Toxin Reviews

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Low molecular mass natural and synthetic

inhibitors of snake venom metalloproteinases

Lina María Preciado & Jaime Andrés Pereañez

To cite this article: Lina María Preciado & Jaime Andrés Pereañez (2018) Low molecular mass
natural and synthetic inhibitors of snake venom metalloproteinases, Toxin Reviews, 37:1, 19-26,
DOI: 10.1080/15569543.2017.1309550

To link to this article: https://doi.org/10.1080/15569543.2017.1309550

Published online: 04 Apr 2017.

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Toxin Rev, 2018; 37(1): 19–26

ß 2017 Informa UK Ltd, trading as Taylor & Francis Group. DOI: 10.1080/15569543.2017.1309550

REVIEW ARTICLE

Low molecular mass natural and synthetic inhibitors of snake venom

metalloproteinases
Lina Marı́a Preciado and Jaime Andrés Pereañez

Programa de Ofidismo/Escorpionismo, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Medellı́n, Colombia

Abstract Keywords
Snake venom metalloproteinases (SVMPs) play an important role in local and systemic Inhibitors, snake venom, metalloproteinases,
manifestations of viperid snakebite envenomations. The therapy available has been based on docking, snake bites, envenomations
the intravenous administration of antivenoms; nevertheless, they have a limited efficacy against
local effects provoked by SVMPs. For this reason, it is important to search for alternative venom History
inhibitors, either synthetic or natural, that would complement the action of antivenoms,
particularly regarding neutralization of local tissue damage caused by SVMPs. In this review, Received 19 January 2017
information about low molecular mass synthetic and natural molecules that have been Revised 17 March 2017
reported to be inhibitors of SVMPs is described, focusing on their ability to abrogate the Accepted 18 March 2017
activities of SVMPs and on the molecular mechanisms involved. Published online 31 March 2017

Introduction edema, myonecrosis, dermonecrosis, disruption of hemostasis

Snake venom metalloproteinases (SVMPs) are major compo-
nents of most viperid and some ‘‘colubrid’’ venoms
(Markland & Swenson, 2013). These enzymes are zinc-
dependent metalloproteinases which belong to the family of
‘‘metzincins’’, together with astacins, serralysins, matrix
metalloproteinases (MMPs) and ADAMs (enzymes with a
disintegrin and metalloproteinase domains). With few excep-
tions, these proteinases contain a similar zinc-binding motif in
their catalytic domain, characterized by the sequence
HEXXHXXGXXH, followed by a Met-turn (Bode et al.,
1993). They are large multi-domain proteins that are classi-
fied as P-I, P-II and P-III based on the presence or absence of
non-catalytic ancillary domains that extend beyond the mature
proteinase domain. While P-I (20–30 kDa proteins) has only
a proteinase domain, the P-II SVMPs (30–60 kDa) contain
proteinase and disintegrin domains (Fox & Serrano, 2008).
Furthermore, the P-III SVMPs (60–100 kDa) contain pro-
teinase, disintegrin-like and cysteine-rich domains.
Additionally, several subclasses have been described within
each class, depending on whether they are monomers or homo
or heterodimers, and also on the variable patterns of post-
translational cleavage of several domains (Fox & Serrano,
2010; Moura-da-Silva et al., 2016).
A diverse spectrum of biochemical and biological
activities has been described for the SVMPs, showing
therefore their critical role in the overall toxicity of the
venom, and their functional versatility. These enzymes are
responsible for the local and systemic hemorrhagic activity,
Address for correspondence: Jaime Andrés Pereañez, Facultad de
Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia,
Calle 70 52-21, Medellı́n, Colombia. E-mail: andrespj20@gmail.com
mediated by procoagulant or anticoagulant effects, platelet
aggregation, apoptosis and pro-inflammatory activities (Dı́az
et al., 2005; Gâz Florea et al., 2016; Gutiérrez & Rucavado,
2000; Gutiérrez et al., 2005). The mechanism of action of
hemorrhagic SVMPs has been investigated in various ways,
and there is consensus in that hydrolysis of basement
membrane (BM) components in microvessels is a key step
in this process (Escalante et al., 2011; Herrera et al., 2015).
In addition, they are able to hydrolyze endothelial cell
membrane proteins, such as integrins and cadherins, involved
in cell–matrix and cell–cell adhesion (Gutiérrez et al., 2005).
Moreover, disintegrin-like and cysteine-rich domains contain
exosites that interact with endothelial cell integrins, interfer-
ing with their adhesion to extracellular matrix (Escalante
et al., 2011; Gutiérrez & Rucavado, 2000; Gutiérrez et al.,
2005).
Inhibitors of snake venom metalloproteinases
Local tissue damage, such as hemorrhage, myonecrosis,
dermonecrosis and edema, constitutes one of the most
common and serious consequences of Viperidae snakebite
envenomation. The pathogenesis of these alterations is
induced by different toxins present in the venoms, such as
myotoxic phospholipases A2 and hemorrhagic metalloprotei-
nases (Gutiérrez et al., 1998). The therapy for snakebite
envenomations has been based on the intravenous adminis-
tration of horse and sheep-derived antivenoms (Gutiérrez
et al., 2003). However, it has been demonstrated that
neutralization of local effects by antivenoms is difficult
to achieve, not only due to the variety of components
inducing these alterations, but also to the rapid onset of
these pathological changes (Gutiérrez & Lomonte, 1989).
20 L. M. Preciado & J. A. Pereañez
Thus, it is important to search for alternative venom
inhibitors, either synthetic or natural, that would complement
the action of antivenoms, particularly regarding neutralization
of local tissue damage (Pereañez et al., 2013). In this work,
the main types of low molecular mass molecules that have
been shown to have inhibitory potential on SVMPs will be
summarized. However, natural inhibitors of SVMPs of
proteinaceous nature were not included, since they have
been reviewed in a recent work (Bastos et al., 2016).
Nevertheless, this information is of value as a starting point
for future research in this field.
Phenolic compounds
Many current studies focus on the health benefits of
phytochemicals, especially their antioxidant and antimicrobial
properties (Gyawali & Ibrahim, 2014; Soobrattee et al., 2005).
It is known that these compounds exert preventive activity
against infectious, degenerative diseases, inflammation and
allergies via antioxidant antimicrobial and proteins/enzymes
neutralization/modulation mechanisms (Ozcan et al., 2014).
The ability of some phenolic compounds to inhibit SVMPs
has been tested; as an example, it has been established that
rosmarinic acid (RA) (Figure 1A) inhibits hemorrhagic
activity induced by the whole venoms of Trimeresurus
flavoviridis, Crotalus atrox and Gloydius blomhoffii, or by
purified toxins, like HTb isolated from the venom of C. atrox,
bilitoxin-2 from Agkistrodon bilineatus, Ac1-proteinase from
Deinagkistrodon acutus and HT-1 from Bitis arietans venom.
This compound also prevents degradation of human fibrino-
gen and abrogates type IV collagen hydrolysis by incubation
with the venom in a concentration of 0.5 mg/mL. Moreover,
Figure 1. Phenolic inhibitors. (A) Rosmarinic acid. (B) 5d compound, apigenin derivate. (C) Gallic acid. (D) Isoquercitrin. (E) Myricetin-3-O-
glucoside. (F) Gallocatechin.
Toxin Rev, 2018; 37(1): 19–26
the pathological study of thigh muscles of the ddY strain
white mice showed that RA inhibited hemorrhage and
neutrophil infiltrations (Aung et al., 2010).
On the other hand, Srinivasa et al. (2014) reported the
inhibitory effect of compound 5d (Figure 1B), an apigenin
based molecule, against SVMPs effects in the venom of Echis
carinatus (EC), both in silico and in vivo. This molecule
effectively abrogated EC venom-induced local hemorrhage,
tissue necrosis and myotoxicity in a dose-dependent fashion.
The histopathological study showed effective inhibition of
BM degradation and accumulation of inflammatory leuco-
cytes at the site of venom injection. The compound also
inhibited venom-induced fibrin and fibrinogen degradation.
Molecular docking studies of compound 5d and bothropasin,
a P-III metalloproteinase from the venom of Bothrops
jararaca, showed that the flavone group of compound 5d
was found buried into the active site grove via strong
hydrogen bonding between the hydroxyl group and Glu146 of
hydrophobic pocket that connects with Ile142 and His145 and
other hydrophobic key amino acids, like Val141, Ile168,
Gly170, Pro171, Thr172 and Ile173. Additionally, acyl
bromide tail of compound 5d formed hydrogen bonding
with residues of active site including Thr110, Ile111 and
Gly112 (Srinivasa et al., 2014).
Other studies with phenolic compounds have been carried
out with gallic acid (Figure 1C) against the local toxicity of
Daboia russelli venom and its purified hemorrhagic complex.
Gallic acid inhibited proteolytic activity in vitro and hemor-
rhage, edema-forming and dermo- and myonecrotic activities
of both D. ruselli venom and hemorrhagic complex in in vivo
experiments. In this study, it was demonstrated that the
inhibition was due to gallic acid induced structural changes in
DOI: 10.1080/15569543.2017.1309550
hemorrhagic complex as revealed by alterations in fluores-
cence emission and Circular Dichroism Spectral Studies
(Mahadeswaraswamy et al., 2011).
Moreover, medicinal plant extracts are rich sources of
natural inhibitors and have been shown to antagonize the
activity of some venoms and metalloproteinases. As an
example, isoquercitrin (Figure 1D), myricetin-3-O-glucoside
(Figure 1E) and gallocatechin (Figure 1F), isolated from
lyophilized aqueous extract of Schizolobium parahyba leaves,
neutralized the biological and enzymatic activities of
Bothrops venoms and the isolated metalloproteinase
BjussuMP-I, from B. jararacussu venom. The flavonol
glycoside isoquercitrin was able to inhibit hemorrhage
induced by the venom, but did not show a high efficiency
to inhibit fibrinogenolytic activity. On the contrary, myricetin-
3-O-glucoside showed a potent inhibition of the fibrinogen
proteolytic activity of B. alternatus venom but it did not
inhibit hemorrhage, which may indicate a specific action
against serine proteinase enzymes. On the other hand,
gallocatechin was efficient in neutralizing the hemorrhagic
and fibrinogenolytic activity induced by B. alternatus, B.
jararacussu venom and BjussuMP-I metalloproteinase (Vale
et al., 2011).
Matrix metalloproteinase inhibitors
Matrix metalloproteinases have outgrown the field of extra-
cellular matrix biology and have progressed toward being
important regulatory molecules in cancer and inflammation.
This rise in status was accompanied by the development of
various types of inhibitors aimed at controlling the role of
these enzymes in disease. Although clinical trials with
synthetic inhibitors for the treatment of cancer were disap-
pointing, recent data indicate that the use of selective
Figure 2. MMP inhibitors. (A) Marimastat. (B) Prinomastat. (C) Tanomastat. (D) CGS-270 23A. (E) Batimastat.
Inhibitors of snake venom metalloproteinases 21
inhibitors might lead to new therapies for acute and chronic
inflammatory and vascular diseases (Hu et al., 2007). MMPs
are grouped together with astacins, serralysins and reproly-
sins, including SVMPs and ADAMs, within the superfamily
of ‘‘metzincins’’, characterized by similar structural features
at the catalytic site and a neighboring loop (Bode et al., 1993).
The high degree of structural and functional homology
between SVMPs and their mammalian relatives, MMPs,
suggests that substrate/inhibitor interactions between these
subfamilies can be similar (Howes et al., 2007). Most MMP
inhibitors developed to date consist of a zinc-binding group
(ZBG), which binds the catalytic metal ion, and a peptido-
mimetic backbone that interacts noncovalently with the active
site of the enzyme. Nevertheless, due to the presence the ZBG
in these molecules, a big part of their activity can be
explained for their capacity to chelate zinc. Several of these
synthetic inhibitors have been shown to effectively neutralize
enzymatic and toxic activities of SVMPs (Escalante et al.,
2000; Rucavado et al., 2004).
The effectiveness of matrix metalloproteinases inhibitors
(MMPIs) such as Marimastat (Figure 2A), AG-3340
(Prinomastat) (Figure 2B), Bay-12 9566 (Tanomastat)
(Figure 2C) and CGS-270 23A (Figure 2D) in abrogating
the hemorrhagic and proteolytic activities of whole
E. ocellatus venom and EoVMP2 (a hemorrhagic P-III
metalloproteinase) was demonstrated (Howes et al., 2007).
The MMPIs Marimastat and CGS-270 23A are both
hydroxamic acid derivatives that are designed to exert
broad-range MMP inhibition by mimicking the cleavage site
of MMP (collagen) substrate. On the other hand, AG-3340
and Bay-12 9566 are both designed to be selective inhibitors
of the gelatinases MMP-2 and MMP-9 (Heath & Grochow,
2000; Hidalgo & Eckhardt, 2001; Yip et al., 1999).
These compounds were effective in neutralizing the
22 L. M. Preciado & J. A. Pereañez
hemorrhagic activity of EoVMP2 in murine assays, but only
AG-3340 successfully inhibited the hemorrhagic activity of
whole E. ocellatus venom.
In a similar way, the peptidomimetic Batimastat (BB-94)
(Figure 2E) was assessed to determinate its ability to
neutralize the local effects induced by the purified B. asper
metalloproteinase BaP1 (Escalante et al., 2000) and the
systemic effects induced by the whole venom (Rucavado
et al., 2004). This compound inhibited proteolytic, hemor-
rhagic, dermonecrotic and edema forming activities of this
metalloproteinase when incubated with the enzyme prior
to the assays. When the inhibitor was administered i.m. at
the site of the toxin injection without preincubation, rapidly
after metalloproteinase administration, it totally abrogated
the hemorrhagic and dermonecrotic effects of BaP1.
Nevertheless, inhibition was less effective as the time lapse
between toxin and batimastat injection increased, due to the
extremely rapid development of BaP1-induced local tissue
damage (Escalante et al., 2000). In addition, Batimastat
inhibited lethality when a venom challenge dose of two LD50s
was used by intraperitoneal and intravenous routes, also
inhibited the hemorrhagic effect induced by the venom in
lungs after intravenous injection, and exerted a significant
inhibition of in vitro coagulant and in vivo defibrinogenating
effects of venom. It is suggested that Batimastat may act
with similar mechanism of peptidomimetic inhibitor with a
hydroxamate moiety, which was co-crystalized with BaP1
(Lingott et al., 2009). One of the most important findings of
this work was that in the non-complexed structure, BaP1 had
a four-fold zinc coordination system, the zinc ion being
tetrahedrally coordinated by the Ne2 atoms of three histidines
(His142, His146 and His152) and a catalytic water. In
contrast, in complexed structure the zinc ion was five-fold
coordinated, with nearly perfect square pyramidal coordin-
ation geometry. The Ne2 atoms of His146 and His152 and
both oxygens of hydroxamate moiety of inhibitor, formed the
basal plane of the pyramid, and the Ne2 atom of His142 was
the apex. The zinc ion was slightly displaced toward the apex,
out of the plane of the basal atoms. In addition, the binding of
the inhibitor resulted in the displacement of several water
molecules at the docking site of native BaP1, i.e. the catalytic
Figure 3. Zinc chelating agents. (A) EDTA. (B) o-phenanthroline. (C) TPEN. (D) DTPA. (E) TTD. (F) Clodronate. (G) Doxycycline.
Toxin Rev, 2018; 37(1): 19–26
Wat67. Wat67 has been proposed to play a critical role as the
catalytic nucleophile in SVMPs, attacking the scissile peptide
bond after polarization by the highly conserved Glu143
(Lingott et al., 2009).
These results demonstrate that MMPIs are a promising
source of compounds with the ability to inhibit the local and
systemic effects of SVMP. Nevertheless, MMPIs show
specificity toward SVMPs and their application to the
neutralization SVMP effects may require a synthetic MMPI
mixture, ensuring that a close structural component for each
SVMP is represented (Howes et al., 2007).
Zinc chelating agents
SVMPs are zinc-containing endopeptidases, and are therefore
dependent on the presence of zinc ions at the catalytic site (for
their enzymatic activity) and calcium ions in other molecular
regions (for the structural stability of the enzyme). Chelation
of these metal ions by agents such as EDTA (Figure 3A)
(ethylenediamine-tetraacetic acid) and o-phenanthroline
(Figure 3B) has shown to inhibit SVMP activity in several
studies (Borkow et al., 1997; Howes et al., 2007; Mazzi et al.,
2004; Patiño et al., 2010).
Specific zing chelating agents such as N, N, N0 , N0 -Tetrakis
(2-pyridylmethyl) ethane-1,2-diamine (TPEN) (Figure 3C),
diethylene triamine pentaacetic acid (DTPA) (Figure 3D) and
tetraethylthiuram disulfide (TTD) (Figure 3E) have been
evaluated in the inhibition of hemorrhage and myotoxicity
induced by E. carinatus (ECV) venom. These compounds
completely blocked the hemorrhagic and myotoxic activities
of ECV in a dose dependent manner upon co-injection;
however, only TPEN successfully neutralized hemorrhage and
myotoxicity following independent injection. Histological
examinations revealed that TPEN effectively prevents deg-
radation of dermis and BM surrounding the blood vessels in
mouse skin sections and prevents muscle necrosis and
accumulation of inflammatory cells at the site of ECV
injections. Subsequent studies confirmed, by spectral and
docking experiments, that TPEN has high affinity for Zn2+.
This specificity explains the potent inhibition of ECV
metalloproteinases (ECVMPs) in vitro (IC50: 6.7 mM)
DOI: 10.1080/15569543.2017.1309550
(Nanjaraj Urs et al., 2015). However, the major limitation of
TPEN is its toxicity as it may sequester physiologically
important Zn2+ containing enzymes at higher doses.
Other two groups of molecules that constitute SVMP
inhibitors are biphosphonates and tetracyclines. Several
studies have addressed their inhibition mechanism in various
MMPs, and they have shown that these compounds sequester
the zinc present at the catalytic site of these enzymes, in
addition to other actions related with MMP expression and
activation (Acharya et al., 2004; Teronen et al., 1999). The
effectiveness of the biphosphonate clodronate (Figure 3F) and
the tetracycline doxycycline (Figure 3G) to inhibit proteo-
lytic, hemorrhagic, coagulant and defibrinogenating effects of
B. asper venom was tested. Both compounds were able to
inhibit these activities, at concentrations in the mM range,
when incubated with venom prior to testing. However, when
inhibition of hemorrhage was assessed in assays involving
independent injection of venom and drug, inhibition was poor,
even when these compounds were injected immediately after
envenomation. These findings support the concept that the
effectiveness of compounds, such as clodronate and doxy-
cycline, whose inhibitory action on SVMPs is based on zinc
chelation alone, is limited, and stress the view that more
specific molecules are required for an effective inhibition of
SVMPs in vivo (Rucavado et al., 2008).
Terpenes
A clerodane diterpenoid known as Bt-CD (7alpha-hydroxy-
3,13-clerodadiene-16,15:18,19-diolide) (Figure 4A), isolated
from the aerial parts of the Brazilian plant Baccharis trimera
(Less), has demonstrated full inhibition of the proteolytic and
hemorrhagic activities of several Bothrops venoms.
Additionally, the inhibitor was able to neutralize the
Figure 4. Terpenes informed as SVMPs inhibitors. (A) Bt-CD. (B) Triacontyl p-coumarate. (C) Macrolobin A, R¼CH2OH Macrolobin B, R¼CH3.
(D) Linearol. (E) Isolinearol. (F) Compound ‘‘8’’. A lupeol acetate derivative.
Inhibitors of snake venom metalloproteinases 23
hemorrhagic, fibrinogenolytic and caseinolytic activities of
class P-I and P-III SVMPs isolated from B. neuwiedi and B.
jararacussu venoms. However, this compound failed to inhibit
the coagulant activity and only partially inhibited the edema
induced by crude venoms (Januário et al., 2004). Molecular
dynamics simulations were used to study the binding of
clerodane diterpenoid to the SVMP (Chinnasamy et al.,
2014).
In the same way, the activity of triacontyl p-coumarate
(PCT) (Figure 4B), isolated from the root bark of
Bombacopsis glabra vegetal extract was assayed against
harmful effects of Bothropoides pauloensis snake venom and
the SVMPs BleucMP and jararhagin from B. leucurus and B.
jararaca venoms, respectively. This compound neutralized
fibrinogenolytic activity and plasmatic fibrinogen depletion
induced by B. pauloensis venom and the isolated SVMPs.
PCT also inhibited the hemorrhagic and myotoxic activities
induced by Jararhagin at 1:5 ratio (toxin:inhibitor, w/w) when
previously incubated with PCT or when PCT was adminis-
tered after toxin injection. Docking studies and molecular
dynamic simulations using the structure of a metalloprotei-
nase (Neuwiedase) suggested that the binding between the
protein and the inhibitor occurs mainly in the active site
region causing blockade of the enzymatic reaction by
displacement of catalytic water molecule (Mendes et al.,
2013).
Furthermore, the antiproteolytic and antihemorrhagic
properties of triterpenoid saponins named macrolobin-A and
B (Figure 4C) from Pentaclethra macroloba have been
reported (da Silva et al., 2007). These compounds were able
to significantly inhibit hemorrhagic and proteolytic activities
of Bothrops venoms and isolated BjussuMP-I metalloprotease
from B. jararacussu venom. Nevertheless, the IC50 of
macrolobin A is approximately 0.5 mM for the proteolytic
24 L. M. Preciado & J. A. Pereañez
activity and 1.8 mM for hemorrhage inhibition, showing a
lower potency when compared, for example, with Bt-CD
(IC50 ¼ 0.3 mM and 1.0 mM for each bioactivity) (Januário
et al., 2004) and batimastat (IC50 ¼ 80 nM for the proteolytic
activity and 1.95 mM for hemorrhage inhibition) (Escalante
et al., 2000).
Moreover, the secodolastane diterpenes linearol (Figure
4D) and isolinearol (Figure 4E), isolated from the Brazilian
marine brown alga, Canistrocarpus cervicornis, were eval-
uated against some of the toxic effects induced by B. jararaca
venom. The mixture of diterpenes inhibited proteolysis in a
concentration-dependent manner and abrogated the hemor-
rhagic activity of B. jararaca venom, but failed to inhibit
clotting and edematogenic activities, and thus, would not
prevent inflammation induced by snake venoms (Domingos
et al., 2015).
Although there are few reports of compounds isolated from
natural products in the inhibition of SVMPs-induced local and
systemic effects, given that most of them are limited to
demonstrate the biological activity of extracts or fractions,
studies whit C. cervicornis, P. macroloba and B. trimera have
demonstrated that natural products are a promising alternative
to finding new and chemically diverse inhibitors of SVMPs.
On the other hand, synthetic terpenes derivatives have been
tested in the inhibition of E. carinatus venom-induced
hemorrhage. Lupeol, lupeol acetate and synthetic derivatives
were screened for inhibition of EC venom-induced hemor-
rhage in a mouse model; compound 8 (Figure 4F) was found
to be the most potent. This compound neutralized EC venom-
induced hemorrhage, dermonecrosis and myonecrosis. It also
protected PMNs (polymorphonuclear leukocytes), PBMCs
(peripheral blood mononuclear cells) and platelets from
venom-induced oxidative stress, and offered protection
against venom-induced proteolytic cleavage of integrin
a2b1, GP VI, DDR1 and CX3CR1 receptors present in
these inflammatory cells, hence favoring their firm binding,
which is critical and essential for resolving inflammation and
tissue repair. Docking studies revealed the binding of
compound 8 in the active site pocket of BaP1 (PDB ID:
Figure 5. Other synthetic compounds described as SVMPs inhibitors. (A) 2-Hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8). (B) Glycolic
acid. (C) N-acetylcysteine. (D) 1-(3-Dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzofuran-5-carbonitrile (DFD). (E) N-(Furan-2-
yl)carbonyl-Leu-l-Trp(P)-(OH)2. (F) Phenazopyridine hydrochloride. (G) Nitrofural. (H) Palmatine chloride. (I) Methoxy-6-harmalan.
Toxin Rev, 2018; 37(1): 19–26
2W12), a SVMP of B. asper venom. The theoric binding
energy was –7.24 kJ/mol. The compound showed hydrophobic
interaction with amino acid residues in the active site pocket
region, which comprises 15 residues of the active site of the
enzyme, which are in close proximity to the ligand and are
hydrophobic in nature. Alteration of the spectral properties of
compound 8 by the added Zn2+ further supports its possible
chelating property (Katkar et al., 2015).
Other synthetic inhibitors
Several synthetic quinolinones were tested for its inhibitory
properties against hemorrhage caused by different Bothrops
venoms and isolated SVMPs. The molecule 2-hydroxymethyl-
6-methoxy-1,4-dihydro-4-quinolinone (Q8) (Figure 5A) was
the most effective compound for inhibiting hemorrhagic and/
or proteolytic activities of the crude venoms of B.
jararacussu, B. moojeni and B. alternatus and the isolated
SVMPs BjussuMP-I, from B. jararacussu, and neuwiedase,
from B. neuwiedi. Docking and molecular dynamic simula-
tions demonstrated that Q8 takes part in a regular square
pyramidal coordination geometry of the cofactor Zn2+, which
is completed by the Ne2 atoms from active site His and the
Glu142/Zn2+-coordinated catalytic water molecule, and pre-
sents a root-mean-square deviation (RMSD) value of only
0.312 Å in relation to the ideal geometry of this type of
coordination (Baraldi et al., 2016). Therefore, the experimen-
tal and theoretical data indicate that quinolinones could be
interesting drug-candidate compounds for snakebite
envenoming therapy, particularly for the treatment of local
effects such as hemorrhage.
On the other hand, the simplest a-hydroxy acid, 2-
hydroxyethanoic acid or glycolic acid (Figure 5B) has been
tested in the inhibition of enzymatic, hemorrhagic and edema-
inducing activities of BaP1, a P-I metalloproteinase isolated
from B. asper venom. Glycolic acid inhibited the proteolytic
activity of BaP1 on azocasein with an IC50 of 1.67 mM, and
also the hemorrhagic activity in skin and muscle in experi-
ments involving preincubation of enzyme and inhibitor prior
to injection. Moreover, glycolic acid inhibited, in a
DOI: 10.1080/15569543.2017.1309550
concentration-dependent manner, edema-forming activity of
BaP1 in the mouse footpad test. Molecular docking studies
suggested that glycolic acid forms hydrogen bonds with
residues Glu143, Arg110 and Ala111 of the enzyme.
Additionally, molecular modeling and UV–vis spectra studies
suggested that the inhibitor chelates Zn2+ and may occupy
part of substrate binding cleft of BaP1 (Pereañez et al., 2013).
These results suggest that glycolic acid is a candidate for the
development of metalloproteinase inhibitors, and it is import-
ant to determinate the activity of wide range of a-hydroxy
acids in order to find out their inhibitory potential.
On the other hand, N-acetyl cysteine (NAC) (Figure 5C)
has been shown to inhibit gelatinase, hyaluronidase, hemor-
rhagic and defibrinogenating activities of Vipera russelli and
E. carinatus venoms. NAC inhibited these activities dose
dependently, showed complete inhibition of hemorrhagic
activity when incubated with venom prior to testing, whereas
little inhibition was observed when venom and NAC were
injected independently. Inhibition of the BM degradation and
accumulation of inflammatory leukocytes at the site of venom
injection in histological sections further corroborated the
inhibitory property of NAC. The observed inhibition of
hemorrhage was likely due to zinc chelation as supported by
spectral studies. Further, docking predictions suggested the
role of –SH and –NH–CO–CH3 groups of NAC in the
inhibition of SVMPs. Future studies related to the protective
role of NAC against hemorrhage induced by purified SVMPs
and crude venoms are needed for determining its therapeutic
potential (Sunitha et al., 2011a).
In addition, the inhibitory effect of the synthetic compound
1-(3-dimethylaminopropyl)-1-(4-fluorophe-nyl)-3-oxo-1,3-
dihydroisobenzofuran-5-carbonitrile (DFD) (Figure 5D) on
viper venom-induced hemorrhagic activity has been tested.
This compound is a derivative of citalopram, a clinically
approved antidepressant drug. DFD effectively neutralized the
hemorrhagic activity of medically important viper venoms
such as E. carinatus, E. ocelatus, E. carinatus sochureki, E.
carinatus leakeyi and Crotalus atrox in a dose-dependent
manner with inhibitory concentrations in the mM range.
Histological examinations revealed that the compound DFD
effectively neutralized degradation of the BM, and the
accumulation of inflammatory leucocytes at the site of E.
carinatus venom injection further confirms the inhibition of
hemorrhagic activity. According to the docking studies, DFD
binds to the hydrophobic pocket of SVMP with the ki of
19.26  109 kcal/mol without chelating Zn2+ in the active
site (Sunitha et al., 2011b).
Other synthetic compounds, such as phosphonate analogs
of the peptidomimetic N-(Furan-2-yl) carbonyl-Leu-Trp-OH,
have been tested. N-(Furan-2-yl) carbonyl-Leu-L-Trp(P)-
(OH)2 (Figure 5E) caused a 75-fold increase of potency of
the inhibitor against adamalysin II, with an IC50 of 0.4 mM. In
this study, it was proposed that the phosphonate group binds
the catalytic zinc ion in an asymmetric bidentate mode (2.0
and 2.8 Å Zn–O distances) (D’Alessio et al., 1999).
New strategies such as compound screening and rational
peptide design have been tested with the aim of developing
new inhibitors of SVMPs. Hence, SVMP BaP1 was used as a
model and two different strategies, a virtual screening on the
Prestwick Chemical Library, and rational peptide design,
Inhibitors of snake venom metalloproteinases 25
were carried out (Villalta-Romero et al., 2012). The virtual
screening of the 880 compounds in the Chemical Library was
developed by a fluorogenic method. Consequently, phenazo-
pyridine HCl (5 F) (IC50 54.3 mM), methoxy 6-harmalan (5 G)
(IC50 70.4 mM), nitrofural (5 H) (IC50 88.1 mM) and palmatine
chloride (5I) (IC50 93.3 mM) were the most active compounds
identified. On the other hand, with the rational peptide design,
the hydroxamate-containing peptides were more efficient
inhibitors of BaP1 than the parental peptides, when Phe-Leu-
Phe (FLF) and Phe-Leu-Arg (FLR) sequences were coupled to
hydroxamate, with IC50 values of 0.5 mM and 4.6 mM,
respectively. Molecular docking analysis suggested different
binding modes evidencing backbone flexibility due to the
rotatable bonds. In addition, the best ranking solutions
generated bidentate binding of the hydroxamate group to
zinc, and was also defined that distances below 3.0 Å
generates an optimal binding affinity. Summarizing, this
study demonstrated that the synthesis of peptides with affinity
for the metalloproteinase active site, coupled to ZBGs, offers
promising possibilities. These results should stimulate future
work based on further structural modifications of the
molecules that exerted inhibition of this SVMP (Villalta-
Romero et al., 2012).
Acknowledgements
The authors are thankful to Professor José Marı́a Gutiérrez,
Instituto Clodomiro Picado, for his suggestions and for
improving the text. The authors acknowledge Comité para
el desarrollo de la investigación (CODI-CIQF-217) and
Universidad de Antioquia (UdeA).
Declaration of interest
The authors report no conflicts of interest.
Geolocation information
Our research is performed in Medellı́n, Colombia
(Geographic coordinates: 6 130 5500 N 75 340 0500 O).
ORCID
Jaime Andrés Pereañez http://orcid.org/0000-0001-9612-
2827
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