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Snake bite
is a severe medical emergency in North Africa region and other parts of the
world. When venom injected into the victim after the bite causes grave
debilitating and life-threatening effects eventually resulting in mortality and
morbidity. Snake venom phospholipases A2 (PLA2) have
local and systemic actions that induce pathophysiological effects in the
victim. 6-Amino-5-bromo-1H-pyrimidine-2,4-dione (6-amino-5-bromouracil) is a
derivative of uracil and has an inhibitory effect on many enzymes. Molecular
docking and fluorescence studies were performed in order to determine the
potential modes of action of 6-amino-5-bromouracil with phospholipases A2.
6-Amino-5-bromouracil showed hydrogen bonds with an active site of enzyme and
changes in the fluorescent spectrum of PLA2. The achieved results
revealed that 6-amino-5-bromouracil treated animals have shown to increase the
mean survival time and the protection fold, but could not
protect mice from death when used alone. The use of 6-amino-5-bromouracil could lead to prevent the snake venom
toxicity and further chemical synthesis analogues and in
vivo studies would be
necessary to substantiate the obtained results.
Keywords: Snake bite, Cerastes cerastes,
6-amino-5-bromouracil, Fluorescence, Phospholipase A2, Molecular
docking
Abbreviations: PLA2: Phospholipases A2; 6A5B:
6-Amino-5-Bromouracil
INTRODUCTION
Snakebite is
a foremost public health problem in numerous African countries including Libya,
Algeria Egypt and Tunisia [1,2]. It is a meticulous confront, even though, in
some parts of Africa, which is home for more than 400 snake species, of which
about 30 venomous species, belonging to four snake families namely: Colubridae,
Viperidae, Elapidae, Atractaspididae and they are appeared to cause human
deaths, as reported by the World Health Organization [2]. Cerastes cerastes is one of the snakes commonly threatening human
life in Libya. The Cerastes cerastes
venom is containing numerous enzymes that have proteolytic activity and leads
to multiple kinds of intoxications [3]. The lethal cause of snake venom mainly
results from its active components mainly PLA2. Cerastes cerastes snake venoms cause proteolysis, shock, blood
clotting, release of bioactive substances such as bradykinin and histamine,
necrosis, hemorrhage and numerous other effects [4-6]. Necrosis might result
from a direct action of myotoxins or myotoxic PLA2s on plasma
membranes of muscle cell or indirectly, as a consequence of blood vessel
degeneration and ischemia caused by hemorrhaging. PLA2s interact
with membrane phospholipid components which lead to promote release of
intracellular creatine kinase, which might be used as a biomarker for myotoxic
activity assessment [7,8]. The snake venom physiopathological processes
encouraged researchers to obtain natural or chemically synthesised inhibitors.
Some
PLA2 inhibitors may be found in different organisms. Manoalide (A)
is a non-steroidal sesquiterpenoid from the marine sponge Luffariella
variabilis, while manoalide (B) was synthetically synthesized based on its
natural analogue. These terpenoids compounds have irreversible inhibitory
effects on numerous PLA2s obtained from snakes [9,10]. The phospholipase A2
(PLA2) activity can be inhibited by some inhibitors as reported in
the literature who have found that a chemical compound called varespladib and
its orally bioavailable prodrug, methyl-varespladib had an inhibitory effect on
PLA2 (sPLA2) at nanomolar and picomolar concentrations
against 28 medically vital snake venoms
from six continents [11]. The aim of the paper was to
study the inhibitory effect of 6-amino-5-bromouracil on phospholipase A2
enzyme to treat snake envenomation using molecular docking and fluorescence
studies.
MATERIALS AND METHOD
Preparation of aqueous 6-amino-5-bromouracil
solution
A weight of 19 mg from
6-amino-5-bromouracil (Matrix Scientific, Columbia, and Catalog Number 078889)
was dissolved in 100 µl DMSO and the solution was completed to 5 ml with 0.9%
sodium chloride to give a final stock solution of 18.5 mM and stored at –20°C
until use.
Venoms
Snake (Cerastes cerastes,)
venom was extracted by manual stimulation and were obtained in liquid forms,
from the Department of Zoology, Faculty of Science, University of Tripoli
(Libya) and stored at –20°C until use. An aliquot of 7.5 μl venom from the
venoms was added to 800 μl of normal saline. A dose of 100 μl (100 ng) was
intraperitoneally injected in 18 ± 2 g male Swiss Albino mice.
Molecular docking
The starting geometry of the
6-amino-5-bromouracil was constructed using chem3D Ultra (version 8.0,
Cambridge soft Com., USA). The optimized geometry of 6-amino-5-bromouracil with
the lowest energy was used for molecular docking. Crystal structures of the
crystal structure of cobra-venom phospholipase A2 in a complex with
a transition-state analogue (1POB) was downloaded from the Protein Data Bank https://www.rcsb.org/structure/1POB. Molecular dockings of
6-amino-5-bromouracil with 1POB was accomplished by Auto Dock 4.2 software from
the Scripps Research Institute (TSRI) (http://autodock.scripps.edu/). Firstly, polar hydrogen atoms
were added into protein molecules. Then, partial atomic charges of the
phospholipase enzymes and 6-amino-5-bromouracil molecules were calculated using
Kollman methods [12]. In the process of molecular
docking, the grid maps of dimensions (62 Å × 62 Å × 62 Å) with a grid-point spacing
of 0.376 Å and the grid boxes are centered. The number of genetic algorithm
runs and the number of evaluations were set to 100. All other parameters were
default settings. Cluster analysis was performed on the results of docking by
using a root mean square (RMS) tolerance of 2.0 Å, which was dependent on the
binding free energy. Lastly, the dominating configuration of the binding
complex of 6-amino-5-bromouracil and phopholipase A2 enzyme
fragments with minimum energy of binding were determined which relied strongly
on the information of 3D structures of the phopholipase A2 binding
site and ultimately generated a series of phopholipase binding complex.
Absorbance
spectra
Absorbance spectra were measured
on a Jenway UV-visible spectrophotometer, model 6505 (London, UK) using quartz
cells of 1.00 cm path length. The UV-Vis absorbance spectra were recorded in
the 200-500 nm range and spectral bandwidth of 3.0 nm. For the final spectrum
of each solution analyzed baseline subtraction of the buffer solution was
performed. The protein content of venoms samples was determined by the
spectrophotometric method as described [13]. Bovine serum albumin (BSA,
Sigma) was used for standard assays.
Fluorescence
spectra
Fluorescence emission and
excitation spectra were measured using Jasco FP-6200 spectrofluorometer (Tokyo,
Japan) using fluorescence 4-sided quartz cuvettes of 1.00 cm path length. The
automatic shutter-on function was used to minimize photo bleaching of the
sample. The selected wavelength chosen provided aggregate excitation of
tryptophan and tyrosine residues. The emission spectrum was corrected for
background fluorescence of the buffer. The changes of fluorescence emission
intensity and fluorescence shifts were monitored in which the formation of the
system was formed by sequential addition of aliquots of Tris buffer, Cerastes
cerastes venom and finally 6-amino-5-bromouracil.
Experimental animals
Swiss Albino male mice (18 ± 2 g)
were used for the experiments. In order to reduce the contact caused by
environmental alterations and handling during behavioral studies, mice were
acclimatized to the Laboratory Animal Holding Center and laboratory surroundings
for three days and at least 1 h before the experiments, respectively. Mice were
kept under standard conditions with food (low protein diet) and water available
ad libitum. The animals were housed six per cage in a light-controlled
room (12 h light/dark cycle, light on 07:00 h) at 27°C and 65% relative
humidity. All experiments were carried out between 11:30 and 14:00 h. Each test
group consisted of at least six mice and each mouse was used only once. All
animal experiments were conducted according to guidelines set by Institutional
Animal Ethics Committee of University of Tripoli. Five groups of mice were used
in this study. The first group of six mice received only 100 μl (100 ng of
total protein) of the Cerastes cerastes venom (LD99 5 μg/kg). Groups 2-4
of six mice each (serving as treatment groups) were given an equivalent amount
of the Cerastes cerastes venom with 100 µl, 200 µl and 300 µl of
6-amino-5-bromouracil (stock 18.5 mM) solution, respectively. Group 5 of six
mice received 100 μl of the Cerastes cerastes venom and ASV. The number
of mortality was recorded within 24 h.
Calculation of LD99 of Cerastes cerastes
venom
The median lethal dose (LD99) of Cerastes
cerastes venom was determined according to the previously developed method [14,15]. A range of doses of venom in 800
μl of physiological saline was injected intraperitoneally using groups of six
mice for each venom dose. The LD99 was calculated with the confidence limit at
99% probability by the analysis of mortality occurring within 24 h of venom
injection. The anti-lethal potentials of 6-amino-5-bromouracil (stock 18.5 mM)
solution were determined against LD99 of Cerastes cerastes venom.
STATISTICAL ANALYSIS
The difference among various
treated groups and control group were analysed using one-way-ANOVA followed
using unpaired Student’s t test. The results were expressed as the mean ± SEM
of the number of experiments done, with P<0.05 indicating significant
difference between groups.
RESULTS AND DISCUSSION
Molecular docking
analysis
Table
1 shows the binding energies of
6-amino-5-bromouracil, Gel and cobra-venom phospholipase A2
(1POB) obtained by the molecular docking strategy. In this study, molecular
dockings of the 6-amino-5-bromouracil and Gel with cobra-venom phospholipase A2
(1POB) were performed using Auto Dock 4.2 to investigate the binding mode of
6-amino-5-bromouracil and Gel with cobra-venom phospholipase A2 (1POB)
and to obtain information about interaction forces between
6-amino-5-bromouracil and cobra-venom phospholipase A2 (1POB).
6-Amino-5-bromouracil, gel and cobra-venom phospholipase A2 (1POB)
was kept as flexible molecules and were docked into seven forms of rigid
phospholipase A2 to obtain the preferential binding site to
6-amino-5-bromouracil and gel on phospholipase A2. The molecular
docking results are shown in Table 1.
The
modeling studies showed that there is van der Waals, hydrogen bonding
and electrostatic interactions between 6-amino-5-bromouracil and gel with phospholipase A2. The contribution of van der Waals and
hydrogen bonding interaction is much greater than that of the electrostatic
interaction because the sum of van der Waals energy, hydrogen bonding
energy and desolvation free energy is larger than the electrostatic energy,
which is consistent with the literature [16,17]. The 6-amino-5-bromouracil, gel and cobra-venom
phospholipase A2 (1POB) interactions are shown in Figure 1. 6-Amino-5-bromouracil showed
a good binding energy (-5.52 kcal/mol) when compared to standard gel (-4.26
kcal/mol) as mentioned in Table 1. Figure 1 shows four hydrogen bonds
between 6-amino-5-bromouracil and cobra-venom phospholipase A2 while
gel shows six hydrogen bonds with cobra-venom phospholipase A2. In
addition, 6-amino-5-bromouracil showed good docking interaction with the
cobra-venom phospholipase A2 binding site (GLY31, TYR63 and ASP48) (Figure 1) and similarly Gel showed
good docking interaction with the cobra-venom phospholipase A2
binding site (ARG30, GLY29 and ASP48). The interaction of both ligands
(6-amino-5-bromouracil and Gel) with the cobra-venom phospholipase A2
binding site of the enzyme is essential for effective inhibition as previously
reported for gel [18,19]. Therefore, 6-amino-5-bromouracil and gel may
be considered as the effective phospholipase A2 inhibitor.
Fluorescence spectra and
6-amino-5-bromouracil
Fluorescence spectroscopy has proved to be
helpful in studies on ligand binding. Steady-state fluorescence quenching and
fluorescence polarization are the primary techniques for studying structure and
function of proteins [20]. The fluorescence spectrum shows
a decrease of fluorescence intensity (Figure
2) of the snake venom due to addition of 105 µM of 6-amino-5-bromouracil
which could be related to various processes. It is well known that a decrease
in fluorescence intensity can be caused by a range of molecular interactions
such as molecular rearrangements, excited-state reactions, ground state complex
formation, collisional quenching or energy transfer. The decrease in
fluorescence emission intensity as shown in Figure 2 was not accompanied any shift which may indicate that Trp
residues buried in a hydrophobic environment have moved into a relatively polar
environment consistent with earlier reports [21]. The decrease in fluorescence
emission intensity (Figure 2) was
accompanied was not accompanied by any shift and this may indicate that binding
of 6-amino-5-bromouracil may have accomplished a conformational change that
moves tryptophan into a relatively more hydrophobic region. This explanation is
consistent with Gorbenko et al.
[22] who found tryptophan fluorescence is quenched by interactions with polar
ligands. Binding of proteins to lipid membranes decreases ease of access to
these polar ligands and consequently decreases the obtained quenching effects.
The obtained fluorescence quenching with
6-amino-5-bromouracil refers to the process that decreases the fluorescence
intensity of the snake venom. Snake venom show fluorescence and the intrinsic
protein fluorescence is due to aromatic amino acids, mainly tryptophan,
considering that phenylalanine has a very low quantum yield and emission by
tyrosine in native proteins is often quenched. The fluorescence of the snake
venom is mainly due to tryptophan of phospholipase, which can be selectively
measured by exciting at 295 nm, because there is no absorption by tyrosine at
this wavelength. Tryptophan fluorescence is extremely responsive to the
environment polarity and shifts in its emission spectrum toward slower
wavelengths (blue shift) can be observed as the increased hydrophobicity [23,24]. Changes in emission spectra from
tryptophan can be seen in response to snake venom phospholipase conformational
transitions, subunit association, 6-amino-5-bromouracil binding or
denaturation, which affect the environment surrounding the indole ring of
tryptophane. In addition, the quenching reaction obtained can be used not only
to probe topological features of the phospholipase structures, but also to
follow protein conformation changes that affect accessibility to tryptophan. It
is reported that any treatment of the native protein that involves a change in
the tryptophan environment can be followed by fluorescence quenching (Figure 2) [25,26].
Calculation LD99 of Cerastes
cerastes venom
Lethality data of Cerastes
cerastes venom was calculated. The LD99 of Cerastes cerastes venom
from this study was 5 μg/kg as reported previously [14].
Acute toxicity of Cerastes
cerastes venom and its neutralization by 6-amino-5-bromouracil and anti-venom
The Cerastes cerastes venom
at the dose 5 μg/kg (LD99) produces 100% mortality in mice. The
6-amino-5-bromouracil significantly increases the mean survival time up to 6.3
± 0.23 h. The 6-amino-5-bromouracil when used at the dose of 100 µl (stock 18.5
mM) solution was found to be more effective against Cerastes cerastes venom
(4.8 h) when compared with 6.2 h produced by 300 µl. ASV (polyvalent anti-snake
venom by Haffkine Bio-Pharmaceuticals Company (India)) was found to be more
effective as compared with the aqueous 6-amino-5-bromouracil showing mean
survival of two days for five mice and complete survival of one mouse and was
consistent to our previously published work [14].
The pharmacological effects of Cerastes
cerastes venoms could be classified into three main types, neurotoxic,
hemotoxic [27-29] and cytotoxic.
The main toxin related to these effects is PLA2s, which is
responsible for many pharmacological effects happening in snakebite victims.
The PLA2s is able to operate on pre- or post-synaptic junctions as
antagonist of ion channels and muscarinic or nicotinic receptors to persuade
cruel neurotoxicity such as paralysis and respiratory failure [29,30]. In addition
PLA2s can cause local tissue damage resulting in blistering,
swelling, necrosis, and bruising. In addition it has systemic effects such as
hypovolemic shock; encourage hemostatic and cardiovascular effects as
coagulopathy, hemorrhage and hypotension. It is also reported that PLA2s
are able of triggering severe pain [31,32].
Because phospholipase A2
(PLA2) activity is a significant part of venom toxicity, it has been
required candidate PLA2 inhibitors by directly testing drugs. It
was observed that 6-amino-5-bromouracil when given to the mice after they
received snake venom of Cerastes cerastes venom significantly increased
mean survival time and the results were found to be better when it was used at
higher dose 300 µl (stock 18.5 mM) of 6-amino-5-bromouracil solution. This
could be possible due to interaction of active venom components mainly PLA2
with 6-amino-5-bromouracil which is consistent with the result obtained by
molecular docking.
In the literature there are few
small molecules can inhibit PLA2 of the snake venom and delay the
toxicity of the snake venom which can support the obtained results in this
paper [33,34]. It has been
reported that Varespladib and methyl-varespladib (it’s orally bioavailable
prodrug) were able to delay the effects of twenty eight medically important
snake venoms from six continents but not for long time. Also, it has been
reported that varespladib and methyl-varespladib were able to suppress host
response safely and are more effective against snake venom PLA2
(picomolar concentrations) than against mammalian sPLA2 [11]. They were able
to perform protection against the injurious effects of hemorrhage, hemolysis
and other tissue obliteration [35].
Results obtained by using
6-amino-5-bromouracil are not very astonishing and are in agreement with some
previous studies [36-38], as one
pyrimidine-2,4,6-trione has been published as a promising efficient and
selective inhibitor of cell matrix metalloproteases [37,38]. Other studies
have also revealed the efficiency of pyrimidine-2,4,6-trione derivatives,
mainly one named RO 28-2653 developed by Hoffman-La Roche research group, in
anticancer therapy [39,40]. However,
6-amino-5-bromouracil has not ever been pointed as PLA2 inhibitor or
been used in the neutralization of snake venom enzymes.
CONCLUSION
Rapid development and use of a broad-spectrum
PLA2 inhibitor alone or in combination with other small molecule
inhibitors of snake toxins might fill the dangerous therapeutic gap spanning
pre-referral and hospital setting. There is an urgent need for economical,
stable and effective snakebite treatments that can be used in places where
medical access is limited. 6-Amino-5-bromouracil has ability to delay the
snakebite envenomation make it reasonable candidates for consideration of
clinical trials and warrant further examination by skilled practitioners and
basic researchers in the field of snakebite. Further elaborative work is
necessary for the better understanding of the mechanism of venom inhibition. We
expect that the results presented herein can motivate future efforts in finding
potent pyrimidine-2,4(1H,3H)-dione derivatives that can be used for snake venom
phospholipases A2 inhibition in vivo.
ACKNOWLEDGEMENT
The authors gratefully acknowledge the technical
support and valuable suggestions obtained from Ms. Amira Abdul Gbaj.
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