13)
had no apparent effect (data not shown). Complexes 1 (V(pic)
and 10 (V10) show inhibition of 20% and 30%, respectively, at 2
hours but in both cases the cells recover at 24 hours. V(imc) (4),
V(anc) (7), and VO(anc) (8) appear to inhibit~25% at 24 hours.
Na VO (Na 11) shows a mild inhibition of 10% of the 0 hour re-
sponse at all other times. Thus, there is no single trend for vanadium
of these complexes; rather the observed effects, although modest, ap-
pear to be more related to the ligand and not the metal or its oxida-
tion state.
3
)
3
.6. Leishmania tarentolae viability studies
3
3
2
It has been reported that exposure of certain vanadate species to
3
4
3
light can induce cleavage in myosin, likely by a redox process [96],
which suggests that photodynamic effects should be evaluated.
Therefore, we conducted some of our viability studies both in the
presence and absence of light.
For L. tarentolae strain #30143, with light exposure and incubation
with 100 μM V10, there is an approximate 50% reduction in the MTT
response by L. tarentolae at 2 hours post addition relative to cells
with no V10 (Fig. 5a,b). However, the culture appeared to recover
by 24 hours. Recovery time has been shown to be important to eval-
uate when considering the effects of intervention [97]. This loss of ef-
fect may be due to degradation of the V10. At 1 μM V10, there was no
apparent inhibition relative to controls. Without the light exposure at
4
. Conclusions
4.1. Leishmania tarentolae viability studies
Evidence of mild inhibition of L. tarentolae viability by V10 was
shown in our studies and this inhibition is dose dependent (Table 3).
At 100 μM of inhibitor, this effect was more pronounced at 24 hours
for strain #30143 with no light exposure and at 2 hours with light expo-
sure cultures than at other times tested. However, light-exposed cells
seemed to develop normally after 12 and 24 hours. We speculate that
the recovery of the V10 culture is due to the decomposition of V10 in
the mildly alkaline BHI culture media. Owing to this instability, use of
V10 as a possible anti-leishmanial drug may require re-dosing every
1
00 μM V10, there was also approximately a 50% reduction in cell vi-
ability, but this was at 24 hours, with no apparent effect at 2 hours.
The LEM-125 strain was much less affected at either V10 concentra-
tion± light (Fig. 5c,d) than the #30143 strain. It is of interest to
note that incubation with 100 μM V10± light treatment, resulted in
an apparent reduction of cell mobility compared to the controls for
both strains when viewed through microscopy (video data are locat-
ed in Supplementary Information). Despite the limited mobility, the
shape and size of cells incubated with V10 were similar to the control
cells. In addition, V10 added to cell culture resulted in an increase in
detectable intracellular superoxide formation in the exposed cells rel-
ative to the control cells and higher levels of superoxide formation are
generally correlated with cell oxidative stress (Fig. S4). Cai and Jones
1
2 hours.
The apparent reduction in mobility of L. tarentolae incubated with
V10± light treatment suggests that V10 may limit the ability for the
cells to move within the host and may decrease infectivity despite
not completely inhibiting the growth of L. tarentolae when assessed
by the MTT viability assay.
Our data agree with Martiny et al. [101] who reported that Na
3 4
VO is
[98] reported that activation of apoptosis is clearly associated with
a mild inhibitor of L. tarentolae growth. Results from our imidazole-con-
taining compounds (imidazole (12), VO(imc) (5), [VO (imc) ] (6)),
2 2 2
production of superoxide in the mitochondria. Also, it has been
reported that Leishmania use apoptotic signals to increase the infec-
tivity of phagocytes such as macrophages [99,100]. Thus the superox-
ide production by Leishmania detected in our studies is not a surprise
and would suggest that the cells are responding to factors such as the
V10 in their environment via apoptotic mechanisms.
–
which appear to initially accelerate cell growth, agree with the work
of Weinrauch et al. [102] who suggest that imidazole-containing
metal complexes (ICCs) fail as anti-leishmanial drugs. We suggest that
imidazole may be a possible growth enhancer for L. tarentolae, encour-
aging their growth as the ICCs are broken down to release free imidaz-
ole species.
When Leishmania strain #30143 promastigotes were exposed to
1
0 μM of compounds 1–13 (Figs. 1 and 2) and assessed by the MTT
assay at 0, 2, 12, and 24 hours post additions, most compounds had
4
.2. Enzyme kinetic studies of ALP, AP, and PTP1B
little effect at any time point as shown in Fig. 6. At 10 μM, imidazole
–
(
12), VO(pic)
2
(2), VO(imc)
2
2
(5), and [VO (imc)
2
]
(6), appear to
We have now shown that V10 affects these three enzymes although
promote L. tarentolae growth by 30, 25, 25 , or 70% , respectively,
likely by different mechanisms. The mixed inhibition of ALP by V10 dif-
fers from the competitive inhibition previously reported for sodium
3 4
orthovanadate (Na VO ) [22]. Further studies are needed to better un-
1
1
1
1
1
80
60
40
20
00
Control
derstand the mechanism behind this inhibition. We speculate that the
rapid decomposition of V10 in the pH 7.8 buffer may partially be the
cause of mixed inhibition, though we suspect that V10 itself plays an
important role, as previously suggested by Aureliano et al. [53,54]. The
different types of inhibition of ALP by V10 and Na VO suggest that en-
3 4
zymes important to Leishmania metabolism be tested with V10 as a po-
1
1
00 µM
0 µM
8
6
4
2
0
0
0
0
0
tential new direction to treat leishmaniasis.
4.3. Decavanadate phosphoglycerate mutase B study
At 10 μM, V10 was the most potent inhibitor of PGAM, with an ap-
parent reduction of about 50% (Fig. 3). At 100 μM, [NH
4 2 2
][VO imc ]
(
[NH ]6) almost completely eliminated the activity of PGAM, but
4
with only ~30% reduction in activity at 10 μM. Our data agree with
Climent et al. [103] who reported PGAM inhibition by orthovanadate
at μM concentrations. Unlike all other compounds tested, which had
Complexes under study
3
only modest effects, Vimc (4) at 100 μM was an apparent activator
of this enzyme, increasing the activity of PGAM to 170% relative to
Fig. 4. Effects of various vanadium complexes on PGAM activity under initial velocity
conditions.
the control enzyme. We speculate that inhibition of L. tarentolae

102
T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
2
1
2
h
a
c
b
2 h
4 h
1
1
1
40
20
00
140
120
100
80
8
6
4
2
0
0
0
0
0
60
40
20
0
Control
1 µM
100 µM
Control
1 µM
100 µM
Concentration of V10
Concentration of V10
d
3
3
2
2
1
1
50
00
50
00
50
00
350
300
250
200
150
100
50
50
0
0
Control
1 µM
100 µM
Control
1 µM
100 µM
Concentration of V10
Concentration of V10
Fig. 5. Decavanadate, V10, inhibition of Leishmania strains determined via MTT assay at 2, 12, and 24 hours for a) #30143 incubated in light, b) #30143 incubated without light, c)
LEM-125 incubated in light, and d) LEM-125 incubated without light. The three bars from left to right indicate the results at 2, 12 and 24 h.
mobility, when cells were directly exposed to V10, is due in part to
the decreased activity of PGAM, resulting in limited function of gly-
colysis and gluconeogenesis. The limited effects of V10 on enolase,
PK, and LDH are expected, as it has been previously reported that van-
adate has no effect on PK [103].
protonated species affect the cells. We speculate that this inhibition
is at least partially due to the diminished function of gluconeogenesis
by a reduction of PGAM activity. Limiting gluconeogenesis may cause
L. tarentolae to shift to other metabolic pathways for energy. This shift
of energy usage can contribute to the apparent reduction of L. tarentolae
motility. Incubation with V10 or breakdown products affects metabo-
lism under certain conditions (at 2 hours with light, at 24 hours without
light), thus inhibiting motility of cells, which may limit spread of leish-
maniasis in an infected individual. However, our phosphatase inhibition
studies also indicate that it is highly likely that multiple enzymes are
moderately affected by the V10. Frequent dosing may also be necessary
because the cultures appear able to recover from a single dose. Taken
4
.4. General conclusions
We have shown that V10 negatively affects L. tarentolae growth at
concentrations of 10 and 100 μM, but this is time and light-exposure
dependent. The time dependency is likely related to the instability of
the V10 as a function of pH and suggests that both V10 and partially
2
1
1
1
1
1
00
80
60
40
20
00
80
60
40
20
0
0
2
1
2
h
h
2 h
4 h
Control Imidazole Na VO4
V(pic)3 VO(pic)2 [VO (pic) ] V(imc)3 VO(imc)2 VO (imc)2 V(anc)3 VO(anc)2 [VO (anc) ] V10
3
2
2
2
2
2
(
12)
(11)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
Compounds (10 µM)
Fig. 6. Effect of added vanadium complexes relative to control #30143 promastigote cells (no additions) and to added imidazole. MTT results are mean and standard deviation of
n=4 replicates reported as a percent of same culture 0 hour. The four bars from left to right indicate the results at 0, 2, 12 and 24 h.

T.L. Turner et al. / Journal of Inorganic Biochemistry 108 (2012) 96–104
103
Table 3
Dose-dependent Leishmania MTT response as a function of V10 concentration.
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