Toxicon 151 (2018) 96–110

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Phoneutria nigriventer venom: A pharmacological treasure T

a,b b,c,∗ a,∗∗
Steve Peigneur , Maria Elena de Lima , Jan Tytgat
a
Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg, PO Box 922, Herestraat 49, 3000 Leuven, Belgium
b
Laboratório de Venenos e Toxinas Animais, Dept de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo-
Horizonte, MG, Brazil
c
Programa de Pós-graduação em Ciências da Saúde, Biomedicina e Medicina, Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Grupo Santa Casa de Belo
Horizonte, Belo Horizonte, MG, Brazil

A B S T R A C T

In millions of years, spiders have optimized their venoms in order to assure successful prey capture and defence against predators. Spider venoms have become
unique cocktails of biological active components enabling potentially interesting application for drug discovery or for agricultural purposes. The venom of Phoneutria
nigriventer has been studied for over 60 years. This spider is responsible for a high number of envenomations with severe clinical manifestations in humans, which
necessitates a comprehensive knowledge of its venom composition. With over 40 different neurotoxic peptides characterized so far and still many more awaiting
identification, this venom is undoubtedly a pharmacological treasure. This review provides an overview of the Phoneutria nigriventer toxins known today and
describes their mechanism of action at a molecular level. We critically discuss the potential of the Phoneutria nigriventer venom peptides as pharmaceutical tools or
lead compounds for drug development.

1. Spider venom as a source of drug discovery channels. A modified synthetic version of this toxin is the basis of the
approved insecticidal agent SPEAR® (Fitches et al., 2012; Herzig and
Spiders can be considered as one of the most successful venomous King, 2015; King and Hardy, 2013). Recently, exciting research has also
animals ever to habitat the planet. So far over 47000 species have been highlighted the potential of spider venom-based drug discovery. Hi1a, a
characterized (Pineda et al., 2014). According to ArachnoServer, only toxin isolated from the Australian funnel-web spider Hadronyche in-
of approximately 100 species the venom has been studied. This has fensa, was found to be highly neuroprotective in acid-mediated neu-
resulted in 1427 characterized spider venom peptides (Herzig et al., ronal injuries. This toxin inhibits acid-sensing ion channel 1a and
2011; Pineda et al., 2017). Since it is believed that spider venoms hereby strongly attenuates for instance brain damage after stroke
contain over 10 million biologically active peptides, no more than (Chassagnon et al., 2017). Hi1a is thus considered as a lead for devel-
0.01% of spider peptides have thus been studied so far (Escoubas, 2006; opment of therapeutics to protect the brain from ischemic injury.
Klint et al., 2012). Therefore, spider venom can be considered as un-
tapped treasure of biological active compounds that are potentially 2. Phoneutria nigriventer
interesting for drug discovery as well as for the development of bio-
insecticides to control pests. Up to date, no spider peptide-based drug The spiders of the genus Phoneutria are members of the family
has been approved by the Food and Drug Administration. However, one Ctenidae, suborder Labidognatha, and order Araneidae. They inhabit
should take into account the small number of spider venom peptides forests of the neotropical region from Southern Central America (Costa
investigated so far. This is a significant lower number compared for Rica) throughout South America, from the East of the Andes to the
instance with scorpion or snake venom components. Several spider North of Argentina. The Phoneutria spiders are also known as the
venom peptides have been characterized as insecticidal peptides with a “armed spider” because of the characteristic posture they adopt when
strong preference for insect over mammalian targets. Illustrative hereof threatened. When in defence position, the Phoneutria will stand on his
is the spider toxin ω/κ-HXTV-Hv1a. This peptide, isolated from the hind legs while typically raising four frontal legs up high, resulting in
venom of Hadronyche versuta, is a potent inhibitor of insect voltage- an “armed position” (Fig. 1). Another popular name is the “banana
gated calcium (Cav) channels devoid of activity on mammalian Cav spider” which relates to their preference for hiding in banana bunches.

∗
Corresponding author. Programa de Pós-graduação em Ciências da Saúde, Biomedicina e Medicina, Instituto de Ensino e Pesquisa da Santa Casa de Belo
Horizonte, Grupo Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil.
∗∗
Corresponding author. Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg, PO Box 922, Herestraat 49, 3000 Leuven,
Belgium.
E-mail addresses: lima.mariaelena@gmail.com (M.E. de Lima), jan.tytgat@kuleuven.be (J. Tytgat).

https://doi.org/10.1016/j.toxicon.2018.07.008
Received 28 February 2018; Received in revised form 27 June 2018; Accepted 5 July 2018
Available online 10 July 2018
0041-0101/ © 2018 Elsevier Ltd. All rights reserved.
S. Peigneur et al. Toxicon 151 (2018) 96–110

very similar to those observed in humans after the accidents with this
spider. An interesting case report is the envenomation of a 52-year-old
man who suffered from a neck bite by a female specimen of Phoneutria
nigriventer. The patient experienced intense local pain, episodes of vo-
miting, tremors, blurred vision and excessive sweating. After 2 h, the
patient was treated with captopril and meperidine because of a strongly
elevated blood pressure. This treatment allowed stabilization of blood
pressure and heart rate. Nevertheless, tachypnea, gentle shaking, cold
extremities, profuse sweating, generalized tremors, and priapism sub-
sisted. Treatment with antivenom resulted in complete recovery within
1 h (Bucaretchi et al., 2008). This case report nicely demonstrated the
presence of venom components with a strong neurotoxic activity, and
herewith, underlined the potential pharmaceutical applicability of
Phoneutria venom. It is thus of no surprise that the venom of P. ni-
griventer is considered as a pharmacological treasure for drug discovery
for over 60 years now. Indeed, this venom is a complex mixture of
proteins and peptides, including neurotoxins, acting on ion channels
and chemical receptors of the nervous and muscular systems of insects
and mammals (Richardson et al., 2006). Notwithstanding that no
Phoneutria venom-derived peptide has made it to the drug market, the
potential pharmacological applicability of these peptides is evidenced
by the ample research studies using these peptides, not only as potent
ligands for specific targets, but also as tools to have a better under-
standing of their physiological function and their involvement in dis-
eases and channelopathies.
The first studies on P. nigriventer venom, report the presence of
biologically active proteins such as peptides, proteases, and hyalur-
onidase. Furthermore, other active compounds such as histamine ser-
Fig. 1. Phoneutria nigriventer, the “armed spider”. The picture shows the spider otonin and some free amino acids were also identified (de Lima et al.,
assuming the typical defensive or “armed” position. (Photographed by Alcides 2016; Gomez et al., 2002). Early work indicated that the whole venom
Sousa, at Fundacão Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil). exhibits a pronounced neurotoxic activity. Injection of whole or par-
tially fractionized venom caused a myriad of excitatory symptoms in
The genus Phoneutria belongs to the Retrolateral Tibial Apophysis experimental animals (Bucherl, 1953, 1969; Schenberg and Lima,
(RTA) clade, whose adaptive and evolutionary process is associated 1966). These observations in animals corroborated well with the re-
with the loss of cribellate silk and prey-capture webs. They only use silk ports on human envenomation (Bucaretchi et al., 2008; Diniz et al.,
for the production of the sacs in which the eggs hatch or for nursery 1990). The very first biochemical and pharmacological characterization
webs. These are wandering spiders with nocturnal habits. They are of an isolated Phoneutria nigriventer toxin was performed in 1990s by Dr.
active hunters, relying on their fast acting and efficient venom for prey Diniz and colleagues (Rezende et al., 1991) at Fundação Ezequiel Dias
capture and defence. Their natural preys are insects although there are (Belo Horizonte, MG, Brazil). Almost 30 years before, Diniz has been
reports of Phoneutria hunting on other spiders and small rodents as well the pioneer in studying this venom (Diniz, 1963). Nowadays P. ni-
(de Lima et al., 2016; Herzig et al., 2002). griventer venom has been extensively studied making it one of the most
Phoneutria nigriventer are very aggressive, solitary spiders. They are studied spider venoms in the world. Besides some non-protein low-
synanthropic species, explaining the high number of human accidents molecular-mass compounds, 41 neurotoxins have been identified from
occurring with this spider. Human evenomations involving Phoneutria the crude venom up to date (Tables 1 and 2) (Herzig et al., 2011; Pineda
spiders occur mainly in Brazil, but there are reports of sporadic cases in et al., 2017). The P. nigriventer venom (PNV) has also been studied for
Central America and in neighbouring countries (de Lima et al., 2016). its potential ability to cross the blood-brain barrier. This has recently
More recently, the export of bananas to Europe has resulted in certain been very nicely reviewed (Cruz-Hofling et al., 2016). This review
cases of Phoneutria envenomation in countries such as the United shows that certain components of the PNV cause activation in multiple
Kingdom and the Netherlands. Most accidents involving humans are brain areas and upregulate the expression of vascular endothelial
mild with only up to 0.5% of severe cases (Hauke and Herzig, 2017). growth factor (VEGF) and its receptors Fms-like tyrosine kinase1 (Flt-1)
Despite the venom being highly neurotoxic, the amount inoculated and fetal liver kinase 1 (Flk-1). The authors suggested that these data of
through the bite is usually too small to induce lethal effects. However, the PNV mechanism in the CNS can contribute to improving the
fatal envenomation usually occurs after a bite by female species. It is treatment in cases of phoneutrism and, in addition, that PNV could be a
reported that females inject a larger amount of venom compared to potential tool for studies on drug permeability across the BBB.
males. Furthermore, as with most venomous animals, intersexual dif-
ferences in venom composition have been demonstrated (Herzig et al., 3. Nomenclature of Phoneutria nigriventer toxins
2002). The clinical manifestations of severe systemic intoxication are
usually seen in elderly and children. In such cases, the penile erection or The nomenclature of Phoneutria nigriventer toxins is rather confusing
priapism is one of the most noted signs of phoneutrism. Other clinical and problematic. Over time, often several names have been given to the
manifestations often reported after a Phoneutria bite are convulsions, same peptide. Historically, the Phoneutria toxins are annotated based on
agitation, somnolence, nausea, profuse sweating and vomiting, lacri- their occurrence in the venom when following the venom purification
mation, excessive salivation, hypertension, tachycardia, tachypnea, methods used in the first studies (Diniz et al., 1990), i.e. based on a
tremors and spastic paralysis (de Lima et al., 2016; Raposo et al., 2016). particular chromatographic step and in the order of elution of the toxin,
Cases of systemic poisoning in adults are uncommon but may happen. in this step. In an attempt to solve the confusing nomenclature of
The effects observed in experimental animals after venom injection are peptides of spiders and of other animal venoms, King et al. (2008) have
proposed a rational nomenclature, which considers the molecular target

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Table 1
Overview for the toxins isolated from the venom of P. nigriventer from the fractions PhTx1-PhTx4. The toxins are ordered numerically according to the original name. AA: number of amino acid residues; P: protein level;
NA: nucleic acid level. Average masses are shown. Data was obtained from Arachnoserver and Uniprot.
Name Alternative Name Rational Evidence Level Uniprot Mass AA Fraction In vivo Activity Molecular Target & Mode of Action Reference
Nomenclature

PnTx1 Toxin Tx1, PNTx1, PhTx1 μ-ctenitoxin-Pn1a P & NA P17727 8597.7 78 PhTx1 LD50 = 5.5 pmol/g (mice) Nav channels, Antagonist Diniz et al., 1990
PnTx2-1 Tx2-1, Tx2-1, PNTx2-1 U2-ctenitoxin-Pn1a P & NA P29423 5841.7 53 PhTx2 Lethal to mice (0.02 pmol/g) Unknown Cordeiro et al., 1992
PnTx2-1A Pn2-1A, U3-ctenitoxin-Pn1a, Pn2-1A U2-ctenitoxin-Pn1b P & NA O76198 6070.0 54 PhTx2 n.a. Unknown Kalapothakis et al.,
1998a,b
PnTx2-5 Tx2-5, PNTx2-5, U4-ctenitoxin-Pn1a δ-ctenitoxin-Pn2b P P29424 5100.9 49 PhTx3 Lethal to mice (2.4 pmol/g) Nav channels, Agonist Rezende et al., 1991
PnTx2-5A Pn2-5A, U4-ctenitoxin-Pn1b δ-ctenitoxin-Pn2c NA O76199 5112.8 48 PhTx2 n.a. Unknown Kalapothakis et al.,
1998a,b
PnTx2-6 Tx2-6, PNTx2-6 δ-ctenitoxin-Pn2a P & NA P29425 5288.2 48 PhTx2 Lethal to mice (7.5 pmol/g) Nav channels, Agonist Cordeiro et al., 1992
PnTx2-9 Neurotoxin Tx2-9, Tx2-9, PNTx2-9 U5-ctenitoxin-Pn1a P P29426 3736.5 32 PhTx2 n.a. Unknown Cordeiro et al., 1992
PnTx3 U26-ctenitoxin-Pn1a, Tx3 U7-ctenitoxin-Pn1b P P31010 unknown n.a. PhTx3 LD50 = 21.9 pmol/g (mice) Unknown Rezende et al., 1991
ED50 = 6.4 pmol/g (mice)
PnTx3A Pn3A U6-ctenitoxin-Pn1a P & NA P81793 3771.4 34 PhTx3 n.a. Unknown Cardoso et al., 2003
PnTx3-1 Tx3-1, PNTx3-1 κ-ctenitoxin-Pn1a P & NA O76200 4575.2 40 PhTx3 Paralysis in mice (0.07 pmol/g) K+ channels, Agonist Cordeiro et al., 1993

98
PnTx3-2 Tx3-2, PNTx3-2 ω-ctenitoxin-Pn1a P & NA O76201 3533.2 34 PhTx3 Paralysis in mice (0.08 pmol/g) L-type Cav channels, Antagonist Cordeiro et al., 1993
PnTx3-3 Tx3-3, PNTx3-3, Omega-PnTx3-3 ω-ctenitoxin-Pn2a P P81789 unknown n.a. PhTx3 Lethal to mice (0.07 pmol/g) L-, P/Q- and R-type Cav channels, Cordeiro et al., 1993
Antagonist
PnTx3-3A Pn3-3A U9-ctenitoxin-Pn1a NA P0C2S6 3873.3 36 PhTx3 n.a. Unknown Kalapothakis et al.,
1998a,b
PnTx3-4 PNTx3-4 ω-ctenitoxin-Pn3a P & NA P81789 8449.6 76 PhTx3 Lethal to mice (5 μg/mouse) N-, P/Q- and R-type Cav channels, Cordeiro et al., 1993
Antagonist
PnTx3-4 PnTx3-4 isoform, Pn3-4A, U8- ω-ctenitoxin-Pn3b P P81789 8332.5 70 PhTx3 n.a. N-, P/Q- and R-type Cav channels Cardoso et al., 2003
ctenitoxin-Pn1a
PnTx3-4 PnTx3-4 isoform, ω-Phonetoxin 2A ω-ctenitoxin-Pn3c P P81789 8362.5 70 PhTx3 n.a. N-, P/Q- and R-type Cav channels Cassola et al., 1998
PnTx3-4 PnTx3-4 isoform, phoneutriatoxin 3-4 ω-ctenitoxin-Pn3d P P81789 8458.5 70 PhTx3 n.a. N-, P/Q- and R-type Cav channels Cordeiro et al., 1993
PnTx3-5 PnTx3-5, Tx3-5, PNTx3-5 U7-ctenitoxin-Pn1a P & NA P81791 4145.8 45 PhTx3 Paralysis in mice (0.07 pmol/g) L-type Cav channels Cordeiro et al., 1993
PnTx3-6 Tx3-6, PNTx3-6, Phα-1β toxin ω-ctenitoxin-Pn4a P & NA P81792 6032.9 55 PhTx3 Paralysis in mice (0.05 pmol/g) N-, P/Q- and L-type Cav channels Cordeiro et al., 1993
PnTx4-3 PNTx4-3 δ-ctenitoxin-Pn1b P P84034 5199.9 48 PhTx4 Non-toxic to mice (288.5 pmol/g) Unknown Oliveira et al., 2003
LD50 = 192.3 pmol/g (house fly)
PnTx4 (5-5) Tx4 (5-5), Pn4A, PNTx4 (5-5) Γ-ctenitoxin-Pn1a P & NA P59367 5174.8 47 PhTx4 Non-toxic to mice (290 pmol/g) NMDAR (Antagonist), Nav channels Penaforte et al., 2000
(Agonist)
LD50 = 9.3 ng/house fly
PnTx4 (6-1) PNTx4 (6-1), Tx4 (6-1) δ-ctenitoxin-Pn1a P & NA P59368 5241.1 48 PhTx4 Non-toxic to mice (286.2 pmol/g) Nav channels, Agonist Figueiredo et al., 1995
ED50 = 36.3 pmol/g (house fly)
Toxicon 151 (2018) 96–110
S. Peigneur et al. Toxicon 151 (2018) 96–110

Table 2
Overview for the toxins isolated from the venom of P. nigriventer from the fractions PhTx5. The toxins are ordered numerically according to the original name. AA:
number of amino acid residues; P: protein level; NA: nucleic acid level. Average masses are shown. PhM refers to the original name given to PhTx5Data was obtained
from Arachnoserver and Uniprot.
Name Alternative Name Rational Nomenclature Evidence Level Uniprot Mass AA In vivo Activity Reference

Pn3-5A U10-ctenitoxin-Pn1a NA P0C2S9 4705.3 39 n.a. Cardoso et al., 2003

Pn3-6A U11-ctenitoxin-Pn1a NA P0C2S7 6589.4 58 n.a. Cardoso et al., 2003
PNTx22C3 U11-ctenitoxin-Pn1b P P84011 6571.3 58 Non-toxic to mice Richardson et al., 2006
Pn3-6B U12-ctenitoxin-Pn1a NA P0C2S8 6370.3 58 n.a. Cardoso et al., 2003
PNTx13C3 U13-ctenitoxin-Pn1a P P83894 3552.1 32 n.a. Richardson et al., 2006
PNTx24A0C3 U13-ctenitoxin-Pn1b P P84017 3481.0 31 n.a. Richardson et al., 2006
PNTx24A0C4 U13-ctenitoxin-Pn1c P P84018 3679.3 33 n.a. Richardson et al., 2006
PNTx22A0C1 U14-ctenitoxin-Pn1a P P83998 4079.9 35 Non-toxic to mice and housefly Richardson et al., 2006
PNTx27C4 U17-ctenitoxin-Pn1a P P83996 4062.8 36 Lethal in mice (3.0 μg/mouse) Richardson et al., 2006
PNTx30C3 U18-ctenitoxin-Pn1a P P83999 7876.6 n.a. Lethal in mice (3.0 μg/mouse) Richardson et al., 2006
PNTx16C1 U19-ctenitoxin-Pn1a P P83997 7577.4 68 n.a. Richardson et al., 2006
Pn4B U1-ctenitoxin-Pn1a NA P61229 5815.4 47 n.a. Penaforte et al., 2000
PNTx22C5 U20-ctenitoxin-Pn1a P P84093 8855.0 80 Lethal in mice (3.0 μg/mouse) Richardson et al., 2006
PN47 U21-ctenitoxin-Pn1a P P84033 22089.0 245 n.a. Richardson et al., 2006
PN10C5 U23-ctenitoxin-Pn1a P P84015 3672.7 33 n.a. Richardson et al., 2006
PN16C3 U24-ctenitoxin-Pn1a P P84032 14775.7 128 n.a. Richardson et al., 2006
PnV2 U27-ctenitoxin-Pn1a P Q9TWR5 unknown n.a. n.a. Marangoni et al., 1993
PnV1 U28-ctenitoxin-Pn1a P Q7M3P1 unknown n.a. n.a. Richardson et al., 2006
PnTkP-I tachykinin peptide-I U29-ctenitoxin-Pn1a P P86298 872.0 7 n.a. Pimenta et al., 2005
PnTkP-II tachykinin peptide-II U29-ctenitoxin-Pn1b P P86299 1000.0 8 n.a. Pimenta et al., 2005
PnTkP-III tachykinin peptide-III U29-ctenitoxin-Pn1c P P86300 1028.0 8 n.a. Pimenta et al., 2005
PnTkP-IV tachykinin peptide-IV U29-ctenitoxin-Pn1d P P86301 1147.3 9 n.a. Pimenta et al., 2005
PnTkP-V tachykinin peptide-V U29-ctenitoxin-Pn1e P P86302 1175.4 8 n.a. Pimenta et al., 2005
PnTkP-VI tachykinin peptide-VI U29-ctenitoxin-Pn1f P P86303 1217.5 10 n.a. Pimenta et al., 2005
PnTkP-VII tachykinin peptide-VII U29-ctenitoxin-Pn1g P P86304 1232.8 10 n.a. Pimenta et al., 2005
PnTkP-VIII tachykinin peptide-VIII U29-ctenitoxin-Pn1h P P86305 1337.9 10 n.a. Pimenta et al., 2005
PnTkP-IX tachykinin peptide-IX U29-ctenitoxin-Pn1i P P86306 1509.9 12 n.a. Pimenta et al., 2005
PnTkP-X tachykinin peptide-X U29-ctenitoxin-Pn1j P P86307 1610.1 13 n.a. Pimenta et al., 2005
PnTkP-XI tachykinin peptide-XI U29-ctenitoxin-Pn1k P P86308 1623.9 13 n.a. Pimenta et al., 2005
PnTkP-XII tachykinin peptide-XII U29-ctenitoxin-Pn1l P P86309 1626.4 13 n.a. Pimenta et al., 2005
PnTkP-XIII tachykinin peptide-XIII U29-ctenitoxin-Pn1m P P86310 1637.9 13 n.a. Pimenta et al., 2005
PnTkP-XIV tachykinin peptide-XIV U29-ctenitoxin-Pn1n P P86311 1653.8 13 n.a. Pimenta et al., 2005
PnTkP-XV tachykinin peptide-XVI U29-ctenitoxin-Pn1o P P86297 1653.9 14 n.a. Pimenta et al., 2005
PhM1 tachykinin peptide PhM1 U29-ctenitoxin-Pn1p P n.a. 1346.6 11 n.a. Pimenta et al., 2005

(including sub-types) of the toxin and also the family, genus and species besides acting on insect sodium channels (de Lima et al., 2002), also can
of the animal from which the toxin was obtained. As an example, the act on mammal sodium channels (Paiva et al., 2016; Silva et al., 2015a),
toxin originally named Tx2-6/PnTx2-6 from P.nigriventer, is also an- although no apparent toxicity was observed when injecting (i.c.) these
notated, following the new nomenclature as: δ-CNTX-Pn2a (delta molecules in rat brain. The fifth fraction (PhM), apparently not toxic to
meaning its modulating activity on Nav inactivation, CNTX = Cteni- mammals, is active on smooth muscle, causing contraction (Rezende
toxin from Ctenidae family; Pn = Phoneutria nigriventer, = isoform). et al., 1991).The average LD50 by i.c. injection in mice for the whole
Some efforts have been done to adopt the new nomenclature of King. venom, PhTx1, PhTx2, PhTx3, and PhTx4, was 47, 45, 1.7, 137, and
However, in general with Phoneutria venom peptides, the name given to 480 μg/kg, respectively (Rezende et al., 1991). PhTx2 is the fraction
the toxin in the first publication has become the common used name of which displayed the strongest neurotoxic activity while PhTx5 or PhM
the toxin. Therefore, in this review we adopted the name as found in the (15 mg/kg, i.v. injection) has no lethal effect in mice (Rezende et al.,
respective publications. The corresponding names suggested by King's 1991). Fig. 2 shows a flow diagram describing the purification of
nomenclature can be easily consulted in ArachoServer and in Tables 1 Phoneutria nigriventer venom fractions.
and 2 Since the venom is a complex mixture, an efficient purification
method was needed. Therefore, an activity-guided purification and
4. PhTx1
fractionation procedure, using gel filtration and reversed-phase chro-
matography had to be developed (Diniz et al., 1990). Following this
The fraction PhTx1 is constituted by just one peptide, initially called
procedure typically generated five distinct venom fractions. Each
Toxin 1 or Tx1. Afterwards, it was given the name Phoneutria nigriventer
fraction showed different activity when assayed in mammals and/or
Toxin 1 or PnTx1. This peptide represents 0.45% of the whole venom
insects, suggesting the presence of toxins with different pharmacolo-
protein content and it was the first purified and sequenced neurotoxin
gical properties (Figueiredo et al., 1995; Rezende et al., 1991). For most
from P. nigriventer venom (Diniz et al., 1990). PnTx1 has a molecular
of the initial studies, toxicity was evaluated in vivo by intracerebral (i.c.)
mass of 8594.6 Da and is constituted of 78 amino acid residues, 14 of
or intrathoracic injections in mice and insects, respectively, and in vitro
which are cysteines (Fig. 3). It is thus a fairly large peptide with a
by smooth muscle assays using guinea pig ileum, besides some assays in
complex disulfide bridge pattern that remains undetermined till today.
synaptosomes from rat brain. Using this activity guided purification
Early research indicated that PnTx1 induces excitation and spastic
method, four distinct venom fractions, named PhTx1, PhTx2, PhTx3,
paralysis in mice upon i.c. injection (Rezende et al., 1991). PnTx1 is
and PhTx4 were identified. PhTx1, PhTx2, and PhTx3 are active on
lethal to rodents and has an astonishing LD50 of 5.5 pmol/g in mice
mammals and differ in their lethality and effects in mice (Rezende et al.,
(Diniz et al., 1993, 2006; Klint et al., 2012). Nevertheless, the exact
1991). PhTx4 produces marked stimulatory effects in insects and is
molecular target of this toxin remained unknown for many years. In-
more toxic to insects than to mammals (Figueiredo et al., 1995). More
itially, it was suggested that PnTx1 could act on calcium channels be-
recently, it was shown that some toxins from the PhTx4 fraction,
cause radio-iodinated 125I-PnTx1 showed partial competition with the

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S. Peigneur et al. Toxicon 151 (2018) 96–110

Fig. 2. Upper panel: Flowchart showing the purification procedure of Phoneutria nigriventer venom fractions. Adapted from de Lima et al., 2016). Lower panel: Elution
profile of reversed-phase (RP-HPLC) fractionation of Phoneutria nigriventer venom. Venom sample was loaded on a preparative Vydac C4 column (2.2 × 25 cm).
Column was eluted at a flow rate of 5 mL/min, monitored at 214 nm, under a gradient of acetonitrile (Richardson et al., 2006). The solid bars indicate the eluted
fractions and (N) nigriventrine. Figure adapted from de Lima et al., 2016.

Fig. 3. Sequence of PnTx1. Cysteines are indicated in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version
of this article.)

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fraction PhTx3, which contains several calcium channel modulating interaction site for local anesthetics or for pyrethroids (Martin-Moutot
peptides. However, labeled PnTx1 did not compete with ω-conotoxin et al., 2006). Furthermore, using mutated Nav1.2 channels devoid of
GVIA, a calcium channel inhibiting toxin isolated from cone snail fast inactivation, it was shown that PnTx1 does not stabilize the
venom (Santos et al., 1999). This rather contrary result was later at- channels in the inactivated state (Silva et al., 2012). It thus can be
tributed to the presence of a small contamination with PnTx3-3. PnTx3- concluded that the PnTx1 induced sodium current inhibition is not a
3 is a well-characterized toxin that potently inhibits high-voltage-acti- result of modified gating and open time probability but rather from a
vated Cav channels, but not low-voltage-activated Cav channels (Leao physical obstruction of the sodium ion pathway through the Nav
et al., 2000). This contamination could explain the partial competition channel. The result of PnTx1 binding is thus a reduction of the single
for Cav channel inhibition (Martin-Moutot et al., 2006). However, the channel conductance.
question whether or not PnTx1 interacts with Cav channels can still not PnTx1 does not inhibit insect Nav channels. Even at higher con-
be answered unambiguously. Experiments using a highly purified centrations, PnTx1 failed to decrease the current peak amplitude of
PnTx1 showed that this toxin partially displaced the calcium-antagonist arthropod Nav channel isoforms such as from the fruit fly Drosophila
dihydropyridine derivative 3H-PN200-110 in GH3 cell membranes. melanogaster (DmNav1), the cockroach Blattella germanica (BgNav1.1)
Furthermore, a 50% inhibition of the calcium influx in GH3 cells was and the mite Varroa destructor (VdNav1). This inactivity on arthropod
observed after application of 1 μM PnTx1 (Santos et al., 1999). On the Nav channels corroborates well with previous studies showing a lack of
other hand, experiments with the recombinant PnTx1 (rPnTx1) showed insecticidal activity of venom fraction PhTx1, when injected in insects
no modification in the calcium currents of dorsal root ganglia (DRG) (Santos et al., 1999).
neurons (Silva et al., 2012). One explanation for these seeming con- Curiously, both native and recombinant toxins were not able to
tradictorily observations could be that PnTx1 does interact with Cav block 100% of the Nav1.2 currents, reaching a maximal inhibition of
channels but only selectively with a certain specific subtype of Cav approximately 85% of the sodium peak current, even at saturating
channels. Nonetheless, PnTx1 awaits further investigation in order to conditions. Subsequent addition of TTX results in completely inhibited
elucidate its Cav channel interaction. sodium channels indicating that TTX can still reach and bind its site of
Using similar competitive binding experiments it was shown that interaction, independent of the presence of PnTx1 (Silva et al., 2012).
125
I-PnTx1 partially blocked the PhTx2 induced activity in myenteric Since these experiments were performed in Xenopus oocytes hetero-
plexus-longitudinal muscle preparations (Santos et al., 1999). Since logously expressing Nav channels it was concluded that PnTx1 in-
PhTX2 is the fraction that contains mainly sodium channel toxins, this completely inhibits the sodium conductance through the channel, a
observation hinted towards an activity of PnTx1 on Nav channels. In- phenomenon shared with the μ-conotoxins (French et al., 2010; Silva
terestingly, 125I-PnTx1 did not compete with PnTx2-6, a toxin that et al., 2012).
modulates the inactivation process of Nav channels (Silva et al., 2015a). Moreover, an interesting sequence comparison can be made be-
The authors consequently concluded that PnTx1 might be interacting tween a central segment of PnTx1 and μ-conotoxins such as GIIIA and
with Nav channels via another binding site on the Nav channel (Santos KIIIA. In this way, the amino acids W33, R35 and K39 of PnTx1 can be
et al., 1999). Later on, these conclusions were shown to be correct when aligned with the similar amino acids W8, R10 and R14, present in the μ-
it was demonstrated that PnTx1 competes with μ-conotoxin GIIIA conotoxin KIIIA (Khoo et al., 2011; Silva et al., 2012; Zhang et al.,
(Martin-Moutot et al., 2006). μ-conotoxins are small peptides isolated 2010). μ-conotoxins have been intensively studied and for several of
from cone snail venom that inhibit Nav channels with great potency them are the key residues determined (Prashanth et al., 2014; Zhang
and selectivity (Green et al., 2014; Green and Olivera, 2016; Prashanth et al., 2010). Single mutations can alter significantly the selectivity of a
et al., 2014). They are known to bind a micro site within the neurotoxin toxin. The substitution of tryptophan at position 8 by arginine de-
binding site 1 of the Nav channel (French et al., 2010). It is interesting creased affinity of μ-conotoxin KIIIA for Nav1.2 subtype, making it
to note that PnTx1 competes with μ-conotoxins for site 1 but not with more selective to Nav1.4 (Van Der Haegen et al., 2011). There are three
the small compound Nav channel inhibitor tetrodotoxin (TTX). Using basic amino acids conserved in μ-conotoxin GIIIA that are putative key
the patch-clamp technique and Chinese hamster ovary cells expressing residues for the interaction with Nav channels: R13, K16, and K19.
Nav1.2 channels, PnTx1 was characterized as a state-dependent PnTx1 has basic residues in two correspondent positions, R35 (instead
channel inhibitor, preferring to interact with the Nav channels in the K16) and K39 (corresponding to K19). However, PnTx1 lacks the first
open state (Martin-Moutot et al., 2006). arginine (R13) and has a glycine (G32) in the corresponding position.
The recombinant PnTx1 was expressed in a bacterial heterologous Arginine-13 was postulated to be a general residue for μ-conotoxins to
system and inhibited a variety of sodium channel isoforms expressed in interact with the receptor site of sodium channels. This residue is par-
Xenopus laevis oocytes (Fig. 4) and native sodium channels in DRG ticularly critical, since it is believed to compete with the guanidinium
neurons (Silva et al., 2012). Surprisingly, recombinant toxin displayed a group of TTX or STX for the binding site 1. The toxin binding sites of
3-fold lower IC50 value compared to native peptide. This could possibly sodium channels were classified based on their ability to compete with
be explained by the presence of three additional amino acids in the other toxins in binding experiments. Site 1 is the binding site of TTX
sequence of the recombinant produced toxin, namely alanine and me- and STX and toxins that can displace them, such as μ-conotoxin GIIIA.
thionine at the N-terminus and a glycine at the C-terminus. The re- Since PnTx1 competes with μ-conotoxin GIIIA but not with TTX, it
combinant toxin, rPnTx1, inhibited mammalian Nav channel isoforms would be more appropriate to consider it as a macro site 1 instead (de
with the following order of potency: rNav1.2 > rNav1.7 rNav1.4 Lima et al., 2016).
rNav1.3 > mNav1.6 hNav1.8 with no effect on Nav1.5 (Silva et al., Based on all information to date, one hypothesis is a mechanism of
2012). Accordingly, rPnTx1 was less effective on TTX-resistant sodium action where PnTx1 binds to the outer mouth of the channel pore, at a
channels in DRG neurons (Silva et al., 2012). From a drug development site similar or at least partially overlapping with the μ-conotoxin
point of view, it is interesting to note that PnTx1 does not inhibit the binding site within neurotoxin binding site 1. Upon binding, PnTx1
cardiac isoform Nav1.5. Indeed, such a significant selectivity towards hinders the flow of sodium ions. Presumably, the inhibition is a result of
neuronal sodium channels is considered appealing as this avoids un- both physical occlusion and repulsion of positive charges, similar to
wanted cardiovascular side effects. Inhibition of Nav channels occurred what has been proposed for the μ-conotoxins (French et al., 2010).
without modification of the biophysical properties of the channels since Therefore, the likely mechanism of action of PnTx1 would be the re-
no alterations of the voltage dependence of activation and steady-state duction of the unitary conductance of the channel, similarly to what is
inactivation were noted (Martin-Moutot et al., 2006; Silva et al., 2012). seen for the mutated toxin μ-conotoxin GIIIA (R13Q) (Becker et al.,
Competitive binding studies have shown that PnTx1 does not compete 1992).
with toxins acting on site 2, 3, 4, 5. Nor does PnTx1 bind to the Spider venom peptides targeting Nav channels (NaSpTxs) have been

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Fig. 4. Effect of rPnTx1 on different subtypes of sodium channels expressed in oocytes. Representative records of Na + currents before (black line) and after (gray
line and *) the addition of 1 μM rPnTx1. Dashed line is the baseline. Holding potential: −90 mV. Test potential: 0 mV. (Inset) Average percentage of Na + current
inhibition by rPnTx1 (1 μM) of different sodium channel subtypes expressed in oocytes. Nav1.2 inhibition was significantly higher when compared with Nav1.3,
Nav1.4 and Nav1.7 and these were higher when compared with Nav1.6 and Nav1.8. No effect was observed for Nav1.5 and for the arthropod isoforms (DmNav1,
BgNav1.1a and VdNav1). The symbols (*), (**) and (***) denote the isoforms on which the toxin effects were not statistically different. (*) indicates that PnTx1
inhibits Nav1.3, Nav1.4 and Nav1.7 with a similar affinity. (**) indicates that Nav1.6 and Nav1.8 are inhibited with a similar affinity. (***) indicates that Nav1.5,
DmNav1, BgNav1 and VdNav1 are not inhibited by PnTx1. Figure adapted from Silva et al. (2012).

divided into 12 families based on sequence identity and intercysteine 5. PhTx2

spacing (Klint et al., 2012). PnTx1 is the representative of family 8
which constitutes the family of the longest NaSpTxs. So far, no struc- The fraction PhTx2 was found to be toxic to both mice and insects
ture, or even disulfide bridge pattern for that matter, has been de- (Figueiredo et al., 1995). Intracerebral injection in mice resulted in
termined for this structural family. Moreover, none of the Phoneutria priapism, salivation, convulsions and spastic paralysis of the anterior
nigriventer peptides has its structure determined so far, emphasizing the and posterior limbs among other manifestations (Rezende et al., 1991).
challenges, and herewith the opportunities from a structural point of Using a frog skeletal muscle preparation, it was shown that fraction
view, to be found in the Phoneutria venom. This is mainly due to peptide PhTx2 alters the Nav channel kinetics. PhTx2 caused a shift in the ac-
scarcity since only small amounts can be isolated from the venom and tivation and steady-state inactivation curves, a slowing down of the
due to the peptide size and complexity. Therefore, many structure- channel inactivation and a partial inhibition of the sodium peak cur-
function questions about PnTx1 remain open, which makes it an at- rent. The same experiments indicated that PhTx2 does not affect the
tractive object for future studies. Elucidation of the structure of PnTx1 potassium current (Araujo et al., 1993). Phoneutria.
would greatly enhance the understanding on how this toxin exerts its Purification of fraction PhTx2 resulted in 9 peptides which were
interesting pharmacology. named PnTx2-1 till PnTx2-9 (Cordeiro et al., 1992). All peptides were
investigated for their toxicity by i.c. injection in mice. PnTx2-2, PnTx2-
3, PnTx2-4, PnTx2-7, and PnTx2-8 showed low toxicity and these
peptides still await justification for their classification as neurotoxin.

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Fig. 5. Sequences of the toxins identified in Phoneutria nigriventer venom fraction 2. Cysteines are indicated in red. (For interpretation of the references to colour in
this figure legend, the reader is referred to the Web version of this article.)

PnTx2-1, PnTx2-5, and PnTx2-6 share up to 77% identity with each with the β-scorpion toxin CssIV (from Centruroides suffusus suffusus) and
other (Fig. 5) and all 3 toxins elicit neurotoxic effects when injected it thus seems unlikely that PnTx2-6 binds to same site as β-scorpion
into mice (Table 1) (Cordeiro et al., 1992). Pruritus, lacrimation, in- toxin (Matavel et al., 2009). As such, structure-function studies are
creased salivation, sweating, and agitation followed by spastic paralysis required to pinpoint which toxin-channel interactions are responsible
of the limbs was observed. PnTx2-9, was much less toxic to mice, for the PnTx2-6 induced channel modulations.
causing pruritus, reduction in motility and tail erection. PnTx2-9 differs The effect of PnTx2-6 on spontaneous penile erection is remarkable
in sequence from the other peptides of PhTx2. It is a rather short and opens perspectives for clinical applications. In a first step to further
peptide of 32 residues with 3 disulfide bridges. Although annotated as a investigate the exact role of PnTx2-6 in erectile function, the toxin was
Nav channel toxin, no functional data is available to justify this clas- expressed recombinantly in E. coli cells (Torres et al., 2010). The re-
sification. combinant toxin was able to produce erection, similar to native toxin
PnTx2-1 remains scarcely studied. The in vivo tests in mice and (Torres et al., 2010). The mechanism of action through which PnTx2-5
based on the sequence homology with PnTx2-5 and PnTx2-6, it can be and PnTx2-6 promote cavernosal relaxation and enhance erectile
assumed that this peptide also targets Nav channels. However, func- function is not completely clarified (Nunes et al., 2010, 2012a;
tional studies are required to investigate the subtype selectivity and Yonamine et al., 2004). However, intensive research in recent years has
species specificity of PnTx2-1 (Richardson et al., 2006). provided a better understanding on how these toxins influence erectile
Among all the polypeptides purified from PhTx2 fraction, PnTx2-5 function (Nunes et al., 2012b). It is suggested that PnTx2-6 intervenes
and PnTx2-6 are the most studied toxins. They have high sequence in the nitric oxide (NO)/cyclic GMP pathway by increasing the release
homology, differing only in five amino acid residues (Cordeiro et al., of NO in the corpus cavernosum tissue (Nunes et al., 2008). The release
1992). Both toxins were shown to modulate sodium channel kinetics by of NO from penile endothelial cells or nitrergic nerves is a key regulator
slowing down the inactivation process and by shifting the voltage de- in erectile function. Increased NO concentrations, triggered by sexual
pendence of activation towards more hyperpolarized potentials. Both stimulation, induces relaxation of penile smooth muscle. This relaxation
peptides were found to induce a painful and persistent penile erection, results in an increased blood flow and intracavernosal pressure which
also known as priapism which is a common clinical manifestation upon leads to penile erection. The PnTx2-6 induced relaxation is neuronal
Phoneutria nigriventer envenomation. Interestingly, despite the high nitric oxide synthase (nNOS) depended (Nunes et al., 2012b). The ac-
homology between both peptides, significant differences in potency tivation of nNOS by PnTx2-6 is most likely indirect. Since PnTx2-6 is
have been reported. PnTx2-5 displayed an approximately 6-fold lower characterized as a modulator of Nav channel inactivation, the binding
potency than PnTx2-6 in electrophysiological assays (Matavel et al., of PnTx2-6 to Nav channels will result in an increased sodium influx.
2002, 2009; Nunes et al., 2010). This will trigger a cascade of cellular reactions, eventually resulting in
Using frog skeletal muscle, an in-depth characterization of PnTx2-6 an activation of nNOS and consequently stimulation of NO release. This
on a molecular level was performed. PnTx2-6 induces a plethora of mechanism of action was supported by the observation that ω-con-
modifications of channel kinetics. PnTx2-6 reduces the sodium peak otoxin GVIA, an inhibitor of N-type calcium channels, could block the
current, slows down the time constant for fast inactivation and causes a relaxation induced by PnTx2-6. Additionally, it was shown that the
hyperpolarizing shift of the voltage dependence of both the activation cavernosal relaxation provoked by PnTx2-6 is not dependent on phos-
and steady-state inactivation curves (Matavel et al., 2002). The di- phodiesterase-5 (PDE5) inhibition (Nunes et al., 2012b). Furthermore,
versity of channel modulations is intriguing from a biophysical point of it was shown that PnTx2-6 induces priapism in mice even after ca-
view. The slowing down of the inactivation process is similar to the vernosal denervation, indicating that the toxin might not depend on
activity of certain scorpion, sea anemone and other spider toxins such cavernosal nerves integrity (Ravelli et al., 2017). Investigating the gene
as the β/δ-agatoxins (Cardoso and Lewis, 2017; Deuis et al., 2017; expression in mice erectile tissue showed overexpressing of two genes
Israel et al., 2017; Nicholson, 2007; Wanke et al., 2009). The subtype potentially related to PnTx2-6 induced priapism (Villanova et al.,
selectivity for mammalian Nav channel isoforms was determined for 2009). One of these genes is directly involved in the activation of the
PnTx2-6 (Fig. 6) (Silva et al., 2015a). These toxins have been char- NO/cGMP pathway.
acterized to bind to the so called neurotoxin binding site 3 Nav channels PnTx2-5 has been less investigated, compared to PnTx2-6, but the
(Catterall et al., 2007). These site 3 toxins slow down sodium channel results that are available do suggest that this toxin is involved in penile
inactivation and in many cases they induce other channel gating potentiation similar to PnTx2-6 (Villanova et al., 2009). PnTx2-5 also
modifications as well (Cardoso and Lewis, 2017; Peigneur et al., 2012; caused penile erection when injected intraperitoneal in male mice.
Zhu et al., 2012). Binding experiments in brain synaptosomes showed These effects are completely abolished by the nNOS-selective inhibitor
that PnTx2-6 partially competes with the typical α-scorpion toxin AaHII 7-nitroindazole, indicating an important involvement of nNOS in this
(from Androctonus australis Hector) (Matavel et al., 2009). A shift of the effect as well (Yonamine et al., 2004). Both toxins, PhTx2-5 and PhTx2-
midpoint of activation potential toward more negative potentials to- 6, represent interesting pharmacological tools to study erectile dys-
gether with a reduction in sodium peak current are characteristics of function. A 19 residues peptide, devoid of disulfide bridges but com-
gating alterations often observed for β-scorpion toxins and certain prising the pharmacophore of PnTx2-6 was designed. This PnTx2-6-
spider toxins (Leipold et al., 2006; Nicholson et al., 2004; Peigneur derived peptide, named PnPP-19, was no longer active on Nav chan-
et al., 2015). However, it was shown that PnTx2-6 does not compete nels. The abolishment of Nav channel activity can be considered as an

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Fig. 6. Representative traces of Nav channel isoforms expressed in Xenopus oocytes in control situation and after the application of 3 μM PnTx2-6. Dotted line
indicates zero current level. Asterisk indicates steady state current traces after toxin application. Figure adapted from Silva et al. (2015a,b).

important amenity since this strongly reduces the peptide toxicity and arrestin2 recruitment by PnPP-19 underlines the potential of this pep-
hereby unwanted side effects. Interestingly however, PnPP-19 potenti- tide as a possible lead compound in the development of improved
ates penile erection with a similar activity as PnTx2-6 (Silva et al., opioid agonists (Freitas et al., 2018).
2015a). Therefore, PnPP-19 can be considered as an exciting lead
compound for drug development related to the treatment of erectile
dysfunction. Moreover, PnPP-19 has also been investigated for its role 6. PhTx3
in both peripheral and central antinociception (Freitas et al., 2016). It
was shown that PnPP-19 inhibits neutral endopeptidase and activates Injection in mice of fraction PhTx3 results in a progressive flaccid
receptors involved in pain pathways such as the cannabinoid receptor 1 paralysis of all legs (Kushmerick et al., 1999; Prado et al., 1996;
and the μ- and δ-opioid receptors (da Fonseca Pacheco et al., 2016; Rezende et al., 1991). Of all venom fractions, PhTx3 must be the most
Freitas et al., 2016). Furthermore, the peripheral antinociceptive effect studied and best characterized fraction. This is a consequence of the
induced by PnPP-19 is resulting from an activation of the NO-cGMP- pharmacological potential present in this fraction. All of the PhTx3
KATP pathway. Hereby, an activation of both endothelial nitric oxide peptides were found to target voltage-gated calcium (Cav) channels or
synthase (eNOS) and nNOS by PnPP-19 occurs in rat paw Freitas et al. Kv channels (Table 1). It is remarkable that up to date only one voltage-
(2017). PnPP-19 selectively activates μ-opioid receptors inducing in- gated potassium channel targeting peptide (KSpTxs) has been char-
directly inhibition of calcium channels and hereby impairing calcium acterized from Phoneutria venom while other spiders are known to
influx in dorsal root ganglion (DRG) neurons. Interestingly, notwith- produce potent and pharmacological interesting KSpTxs (Priest et al.,
standing the activation of opioid receptors, PnPP-19 does not induce β- 2007; Swartz, 2007). The genes encoding for all PhTx3 toxins are
arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, identified. It was shown that these toxins are encoded as a precursor
among opioid receptors, selectively activates μ-opioid receptors. The peptide composed of a signal peptide, an intervening propeptide, and
observed lack of β-arrestin2 recruitment highlights the potential of this the mature toxin (Cardoso et al., 2003; Carneiro et al., 2003;
peptide for the design of new improved opioid agonists (Freitas et al., Kalapothakis et al., 1998b). Interestingly, certain isoforms identified by
2018). One of the very serious and life-threatening conditions devel- molecular cloning have never been purified from the crude venom.
oped following the use of the usual opioid agonist medicines is re- However, some of these peptides were functionally expressed in het-
spiratory paralysis. It has been demonstrated that the induction of re- erologous systems in order to allow functional characterization
spiratory paralysis, as well as other side effects, after the use of opioids (Carneiro et al., 2003; Souza et al., 2008). Many studies have been
may be linked with the recruitment of the β-arrestin pathway, which is devoted to the identification of the molecular target of PhTx3. First
stimulated downstream following activation of μ-opioid receptor. Since studies showed that this venom fraction reduces the release of tritiated
opioid receptors are still one of the most relevant targets for pain acetylcholine in the brain indicating a potential target involved in the
treatment, great effort is being put in the development of new opioid acetylcholine release in the brain and in the autonomic nervous system.
agonists that elicit fewer negative side effects. In this way, the lack of β- Therefore, the target could be a calcium channel (de Lima et al., 2016;
Gomez et al., 1995). This hypothesis was further supported by the

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Fig. 7. Sequences of the toxins identified in Phoneutria nigriventer venom fraction 3. Cysteines are indicated in red. (For interpretation of the references to colour in
this figure legend, the reader is referred to the Web version of this article.)

observation that PhTx3 abolishes calcium-dependent glutamate release Ample ω-conotoxins have been identified from cone snail venom
in rat brain cortical synaptosomes without influencing the calcium-in- (Prashanth et al., 2014). Noteworthy, all studies with PhTx3 peptides
dependent exocytosis. PhTx3 partially inhibited the glutamate release acting on Cav channels have used either GVIA or MVIIC ω-conotoxins to
without affecting the glutamate release triggered by intracellular cal- compare the analgesic activity. A more recent study investigated the
cium stocks. This hinted towards the presence of toxins in PhTx3 which effects of PnTx3-3 on sensory transmission in the spinal cord of rats
interfere with the calcium influx in synaptosomes (Prado et al., 1996). (Dalmolin et al., 2017). Using in vivo electrophysiological recordings, it
From this fraction six toxins, named PnTx3-1 to PnTx3-6, have been was shown that PnTx3-3 exerts a prevalent antinociceptive effect
characterized (Fig. 7). PhTx3 is the venom fraction composed of the mainly by inhibiting R-type Cav channels (Dalmolin et al., 2017).
most heterogeneous group of toxins. The peptides found in PhTx3 share PnTx3-4 affects the neurotransmission by blocking presynaptic
little sequence identity, and therefore, display a wide array of differing calcium channels associated with exocytosis in mammals, lower ver-
pharmacological activities when injected in vivo. For example, PnTx3-1, tebrates and arthropods (de Lima et al., 2016; Troncone et al., 2003).
PnTx3-5, and PnTx3-6 induce paralysis of the posterior limbs. PnTx3-2 PnTx3-4 potently inhibits high-voltage-activated Cav channels in the
induces immediate clockwise gyration and flaccid paralysis. PnTx3-3 sensory neurons of dorsal root. No activity was observed on low-vol-
and PnTx3-4 are the most toxic: at 5 μg/mouse they reproduce the fast tage-activated Cav channels (Cassola et al., 1998). Moreover, PnTx3-4
flaccid paralysis followed by death observed for the whole PhTx3 is suggested to act on P/Q-type Cav channels (Miranda et al., 2001).
fraction (Cordeiro et al., 1993; de Lima et al., 2016). PnTx3-6 also in- Using heterologous expressed channels in HEK293 cells, it was shown
duces analgesia models pain in rodents (Souza et al., 2008). that PnTx3-4 produced a potent and almost irreversible inhibition of P/
It was shown that PnTx3-1 increases calcium oscillation in GH3 Q-type Cav2.1 channels and N-type Cav2.2. Only a partial and re-
cells, presumably by blocking potassium currents (Kushmerick et al., versible inhibition of R-type Cav2.3 channels was observed (Dos Santos
1999). Whole-cell patch clamp experiments showed that PnTx3-1 re- et al., 2002). Furthermore, this toxin blocked potassium-induced and
versibly and selectively inhibits type-A potassium current (IA) without capsaicin-induced glutamate release from rat brain synaptosomes
affecting the delayed or the inward rectifying potassium current. (Goncaves et al., 2011; Reis et al., 1999). Incubation of synaptosomes
PnTx3-1 has no effect on large conductance calcium sensitive potassium with PnTx3-4 in the presence of the calcium chelator EGTA blocked
channels. Furthermore, this toxin does not interact with T- and L-type calcium-independent glutamate release, contrasting with the observa-
Cav channels. The inhibition of IA favors cell depolarization and Cav tion that the fraction PhTx3 did not inhibit calcium-independent com-
channel activation, increasing the frequency of calcium oscillation ponents of glutamate release (de Lima et al., 2016). This seemingly
(Kushmerick et al., 1999). It is important to note that these patch clamp contradiction could be explained by the low amount of PnTx3-4 present
experiments in GH3 cells do not rule out that the observed effects on in the venom fraction PhTx3 (de Lima et al., 2016). The fraction PhTx3
oscillation frequency are following upon interaction of PnTx3-1 with an exhibits a strong neuroprotective effect against neuronal damage in-
unknown target. Further research is required to verify that Tx3-1 is duced by oxygen deprivation and low glucose (ODLG) insults on hip-
indeed a potassium channel toxin and to determine which potassium pocampal slices (Pinheiro et al., 2006). In an in vitro model of retinal
channel isoforms this peptide is targeting. In the heart, PnTx3-1 had an ischemia induced by ODLG, the fraction PhTx3 protected approxi-
anti-arrhythmogenic effect, decreasing the ACh-mediated heart rate by mately 80% of the cells from injury (Agostini et al., 2011). PnTx3-3 and
doubling the frequency of spontaneous miniature end plate potential PnTx3-4 were identified as the peptides responsible for this neuropro-
protecting ischemia/reperfusion heart against arrhythmia (Almeida tective effect. Both toxins were effective in preventing cell death after
et al., 2011; de Lima et al., 2016). More recently, it was shown that ischemic injury. It has been shown that ischemic injury induced by
PnTx3-1 causes an antinociception by interfering with the choline es- ODLG insults in retinal slices leads to increased levels of glutamate.
terase activating cholinergic system (Rigo et al., 2017). These increased concentrations of glutamate cause retinal cell death.
PnTx3-2 is characterized as a selective inhibitor of L-type Cav PnTx3-3 and PnTx3-4 exert their neuroprotective effect by indirectly
channels (Kalapothakis et al., 1998a). PnTx3-2 does not show affinity inhibiting the glutamate increase through the inhibition of N and P/Q
for N- or P/Q-type Cav channels since the presence of this toxin did not type Cav channels. PnTx3-4 showed superior protection when com-
modify the KCl-evoked glutamate release nor the rise of intracellular pared to PnTx3-3, the fraction PhTx3, or the Cav channel inhibiting
calcium in synaptosomes (Prado et al., 1996). cone snail toxins ω-conotoxin GVIA and ω-conotoxin MVIIC (Agostini
PnTx3-3 is considered as the most potent toxin from the venom et al., 2011; Binda et al., 2016; Pinheiro et al., 2009). A similar neu-
fraction PhTx3. Many of the toxic properties of fraction PhTx3 are roprotective activity was observed for both toxins in an in vitro model of
produced by this toxin (Guatimosim et al., 1997; Prado et al., 1996). brain ischemia. By using electrophysiology and the glutamate release
PnTx3-3 inhibits P/Q- and R-type Cav channels with high affinity. At assay in hippocampal slices, it was observed that both PnTx3-3 and
higher concentrations, PnTx3-3 also inhibits L- and N-type Cav chan- PnTx3-4 blocked the glutamate release (Pinheiro et al., 2009). Fur-
nels (Leao et al., 2000). Interestingly, PnTx3-3 induces a different thermore, the neuroprotective effect of PnTx3-4 was also evidenced in
profile of behavioral effects compared to the well characterized ω- vivo using a rat model of NMDA-induced injury of the retina (Binda
conotoxin MVIIC which is also an inhibitor of P/Q-type Cav channels. et al., 2016). These results underline the role of PnTx3-4 as a novel

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S. Peigneur et al. Toxicon 151 (2018) 96–110

Fig. 8. Sequences of the toxins identified in

Phoneutria nigriventer venom fraction 4.
Cysteines are indicated in red. (For inter-
pretation of the references to colour in this
figure legend, the reader is referred to the
Web version of this article.)

potential tool to study or treat retinal injury. It is remarkable to note 7. PhTx4

that the complete, correct sequence for PnTx3-4 matching the mass of
the native toxin has still not been published despite the growing PhTx4 is historically referred to as the insecticidal fraction. It earnt
number of studies using this toxin (Herzig, 2016). As can be seen in this reference based on its high toxicity and lethality toward insects
Table 1, several PnTx3-4 isoforms have been reported. while displaying a minor toxicity when injected in mice. This fraction
The least studied toxin of the venom fraction PhTx3 is the toxin causes hyperactivity such as cramps, quivering, jerking of the limbs,
PnTx3-5. However, this peptide is known to potently block L-type Cav and violent trembling of the body and the legs, leading to muscle fa-
channels (Leao et al., 2000). PnTx3-5 demonstrated promising anti- tigue and therefore causing paralysis in insects (de Lima et al., 2016;
nociceptive activity in clinically relevant pain models of postoperative, Figueiredo et al., 1995). It is suggested that PhTx4 acts on the gluta-
neuropathic and cancer-related pain (Oliveira et al., 2016). Interesting matergic system of both insects and mammals. Three toxins have been
to note is that PnTx3-5 also displayed efficacy in opioid tolerant ani- characterized from the venom fraction PhTx4 (Fig. 8 and Table 1). They
mals. Indeed, the clinical applicability potential of L-type, as well as N- were named PnTx4 (6-1), PnTx4 (5-5), and PnTx4 (4-3) (de Figueiredo
type, Cav channel inhibitors such as PnTx3-5, can be found in their et al., 2001; Figueiredo et al., 1995; Oliveira et al., 2003). These 3 in-
apparent synergism with morphine. Although the exact mechanism is secticidal toxins share high sequence identity and as such are believed
still under investigation, it is suggested that long-term use of morphine to exhibit similar pharmacological properties.
results in an increased expression of L- and N-type Cav channels (Verma PnTx4 (4-3) is not very well studied compared to the other toxins in
et al., 2009). Therefore, inhibitors of L-type Cav channels can induce this venom fraction. However, this toxin induces excitatory effects
analgesia but also potentiate the morphine-induced analgesia. This when injected in houseflies and cockroaches. Furthermore, it inhibits
could significantly reduce the consumption of morphine in patients the glutamate uptake in rat brain synaptosomes (Oliveira et al., 2003).
with chronic pain (Oliveira et al., 2016). PnTx4 (6-1) and PnTx4 (5-5) act on insect sodium channels (De
The toxin PnTx3-6 (also known as Phα1b) binds to a broad range of Lima et al., 2007; de Lima et al., 2002). Despite their apparent lack of
high-voltage-activated Cav channels. It inhibits N-, R- and P/Q-type Cav toxicity to mammals, they have been shown to inhibit glutamate uptake
channels with a comparable affinity. At higher concentrations, this in the mammalian central nervous system and to modulate the in-
toxin also blocks L-type Cav channels. However, PnTx3-6 has no ac- activation process of mammalian Nav channels (Mafra et al., 1999;
tivity on T-type Cav channels (Vieira et al., 2005). More recent, it was Oliveira et al., 2003; Paiva et al., 2016). It was evidenced that PnTx4
discovered that the analgesic activity of PnTx3-6 results, besides of the (5-5) is a reversible antagonist of NMDA ionotropic glutamate receptor
inhibition of Cav channels, from the interaction with TRPA1 receptors. in rat brain neurons (de Figueiredo et al., 2001). Since inhibition of
PnTx3-6 is a potent TRPA1 antagonist while having no affinity for NMDA receptors has been proposed as a therapeutic mechanism of
TRPV1 or TRPV4 receptors (Tonello et al., 2017). To overcome scar- neuroprotection, PnTx4 (5-5) has been investigated for its potential to
ceness of the native PnTx3-6, a recombinant form of the peptide has promote neuronal survival by blocking NMDA receptors. Nevertheless,
been produced. This recombinant peptide, annotated CTK 01512-2, more research is required to determine the NMDA receptor subtype
showed similar activity as the native PnTx3-6 (Tonello et al., 2017). selectivity of PnTx4 (5-5) (Silva et al., 2016). The activity of PnTx4 (5-
PnTx3-6 inhibited potassium-induced calcium-dependent glutamate 5) on insect and mammalian Nav channels was characterized in depth
release by blocking voltage-gated calcium channels, but it was not able (Paiva et al., 2016). The subtype selectivity among mammalian Nav
to modify the calcium-independent process (de Lima et al., 2016). channels was determined using the Xenopus oocyte expression system
PnTx3-6 has been investigated for its analgesic proprieties. Using both and recombinant produced PnTx4 (5-5). Among mammalian Nav
native and recombinant peptide, it was shown that PnTx3-6 was effi- channels, the toxin caused a delay in the channel inactivation and a
cient for the treatment of persistent pathological pain mediated by ei- reduction of the sodium peak current. This reduction in peak current is
ther glutamate release or capsaicin-induced calcium influx, but not the result of a toxin induced shift of the channel activation towards
involving capsaicin receptor inhibition (Castro-Junior et al., 2013; more depolarized membrane potentials. PnTx4 (5-5) has the most
Souza et al., 2008). The therapeutic potential of PnTx3-6 was evidenced pronounced effect on insect Nav channels. Using the cockroach Nav
by the observation that in preclinical models of chronic neuropathic channel BgNav1, it was illustrated that PnTx4 (5-5) has a devastating
pain, this toxin has a similar efficacy but a higher therapeutic index modulatory effect on these channels (Fig. 9). Upon binding, this toxin
than the clinically used ziconotide (McGivern, 2007; Tonello et al., causes a complete inhibition of the inactivation of the channel and an
2014). PnTx3-6 was effective in potentiating the analgesic effect of increase of the sodium peak amplitude. However, contrary to what was
morphine in mice. Furthermore, the same study showed that this pep- observed for mammalian Nav channels, no shift in the midpoint of
tide reduced the hyperalgesia, tolerance, constipation and withdrawal activation was noted for insect Nav channels (Fig. 9) (Paiva et al.,
syndrome induced by repeated morphine injection (Tonello et al., 2016).
2014). Similar, PnTx3-6 also potentiates the analgesic action of TRPV1 The most active toxin of this fraction is the anti-insect neurotoxin
blockers, underlining the potential of Cav channel inhibitors as ad- PnTx4 (6-1). A detailed mode of action of PnTx4 (6-1) and PnTx4 (5-5)
juvant analgesic agents (Palhares et al., 2017). Because of its action on has been acquired with the double-oil-gap method using axonal pre-
N- and P/Q-type Cav channels, PnTx3-6 has also been used as a tool to parations of the cockroach Periplaneta americana (de Lima et al., 2002).
study the involvement of these channels in bladder dysfunctions such as Both toxins induced evoked action potential prolongation in axonal
cystitis (Silva et al., 2015b). It should be noted that Cav channel in- preparations. This effect was stronger after PnTx4 (6-1) administration
hibitors, such as the PnTx3 toxins, have not only a potential in anti- than after PnTx4 (5-5). Tests performed in a voltage-clamp configura-
nociception. Because of the relevance of Cav channels in tumor pro- tion showed that both PnTx4 (6-1) and PnTx4 (5-5) prolonged the ax-
gression, these peptides could also be useful in cancer related studies onal sodium current in a manner similar to toxins binding to neurotoxin
(Nicoletti et al., 2017). site 3 of Nav channels. Similar tests in Xenopus oocytes indicated that
PnTx4 (6-1) has no effect on Nav1.2 and Nav1.4 channels (de Lima

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S. Peigneur et al. Toxicon 151 (2018) 96–110

Fig. 9. Effects of PnTx4 (5-5) on the cockcroach B. germanica sodium channel (BgNav1) expressed in X. leavis oocytes. A) Representative whole-cell current traces in
control and in presence of 1 μM rPnTx4 (5-5) (*). B) Current–voltage (I x V) relationships in control (○) and in presence of 1 μM rPnTx4 (5-5) (●). C) Activation of the
conductance in control (○) and in presence of 1 μM rPnTx4 (5-5) (●), fitted with the Boltzmann equation. Figure adapted from Paiva et al. (2016).

et al., 2016; de Lima et al., 2002).

However, in DUM cells, PnTx4 (6-1) changed the regular sponta-
neous firing pattern of action potential generation into an irregular
activity, evidencing its activity on insect Nav channels. The results
obtained with electrophysiological experiments suggested that PnTx4
(6-1) is most likely binding to the neurotoxin binding site 3 of Nav
channels. This was later on confirmed with binding assays using Bom
IV, an α-like scorpion toxin that binds to receptor site 3 on insect so-
dium channels. Bom IV was displaced by PnTx4 (6-1) (Figueiredo et al.,
1995). More recently, it was found that PnTx4 (6-1) induces anti-
nociception in inflammatory, neuropathic and acute pain models in
rats. It is suggested that this antinociceptive effect involves the can-
nabinoid system, through cannabinoid receptor 1, and the opioid
system, through the μ- and δ-opioid subtype receptors (Emerich et al.,
2016).

8. PhTx5

The fifth Phoneutria venom fraction is also known as fraction PhM

(Fig. 2, Table 2). An improved purification method of P. nigriventer
venom combined with mass spectrometry analysis contributed for the
identification of the fraction PhM (Pimenta et al., 2005; Richardson
et al., 2006). This fraction was characterized for its activity on smooth
muscle. However, the previous described purification method did not
allow identification of these peptides because of their low levels in the
whole venom and because their N-termini were somehow blocked
which prevented sequencing by Edman degradation. It is also probable Fig. 10. Amino acid sequences of the tachykinin family identified in the
that some of these small molecules do not represent mature peptides, Phoneutria nigriventer venom.
but are result of precursor processing and/or degradation of mature
toxins (Pimenta et al., 2005). PhM consists of a pool of similar isoforms
toxins targets Cav channels (Lucio et al., 2008). Two different isoforms
of smaller (< 2 kDa) peptides. The amino acid sequences of 15 of these
PnTx27C4 and PnTx26AN0C3 were isolated and characterized. Both
isoforms (Fig. 10) were determined by mass spectrometry (Pimenta
peptides share 92% sequence identity. These toxins produce spastic
et al., 2005). These muscle-active peptides contain 7–14 amino acid
paralysis and death in mice when injected intracerebral. Three very
residues and have a common scaffold composed of basic and acid amino
similar peptides, PnTx13C3, PnTx24An0C3 and PnTx24An0C4, were
acids (PyrKKDKKDx), where x can be either K or R. Since all of these
identified in this fraction. These peptides share 79% amino acid se-
molecules are structurally related to the tachykinin family of neuro-
quence identity. Similar to the PhTx4 toxins, these 3.5 kDa toxins are
hormone peptides, which possess N-terminal pyroglutamate residues,
very toxic to houseflies and induce no observable toxic effects in mice
they were named Phoneutria nigriventer tachykinin peptides PnTkPs
by i.c. injection (Richardson et al., 2006).
(Pimenta et al., 2005). The PnTkPs display a variation of post-transla-
Two proteases could be identified in the Phoneutria nigiriventer
tional modifications such as proteolysis, C-terminal amidation, and
venom. They were named proteinase PN44 and proteinase PN47. The
cyclization (Pimenta et al., 2005). The new procedure also resulted in
complete amino acid sequence of the PN47 and the N-terminal se-
the discovery of two new structural families of Phoneutria peptides
quence of have been determined. Both proteases are serine proteases
(Penaforte et al., 2000; Richardson et al., 2006). The family of 4 kDa

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S. Peigneur et al. Toxicon 151 (2018) 96–110

Fig. 11. Proposed structure of nigriventrine (Gomes et al., 2011).

belonging to the peptidase S1 family. It has been suggested that the Acknowledgements
endogenous proteolytic enzymes in the venom may be responsible for
the post-translational modification observed in some of the venom M.E. de Lima received grants from Brazilian Agencies FAPEMIG
components (de Lima et al., 2016). (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and
CNPq (Conselho Nacional de Desenvolvimento Científico e
9. Non-protein P. nigirventer venom components Tecnológico).

In addition, a novel non-protein low-molecular-mass neurotoxin Transparency document

named nigriventrine was isolated from the hydrophilic fractions ob-
tained from the venom by RP-HPLC purifications (Gomes et al., 2011). Transparency document related to this article can be found online at
Nigriventrine, a piperidine derivative, has neuroactive properties and https://doi.org/10.1016/j.toxicon.2018.07.008.
causes convulsion when injected in mice, intracerebrally or periph-
erally by intravenous injection (i.v.). Its structure was determined and it References
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