Drug Discovery Today  Volume 00, Number 00  September 2019 REVIEWS

Reviews  POST SCREEN

Animal venoms: therapeutic tools for
tackling Parkinson’s disease
Henrique de Oliveira Amaral, Victoria Monge-Fuentes, Andréia Biolchi Mayer,
Gabriel Avohay Alves Campos, Kamila Soares Lopes, Luana C. Camargo,
Matheus Ferroni Schwartz, Priscilla Galante and Márcia R. Mortari
Laboratory of Neuropharmacology, Department of Physiological Sciences, Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil

Parkinson’s disease (PD) is a neurodegenerative pathology of the central nervous system, mainly
involving the selective and progressive loss of dopaminergic neurons from the substantia nigra, resulting
in motor and non-motor symptoms. PD remains an incurable ailment; thus, treatments are limited to
symptom alleviation. With long-term use, conventional treatments can become inefficient, often
triggering possible side effects. Considering these drawbacks, drug discovery constantly turns to nature
as a source of efficient therapeutics. Thus, this review explores animal venoms as a rich source of
bioactive compounds with potent neuropharmacological profiles for the development of effective
adjuvant treatments with fewer side effects, ultimately aiming for the neuroprotection of dopaminergic
neurons and the symptomatic relief of PD.

Introduction Despite the efforts made through arduous research, PD remains

Parkinson’s disease (PD) is a neurodegenerative and progressive an incurable ailment and available pharmacological treatments
disorder that affects the central nervous system (CNS) of 2–3% of are mainly focused on treating classical motor symptoms and
the population over the age of 65 years. PD symptomatology is improving patient quality of life [5]. Moreover, current treatment
commonly associated with cardinal motor complications, such as is unable to slow or halt the progression of the disease and, with
bradykinesia, postural instability, resting tremor and rigidity. long-term use, some of these drugs can become inefficient and/or
Additionally, PD is accompanied by several non-motor symptoms, even trigger adverse effects [6]. In view of these limitations, drug
bringing patients to overall disability [1]. Classical neuropatho- discovery repeatedly turns to nature as a source of more-efficient
logical hallmarks that are linked to PD include the selective and therapeutics. Thus, herein, we assess animal-venom-derived com-
progressive loss of dopaminergic neurons from the substantia pounds as promising lead candidates for the development of
nigra (SN) and the histopathological presence of Lewy bodies, pharmacological probes and drugs able to translate as effective
composed mainly by protein aggregates of a-synuclein [2]. Fur- adjuvant treatments with fewer side effects, ultimately aiming for
thermore, available knowledge on PD lacks a well-defined etio- the neuroprotection of dopaminergic neurons and the symptom-
pathogenic mechanism; thus, several hypotheses have linked atic relief of PD [7].
genetic factors, environmental toxins, neuroinflammation, mito-
chondrial disturbances, excitotoxicity, protein misfolding and Animal venoms as a promising adjuvant treatment for
aggregation, impairment of protein clearance pathways, cell-au- Parkinson’s disease
tonomous mechanisms and advanced age [3,4] as possible causes. Animal venoms translate as the result from millions of years of
evolutionary pressure, which led to the development of several
venom-derived compounds with binding specificity, high affinity
and ample potency profiles [8]. Considering that the extraordinary
Corresponding author: Monge-Fuentes, V. ([email protected])

1359-6446/ã 2019 Elsevier Ltd. All rights reserved.

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repertoire of animal venoms evolved as predation and defense proapoptotic factor Bax production and increasing the expression
tools, they often act on the nervous and cardiovascular systems of antiapoptotic factor Bcl-2 in SH-SY5Y neuroblastoma cells [23].
with the ability to disrupt a range of vital physiological and BV also exhibited effects against glutamate excitotoxicity [24] and,
biochemical processes [9]. Such a disruptive neurological effect in a 6-hydroxidopamine (6-OHDA) rat model for PD, it diminished
can be attributed to the ability that many of these venom-derived contralateral forelimb akinesia and improved the function of
compounds have to modulate synapses by acting with remarkable cortico-basal ganglia circuits [25].
potency and selectivity on various biological targets, including Interestingly, although the therapeutic landscape of BV for the
receptors, ion channels and enzymes [10]. treatment of parkinsonism in preclinical studies seems vast and
Reviews  POST SCREEN

Given that animal venoms exhibit chemical diversity of bioactive promising, unfortunately it does not mirror results obtained in
substances [inorganic salts, small organic molecules (<1 kDa), poly- clinical studies developed in this field, where few trials have been
peptides (2–9 kDa) and high-molecular-weight molecules (>10 registered (NCT01341431 and NCT01970813) (https://
kDa) such as proteins and enzymes], several studies have investigat- clinicaltrials.gov/; information on clinical trials was obtained with
ed their mode of action on targets, exploring the biochemical and the search criteria: ‘melittin and Parkinson’s disease’; ‘apamin and
genomic composition of the compounds, and potential to generate Parkinson’s disease’; ‘bee venom and Parkinson’s disease’; ‘snake
new pharmaceuticals. In addition, with the ever-increasing venom and Parkinson’s disease’; ‘Exendin-4 and Parkinson’s dis-
advances in the development of new analytical tools (i.e., mass ease’; ‘exenatide and Parkinson’s disease’). Summarizing the avail-
spectrometry, transcriptomics and proteomics screenings), the able clinical evidence, first, the efficacy of repeated subcutaneous
use of venom components as therapeutics represents a growing injections of BV on PD motor symptoms and disease progression
and promising approach [11]. Most neurological disorders are cur- was investigated [26]. However, owing to the lack of clear symp-
rently treated with symptomatic-only drugs, which can often be tomatic or disease-modifying effects compared with placebo, fu-
associated with a range of adverse peripheral effects. Therefore, the ture studies were suggested using higher BV individual doses and
selectivity seen in a variety of venom-derived components obtained administration frequency. Following this pilot study, consequent
from bees, scorpions, snakes and lizards could potentially lead to the studies treated patients with BV acupuncture (BVA) or acupunc-
development of new pharmacological probes and drugs with effi- ture, used as adjuvant therapy, reporting that acupuncture and
ciency and fewer side effects for various diseases, including PD [7], as BVA groups showed significant improvement in several motor
we shall examine in detail below. tests; thus, effectiveness remained uncertain as it could be attrib-
uted either to the pharmacological actions of the venom or to the
Bee venom mechanical effect induced by the acupuncture stimulation [14].
Bee venom (BV) is a complex fluid comprising a variety of com- Another study developed by the same research group assessed the
ponents, including peptides (mellitin, apamin, adolapin), combination of BVA with manual acupuncture, revealing that the
enzymes (phospholipase A2, hyaluronidase, acid phosphomono- combined treatment was safe with significant motor function
esterase, lysophospholipase and -D-glucosidase) and lower-molec- improvement [27]. Later, a triple-armed study reported a promi-
ular-weight compounds (biogenic amines and histamine), nent placebo effect; however, long-term effects on symptom im-
secreted from the gland of honey bee, Apis mellifera, exhibiting provement were detected for the active treatment group (BVA and
a wide range of pharmacological activities [12,13]. BV has been acupuncture); suggesting that BV seems to have an actual thera-
considered a possible medicine for several conditions, such as peutic biological effect [28]. As further noted by Awad et al., BV
neurological and neurodegenerative disorders, which includes could be a promising adjuvant treatment for PD; however, there is
PD [13–16] (Table 1). In view of this, several experimental studies still lack of enough evidence to validate the efficacy of BV and/or
have elucidated its possible activity as a neuroinflammatory mod- its components for PD treatment in a clinical setting [16]. In our
ulator and neuroprotector. Research performed with lipopolysac- point of view, trial design for BV investigation needs to be im-
charide (LPS)-stimulated BV2 microglial cells showed that BV proved, using larger sample sizes, higher doses and frequency,
inhibited the production of nitric oxide (NO), inducible nitric additional robust control groups covering all possible variables,
oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 considering interactions when combined (or not) with PD con-
(PGE2), nuclear factor (NF)-kB and proinflammatory cytokines ventional treatments. Because BV contains a series of compounds
[tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6] with different pharmacological effects and the increasing impor-
[17–19]. In addition, BV increased the proportion of tance given to the research and development of new strategies to
CD4*CD25*Fosp3 regulatory T cells (Tregs) and it also reduced tackle neurological disorders, we will further explore the main
microglia activation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro- components of BV and assess their potential effect for PD treat-
pyridine-induced (MPTP) mouse model for PD [20]. Further, the ment as reported, to our knowledge, so far only in preclinical
neuroprotective effect of BV was described as it reduced astrocyte studies.
activation in a subchronic MPTP-induced model, attenuating
dopaminergic neuronal loss [21]. Melittin
Moreover, BV inhibits apoptosis, as noted by Jung et al. who Among the main components of BV we highlight melittin – an
evaluated its neuroprotective effect on NSC34 cells after rotenone alkaline and amphiphilic peptide comprising 26 amino acid resi-
treatment. It was further observed that BV decreased caspase-3 dues [16]. Melittin makes up 40–60% of the total of dry BV and it is
expression and blocked the JNK and ERK1/2 signaling pathways recognized as one of the compounds with more pharmacological
[22]. Additionally, the neuroprotective mechanisms of BV de- activities. Thereby, recent studies are attempting to elucidate the
creased caspase-3 activity and DNA fragmentation, suppressing mechanism of action of melittin and actual effect for the treat-

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TABLE 1
Pharmacological effects studied in whole bee venom (Apis mellifera) and its main components for the treatment of Parkinson’s disease.
Venom or Experimental Treatment dose Outcome Refs
compound model
BV LPS-stimulated BV2 microglial cells 1, 10, 100 ng/ml Potential anti-inflammatory effect with reduction of [17]
TNF-a, iNOS and NO production in activated microglia
in dose-dependent manner
BV and melittin LPS-stimulated BV2 microglial cells 0.5, 1, 2 mg/ml Suppression of LPS-induced inflammation by [18]
reducing NO, PGE2 and cytokines (IL-1b, IL-6 and TNF-

Reviews  POST SCREEN

a) production.
Both compounds regulated different pathways (JNK
and Akt pathways and NF-kB activation via regulation
of the IkBa pathway) attenuating NO production
BV LPS-stimulated BV2 microglial cells 0.625, 1.25, 2.5 mg/ml Suppression of NO production and proinflammatory [19]
cytokines expression (TNF-a and IL-6) in a dose-
dependent manner. Inhibited NF-kB transcriptional
activity in MyD88-dependent manner
BV MPTP-induced PD model 1 mg/kg Increased Treg infiltration in vivo and reduced [20]
microglia activation. Treg infiltration in brain can
induce neuroprotection effect
BV Subchronic MPTP-induced PD 200 mg/kg Attenuated astrocyte activation in SN in subchronic [21]
model MPTP-induced PD model, preventing dopaminergic
neurons from death
BV Rotenone-induced cell death in 2.5 mg/ml Neuroprotective effect by blocking the JNK and ERK1/ [22]
motor neuron line NSC34 2 signaling pathways
Suppressed rotenone-induced activation of caspase-3
and mitochondria impairment
BV SH-SY5Y human neuroblastoma cell 1, 10 and 100 ng/ml Reduced expression of Bax and increased expression [23]
of Bcl-2 and caspase-3 activation
Attenuated PI3K/Akt-mediated signaling pathway
BV N2a neuroblastoma cell/BV2 murine 2.5 and 5 mg/ml Inhibited phosphorylation of ERK, p38 and JNK in N2a [24]
microglial cell neuroblastoma cells and JNK phosphorylation in BV2
microglial cells
Antiapoptotic effect and protected cells from
glutamate-induced toxicity
BV and apamin 6-OHDA- BV (1 and 3 mg/kg) Reversed catalepsy behavior induced by haloperidol [25]
induced PD model/haloperidol- Apamin (0.1, 0.2 or Modulated the basal ganglia signaling by
induced catalepsy model 0.4 mg/kg) counteracting imbalanced activity of the trans-striatal
pathways. Results implicate in an efficacy of BV on
motor PD symptoms, which could be exerted by
apamin
BV Randomized, double-blind, 100 mg (Alyostal1) (in 1 ml BV group did not show clear symptomatic or disease- [26]
placebo-controlled, parallel-group of NaCl 0.9%) modifying effects when compared to placebo group
single-center trial
BV Randomized controlled clinical trial 0.1 ml diluted to 0.005% in Patients undergoing BVA and acupuncture as [14]
distilled water adjuvant treatment twice a week for 8 weeks
improved motor symptoms, as evaluated by UPDRS
score
No significant difference was observed between
treatment groups (acupuncture and BVA)
BV Prospective open-label clinical 0.1 ml diluted to 0.005% in Patients undergoing BVA and acupuncture as [27]
study normal saline adjuvant treatment twice a week for 12 weeks
improved motor symptoms, as evaluated by UPDRS
score
BV Double-blind, randomized 0.05 mg/ml BVA and acupuncture improved motor symptoms [28]
controlled clinical trial when administered twice a week for 12 weeks as an
adjuvant treatment
Prominent placebo effect
Melittin ALS mouse model (hSOD1G93A 0.1 mg/g Improvement of motor behavior in the ALS mouse [30]
mice) model and mitigation of microglial activity, reducing
TNF-a expression
Attenuated post-translational modification of
⍺-synuclein, which could be related to the restoration
of proteasome activity, as seen in brainstem and spinal
cord of the transgenic mice
Melittin H2O2-induced neurotoxicity SH- 0.5, 1 and 2 mg/ml Suppressed expression and activation of caspase-3 [31]
SY5Y human neuroblastoma cell and modulated expression of the Bcl-2 protein family
(Bax and Bcl-2)

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TABLE 1 (Continued )
Venom or Experimental Treatment dose Outcome Refs
compound model
PLA2 MPTP-induced PD model 0.5 mg/kg Attenuated microglia activation and diminished CD4+ [41]
T cell infiltration in the brain of mouse with
parkinsonism
Affinity for mannose receptor (CD206) on dendritic
cells, promoting Treg induction in CD4 T cells
PLA2 A53 T a-Syn transgenic mouse 0.2 and 1 mg/kg Increased microglia M2 phenotype, changing the M1: [42]
model of ALS M2 ratio, promoting an anti-inflammatory response
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(A53 T a-Syn transgenic mice) Improved motor behavior and reduced a-Synuclein in
the spinal cord of transgenic mice
PLA2 and melittin MPTP-induced PD model Extracts with diverse PLA2 improved motor behavior and protected loss of [43]
composition of PLA2 and dopaminergic neurons. Melittin did not exert the
melittin same effects
PLA2 induced Treg cell differentiation and decreased
CD4+ T cells with Th1 and Th17 phenotypes. Melittin
did not exert the same effects
Apamin 6-OHDA-induced PD model Bilateral injections of apamin Improvement of akinetic deficits produced by [47]
(0.05 or 0.1 ng per side, nigrostriatal dopaminergic lesions when injected
dissolved in a volume of 0.5 directly into the subthalamic nucleus of parkinsonian
ml, in 0.9% sterile NaCl) rats
Apamin 6-OHDA-induced PD model 0.1 or 0.3 mg/kg Alleviation of non-motor symptoms induced by 6- [50]
OHDA, reducing anhedonia and anxiety, preserving
short-term social and spatial memories
Reduced motor symptoms, playing an important part
in cellular mechanisms of emotional behaviors and
spatial memory mediated in the basal ganglia
Apamin MPTP-induced PD model BV (12 mg/kg/BW and Protection of dopaminergic neurons from SNpc [51]
and 120 mg/kg/BW) and apamin against death in a chronic parkinsonian model
BV (0.5 mg/kg/BW, 1.0 mg/kg/ Apamin did not preserve dopaminergic nerve
BW and 1.0 mg/kg/BW) terminals, suggesting that another molecule in BV
might enhance apamin activity
Abbreviations: 6-OHDA: 6-hydroxidopamine; ALS: amyotrophic lateral sclerosis; Akt: protein kinase B; BV: bee venom; BVA: bee venom acupuncture; BW: body weight; ERK extracellular
signal-regulated kinase; IL: interleukin; iNOS: inducible nitric oxide synthase; JNK: C-jun N-terminal kinase; LPS: lipopolysaccharides; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;
NO: nitric oxide; PD: Parkinson's disease; PGE2: prostaglandin E2; PLA2: phospholipase A2; SN: substantia nigra; SNpc: substantia nigra pars compacta, TNF: tumor necrosis factor; Treg:
regulatory T cell; UPDRS: Unified Parkinson's Disease Rating Scale.

ment of neurodegenerative diseases (Table 1). Moon et al. reported considering issues on selectivity, cell membrane morphological
that melittin reduced neuroinflammation (decreased IL-1b, IL-6, alterations and possible cytotoxic activity [33,34], experiments in
TNF-a and NO production, and iNOS, PGE2 and COX-2 expres- animal models need to be more conclusive to elucidate the
sion) in a LPS-stimulated BV2 microglial model [18]. Other experi- effects and properties of melittin with emphasis on the route
mental studies have also contributed to the understanding of these of administration, possible side effects as well as target cells and/
mechanisms [29,30]. or organs before considering their application in clinical trials.
Considering the strategies that might be effective for the treat-
ment of PD, melittin has been shown to reduce neuroinflamma- Phospholipase A2
tion (TNF-a expression reduction), decrease -synuclein misfolding Phospholipase A2 (PLA2) is a catalytic enzyme that breaks down
and restore proteasomal activity in the brainstem and spinal cord phospholipids into phosphate and fatty acids [35]. Interestingly,
in an amyotrophic lateral sclerosis model (hSOD1G93A transgenic PLA2 has been isolated from the venom of a number of animals,
mice) [30]. Moreover, melittin exhibited antiapoptotic effects by such as snakes, spiders, lizards and bees (bvPLA2) [36]. Besides its
increasing antiapoptotic factor Bcl-2 production while inhibiting enzymatic activity, bvPLA2 has the ability to bind to the CD206
proapoptotic factor Bax and caspase-3 synthesis in a model of receptor in dendritic cells [37].
neuroblastoma (cell line SH-SY5Y) [31]. In addition, the pharma- The adaptive immune response has an important role in PD, and
cological effects of melittin and BV with emphasis on dopaminer- recent studies demonstrated that infiltrating T cells were sur-
gic related behavior were evaluated, showing that both rounded by dopaminergic neurons, attenuating neuronal death
compounds caused catalepsy and reduced motor stereotypies in- [38]. By contrast, Tregs are known for protecting neurons from
duced by apomorphine in rodents. Melittin further exhibited degeneration [39,40]. In this sense, studies have reported that PLA2
antipsychotic properties, without inducing side effects, suggesting increases the Treg population and decreases neuronal death in the
an interaction of BV and melittin with the dopaminergic system MPTP model [41] and the -synuclein transgenic model [42]. When
[32] (Fig. 1). PLA2 binds to CD206 in dendritic cells, PGE2 is activated, inducing
Overall, at the preclinical level, melittin shows several desired T-cell differentiation into T-cell regulation via Foxp3 expression
characteristics that depict it as an interesting candidate to [41]. Moreover, treatment with PLA2 changes the M1:M2 ratio by
tackle some PD neuropathogenic mechanisms. Nevertheless, decreasing the M1 phenotype of microglia and increasing the M2

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Apis mellifera
microglia
(phenotype M1) Treg

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proinflammatory
factors

microglia
neuroprotection mellitin PLA2 IL-13
(phenotype M2)
IL-4
dopaminergic
neuron IL-10 TGF-β
apamin
potassium
channel
blocker
dopaminergic
neuron

potassium
channel

Drug Discovery Today

FIGURE 1
Mechanisms of action for the main components found in bee venom (BV) (Apis mellifera) on the central nervous system. Left: Mellitin presents a suppressive
effect over inflammatory responses in M1 microglia by reducing the release of proinflammatory factors [decreases production of interleukin (IL)-1b, IL-6, tumor
necrosis factor-alpha (TNF-a) and nitric oxide (NO), and expression of nitric oxide synthase (iNOS), prostaglandin E2 (PGE2) and cyclooxigenase 2 (COX-2)].
Moreover, mellitin shows a neuroprotective effect by inducing an antiapoptotic effect [increases B-cell lymphoma protein 2 (Bcl-2) and inhibits Bcl-2-like protein
4 (Bax)]. Bottom: Apamin binds to small conductance Ca2+-activated K+ channels (SK-channels), selectively blocking their activation, serving as a dopaminergic
neuron regulator. Right: phospholipase A2 (PLA2) increases Treg cell population at the central nervous system, causing a neuroprotective effect. PLA2 also
increases M2 microglia, producing an anti-inflammatory response.

phenotype, which produces an anti-inflammatory response [43] (Table 1). Although bvPLA2 might possess a neuroprotective
(Fig. 1). Besides, in the -synuclein transgenic model, PLA2 anti-inflammatory effect, its mechanism of action still needs to be
decreases the -synuclein inclusions in the spinal cord and mice elucidated.
treated with PLA2 improve their performance in the Pole locomo- bvPLA2 presents several attractive characteristics for PD treat-
tion test; however, the mechanism by which PLA2 acts on -synu- ment; nevertheless, it also interacts with cell membranes by in-
clein is still unclear [42]. ducing the breakage of phospholipid bilayers, triggering damage
In addition, another study compared the effects of bvPLA2 and in cell and organelle membranes, implying a nonselective effect of
melittin (combined or not) extracted directly from BV in an MPTP- this enzyme [36]. Considering the variety of targets where PLA2
induced mouse model of PD. Kim et al. observed that bvPLA2 could interact, preclinical experiments need to work with a critical
exerted a potent neuroprotective effect, with improved motor approach concerning nonselectivity issues and possible PLA2 un-
function. Moreover, bvPLA2 inhibited proinflammatory T cell wanted effects and properties before entering clinical develop-
phenotypes and induced Treg cell differentiation in a dose-depen- ment.
dent manner. Melittin alone exhibited a limited therapeutic effect
on motor behavior and neuroprotection. However, when a melit- Apamin
tin (15%) and PLA2 (78%) combined extract was applied, a con- Apamin stands as another important component isolated from BV
siderable neuroprotective effect with motor function with relevant activity in PD models (Table 1). Apamin is a peptide
improvement was seen. As discussed by the authors, melittin could composed of 18 amino acid residues and the ability to cross the
have a synergistic effect, contributing to PLA2 therapeutic activity blood–brain barrier (BBB) [44,45]. Furthermore, apamin binds to

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small-conductance Ca2+-activated K+ channels, also known as SK nanobiotechnological strategies to create stealth liposomes modi-
channels (composed of either SK2 or SK3 subunits), selectively fied with ClTx (ClTx-LS) to serve as L-Dopa carriers in an MPTP-
blocking their activation [46] (Fig. 1). In the context of PD treat- induced PD model in mice. This enabled a system that reverses
ment, the selectivity of apamin to bind to either subunit is relevant coordinated movement impairments caused by MPTP through
considering that SK channels regulate dopaminergic neurons and successful delivery of L-Dopa across the BBB to the brain microvas-
are found in high density in the basal ganglia [47]. SK channels are cular endothelial cells, consequently increasing dopaminergic
also responsible for maintaining neuronal excitability after hyper- levels in the SN and striatum [60].
polarization; thus, channel blockade by apamin facilitates neuro- In view of the capacity of compounds to work as neuroprotec-
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nal firing [48,49]. Therefore, when 6-OHDA-lesioned animals were tors, a heat-resistant peptide, named SVHRP, was isolated from the
treated with apamin, motor and non-motor symptoms were coun- venom of Chinese scorpion Mesobuthus martensii (previously
teracted and extracellular dopamine concentration in the striatum Buthus marthensii Karsch). SVHRP protects dopaminergic neurons
was increased [50]. Likewise, when apamin was directly injected in the SN, improving behavioral deficits in parkinsonian rats and
into the CNS in a 6-OHDA animal model, akynetic symptoms were mice [62]. In a subsequent study, additional beneficial actions were
reduced [47]. In an MPTP model, apamin protected dopaminergic reported for SVHRP when tested in an early-stage 6-OHDA-PD rat
cell body neurons in SN pars compacta (SNpc); however, dopami- model. SVHRP significantly reversed the effects caused by neuro-
nergic neuronal terminals were not spared [51]. Electrophysiolog- toxin 6-OHDA, acting as a neuroprotector that prevents biochem-
ical studies have further demonstrated that when afterpolarization ical (reversed abnormal activities of monoamine oxidase-B,
is blocked by apamin in dopaminergic neurons, an increase in the superoxide dismutase and malondialdehyde in the mitochondria
number and frequency of action potentials is observed [48]. These of neurons in the midbrain) and ultrastructural damage (improved
changes in firing patterns decrease the interaction between N- optical density of dopaminergic neurons) in midbrain neurons
methyl-D-aspartate (NMDA) receptors and SK channels, contribut- [63] (Table 2).
ing to an increase in dopamine release [52].
Snake venom
Scorpion venom Originally, snake venoms were used exclusively for antivenom
Scorpion venoms are composed of a variety of different chemical production; however, as their components were isolated and
molecules, including inorganic salts, free amino acids, peptides, biological activity tested, application was extended for diagnostic
heterocyclic components, proteins (mainly enzymes) and other tests, biotechnological tools and drug development, becoming the
substances that are still unknown [53,54]. Scorpion venoms as well main source of venom-derived drugs [11,64,65]. Snake venoms
as the animal’s organs have been used since medieval times to cure involve a complex mixture of peptides, proteins and small mole-
several pathologies [55]. Indeed, peptides isolated from scorpion cules that present an effect particularly in the CNS, cardiovascular,
venoms exhibit multiple pharmacological effects, including anal- muscular and vascular systems [64,66]. Additionally, minor com-
gesic, anticancer, cardiovascular and immunosuppressive actions ponents in snake venoms might be accountable for potential
[56]. therapeutic use as antiparasitic, antitumor, neuro and ischemic
Aiming to elucidate whether any of these components present tissue protection [65].
useful activity for PD treatment, some studies have analyzed their As an approach to identify novel therapeutic activities derived
direct or indirect interaction with ion-channel function mecha- from snake venoms, the use of C-Map (Connectivity Map com-
nisms. It is notable that venom-derived peptides obtained from posed of a database of gene expression patterns) has been proposed
scorpions exert their effect mainly by the blockage of Nav channels as a biological activity screening tool. Through the use of C-Map,
[57]. In this sense, it has been demonstrated in an MPTP non- venom of the South American pit viper Bothrops jararaca, incubat-
human primate model of PD that motor cortex stimulation by ed with MCF7 cells, was associated to 19 drugs used for the
high-frequency electrical interference modulates the subthalamo- treatment of neuropsychiatric disorders, including hits for two
pallido-cortical loop, alleviating parkinsonian symptoms [58]. antiparkinsonian drugs: metixene and lisuride. The antiparkinso-
This type of stimulus is more specifically related to the blockage nian activity seen in B. jararaca might be related to the presence of
of Na+ and Ca2+ voltage-gated currents, leading to the interruption neurotoxins with affinity for muscarinic receptors [67]. In this
of spontaneous activities in subthalamic neurons [53]. In this respect, snake neurotoxins (polypeptides MT1 and MT2) that bind
context, Cn2 toxin, purified from the venom of Mexican scorpion to muscarinic acetylcholine receptors (mAChRs) were isolated
Centruroides noxius, induces a voltage-sensor-trapping mechanism from green mamba venom [68]. The selectivity observed in these
in the Na+ channel, isoform NaV1.6 current, as recorded in wild- polypeptides is of great interest for the treatment of neurodegen-
type cerebellar Purkinje cells, blocking firing with selectivity and erative diseases, such as PD, considering that the selective blocking
binding stability at the channel site [59]. Thus, Cn2 scorpion toxin of these muscarinic receptors might be of great aid for restoring
represents a promising pharmaceutical candidate for further stud- normal movement [68,69].
ies based on PD motor symptom relief. Trophic factors have also been suggested as important thera-
Molecules derived from venoms can also be applied for the peutics for the treatment of neurodegenerative conditions. Among
construction of ligand-modified targeting delivery systems [60]. their roles, trophic factors are in charge of supporting and protect-
Such is the case of a 36-amino-acid peptide originally isolated from ing cellular subpopulations [70]. Studies have also specifically
Leiurus quinquestriatus scorpion venom: chlorotoxin (ClTx) [61]. reported that trophic factors act on dopaminergic neurons in vitro
Considering the various obstacles to effectively delivering L-Dopa and in vivo, protecting them or restoring their function after the
to the CNS for PD therapy, Xiang and co-workers applied acute effects of neurotoxins; thus, turning them into potential

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TABLE 2
Pharmacological effects investigated in scorpion and snake venoms for the treatment of Parkinson’s disease
Animal Venom or compound Experiment model Treatment dose Outcome Refs
Leiurusquin Chlorotoxin (ClTx) MPTP-induced PD mouse model 20 mg/kg of Liposome modified with ClTx actively delivers L-Dopa [60]
questriatus levodopa (L-Dopa) to the brain, increasing dopaminergic levels in the SN
and striatum
Improved motor behavior in MPTP parkinsonism
mouse model
Mesobuthus Scorpion venom 6-OHDA-induced early 0.05 mg/kg Protected dopaminergic neurons from death [63]

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martensii heat-resistant PD rat model Attenuated mitochondrial damage and reversed
peptide (SVHRP) abnormal activities of MAO-B, SOD and MDA,
supporting antioxidant activity of the compound
Bothrops atrox Tripeptide isolated Rat pheochromocytoma cell line 350 mg/ml Decreased activity of proteases caspase-3 and [72]
from the venom (PC12) treated with caspase-9
dopaminergic neurotoxin MPP+ Neuroprotection mechanism might be related with
neurotrophic effect
Abbreviations: 6-OHDA: 6-hydroxidopamine; MAO-B: monoamine oxidase; MDA: malondialdehyde; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine; PLA2: SOD: superoxide dismutase; SN: substantia nigra.

therapeutic candidates for PD [70,71]. Brain-derived neurotrophic amylase secretion, but increased intracellular cAMP [78], an effect
factor (BDNF), a key growth factor, acts as a neuroprotective and later associated with the binding of Exendin-4 to glucagon-like
pro-neuroplasticity agent, improving the therapeutic outlook of peptide-1 receptor (GLP-1R) [79]. These receptors are expressed
patients with neuronal pathological degeneration [62]. With this and distributed in pancreatic b cells and within the brain, in
in mind, and with the aim of investigating the neuroprotective particular in the midbrain and striatum, and their activation on
function and neurotrophic ability of a tripeptide (Glu-Val-Trp) neurons leads to important neurotrophic and neuroprotective
isolated from Bothrops atrox venom, Martins and co-workers used effects, such as neurogenesis, neuroinflammation reduction,
PC12 cells treated with dopaminergic neurotoxin MPPT. Results oxidative stress protection, apoptosis inhibition and synaptic
showed an increase in cell viability and proliferation, as well as a plasticity enhancement [80].
decrease in apoptotic protease caspase-3 and caspase-9, both acti- In this sense, several studies were first conducted in cellular and
vated in PD. Additionally, the tripeptide tested on the PC12 cells animal models [81–85] (Table 3), evidencing neuroprotection after
treated with the dopaminergic toxins and the nontreated PC12 administration of Exendin-4. Considering that Exendin-4 safety
cells increased neuritogenesis and protected against differentia- has already been established and as a result of the growing evi-
tion impairment [72]. dence of its efficacy in animal models, a proof-of-concept single-
Taken together, these studies support the notion that this blind study with 45 moderate PD patients was conducted, dem-
tripeptide offers a promising mechanism of neuroprotection onstrating that twice-daily injections of Exendin-4 resulted in
against dopaminergic cell death, useful as a platform for the design improvement in the Movement Disorder Society-Sponsored Revi-
of novel pharmacological agents for PD. Moreover, because the sion of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS)
composition of B. atrox and B. jararaca venoms are related [73], the after 12 months of treatment [86]. In addition, the effect was
presence of this tripeptide found in B. atrox should be further maintained after a 12-month washout period [87], indicating a
investigated in the venom of B. jararaca to understand its role as a possible neuroprotective effect. A second, double-blind, placebo-
neuroprotector agent (Table 2). controlled trial was performed in 62 patients treated with weekly
injections of Exendin-4 for 48 weeks, followed by a 12-week
Lizard venom washout period, revealing improved motor skills in the Exen-
Venom of Heloderma suspectum, known as the Gila monster, din-4-treated group compared with placebo [88]. Nevertheless,
originally isolated from its saliva and now synthesized on a large considering that Exendin-4 is immediately available once it is
scale, has been of particular interest for drug design and the injected subcutaneously and its elimination half-life is 2.4 h
pharmaceutical industry, particularly since the development of [89], Chen et al. worked on a formulation, named PT302, that
licensed drugs for the treatment of type 2 diabetes mellitus encapsulated Exendin-4 in biodegradable poly lactic-co-glycolic
[74,75]. Some of the components identified in Heloderma venom acid (PLGA) microspheres, providing sustained release in a 6-
include cysteine-rich secretory protein, exendin, helofensin, kal- OHDA hemiparkinsonism rat model. Pre- and post-treatment with
likrein, natriuretic peptide and PLA2 [76]. Initial studies demon- PT302 significantly reduced methamphetamine-induced rotation
strated that the crude venom of H. suspectum induced amylase after lesion and post-treatment significantly increased tyrosine
release from dispersed acini from guinea pig pancreas, greatly hydroxylase in the lesioned SN and striatum, suggesting that
increasing intracellular cyclic adenosine monophosphate (cAMP) PT302 has a neuroprotective effect for nigrostriatal dopaminergic
[77]. Those findings motivated the search for secretory compo- neurons at a clinically relevant dose [90]. Collectively, these results
nents in the venom, leading to the discovery of a 39-amino-acid indicate relevant potential of Exendin-4 and other GLP-1 receptor
peptide, named Exendin-4. This peptide did not stimulate agonists in the treatment of PD (Table 3).

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TABLE 3
Pharmacological effects researched in Gila monster lizard (Heloderma suspectum) venom for the treatment of Parkinson’s disease
Venom or compound Experimental model Treatment dose Outcome Refs
Exendin-4 In vitro [anterior wall of the lateral In vitro (100 nM) Neurogenesis modulator effect as it [81]
ventricle of 5-week-old mice (C57 black)] In vivo (0.1 mg/kg) induced proliferation and
In vivo (6-OHDA-induced PD model) differentiation of in vitro and in vivo
neural progenitor/stem cells (NSCs)
Improved locomotor function in
parkinsonian rats
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Increased TH/VMAT2-positive cells

in the SN
Exendin-4 6-OHDA-induced PD model and LPS 0.1 mg/kg and 0.5 mg/kg Reduced amphetamine-induced [82]
injected into the SNpc rotations
Increased striatal dopamine and
protected TH + neurons in SNpc in
both models tested, reversing the
loss of these neurons
Exendin-4 MPTP-induced PD model 10 mg/kg Prevented loss of TH + SNpc [83]
neurons and TH + striatal fibers
Mechanism of neuroprotection is
suggested by inhibition of
microglial activation, reducing
release of proinflammatory
mediators
Exendin-4 MPTP-induced PD model 20 nM, 0.25 ml/h in the lateral Neuroprotection over the [84]
ventricle dopaminergic system, because it
prevented the loss of TH + SNpc
neurons and TH + striatal fibers
Preserved dopaminergic levels and
improved motor function in animals
treated with MPTP
Exenatide Single-blind trial design 5-mg exenatide pen device (Byetta Improvement of motor and [87]
5 mg) self-administrated twice-daily cognitive behavior as assessed by
for 1 month, then 10 mg exenatide MDS-UPDRS scale after 12 months
pen device (Byetta1 10 mg) self- of treatment
administrated twice-daily for the Good tolerability
subsequent 11 months
Exenatide Single-center, randomized, Subcutaneous injections of Treatment improved motor [88]
double-blind, placebo-controlled trial exenatide 2 mg once-weekly for 48 behavior as assessed by MDS-
weeks UPDRS scale
PT302 (long-acting 6-OHDA-induced PD model 0.4 mg/kg and 2 mg/kg Post-treatment of PT302 improved [90]
Exendin-4 sustained r motor behavior and significantly
elease formulation) increased TH + in the lesioned SN
and striatum
6-OHDA: 6-hydroxidopamine; LPS: lipopolysaccharides; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; SNpc: substantia nigra pars compacta. MDS-UPDRS:
Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale.

Concluding remarks these promising and naturally occurring peptides possess restric-
Medical and pharmaceutical alternatives for the treatment of tive intrinsic characteristics that hamper their human clinical
neurodegenerative disorders are currently limited to the manage- development [91,92]. Strategies involving the use of rational
ment of symptomatic manifestations. In the case of PD, the use of peptide design, engineered nanomaterials (drug delivery systems),
gold-standard medications causes a series of adverse effects multifunctional and cell-penetrating peptides, peptide drug con-
through their continued and prolonged use. In light of this jugates and technologies for alternative routes of administration
limitation, the search for new and effective compounds obtained are proposed to broaden the application of peptides as therapeutics
from novel sources is imperative as a way to provide hope for by improving circulating plasma half-life, chemical and physical
patients and their families. stability under physiological conditions, cell or tissue selectivity,
Scientific advances, especially in the development of omic molecular target specificity and oral bioavailability [91–93].
analytical tools seeking the isolation, purification and identifica- Counteracting limitations and generic pharmacokinetic disad-
tion of compounds, have evidenced the pharmaceutical potential vantages, >60 peptide drugs have been approved, >150 are in
found in the many bioactive components present in animal active clinical development and an additional 260 have been
venoms. As discussed, these compounds possess antioxidant, anti- tested in human clinical trials [92]. However, the volume of data
apoptotic, anti-inflammatory and neuroprotective properties, all supporting laboratory positive results for PD does not translate
of which are fundamental to target PD. Nevertheless, several of into the current number of clinical trials testing animal venoms.

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This failure to translate could be linked to an incomplete under- weapons for a variety of purposes, many of which are still to be
standing of PD pathogenic mechanisms, excessive reliance on explored. We expect that the information presented in this paper
results obtained from toxin-based animal models, the absence and additional future discoveries in this field will awaken the
of validated biomarkers of disease progression and/or unsuccessful interest of more researchers to invest in the discovery of new
trial design [94]. Thus, more research is needed to explore bio- bioactive compounds isolated from animal venoms and explore
chemical, genetic and pathological mechanisms involved in PD to their potential applications for the treatment of PD, bringing hope
offer a true differential therapeutic intervention for patients. We to patients who suffer from this ailment.
also foresee that, in the coming years, clinical development of
Acknowledgements

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these venom-derived peptides for PD will flourish, considering the
emergence of peptide technologies, nanobiotechnological solu- This work was supported by the following government Brazilian
tions and improved pharmaceutical properties that will further set agencies: Conselho Nacional de Desenvolvimento Cientı́fico e
the bases to ameliorate venom-derived therapeutic efficacy. Tecnológico (CNPq - 444292/2014-4) and the Fundação de Apoio à
Overall, this review further supports the idea that the develop- Pesquisa do Distrito Federal (FAPDF - 0193000949/2015 and
ment of new adjunctive treatments for PD could be hidden in 193001612/2016).
small and powerful venomous animals that use their poisonous

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