Elucidation of the Structure and Synthesis of Neuroprotective Low Molecular Mass Components of the Parawixia bistriata Spider Venom

Article abstract of DOI:10.1021/acschemneuro.0c00007

The South American social spider Parawixia bistriata produces a venom containing complex organic compounds with intriguing biological activities. The crude venom leads to paralysis in termites and stimulates l-glutamate uptake and inhibits GABA uptake in rat brain synaptosomes. Glutamate is the major neurotransmitter at the insect neuromuscular junction and at the mammalian central nervous system, suggesting a modulation of the glutamatergic system by the venom. Parawixin1, 2, and 10 (Pwx1, 2 and 10) are HPLC fractions that demonstrate this bioactivity. Pwx1 stimulates l-glutamate uptake through the main transporter in the brain, EAAT2, and is neuroprotective in in vivo glaucoma models. Pxw2 inhibits GABA and glycine uptake in synaptosomes and inhibits seizures and neurodegeneration, and Pwx10 increases l-glutamate uptake in synaptosomes and is neuroprotective and anticonvulsant, shown in in vivo epilepsy models. Herein, we investigated the low molecular mass compounds in this venom and have found over 20 small compounds and 36 unique acylpolyamines with and without amino acid linkers. The active substances in fractions Pwx1 and Pwx2 require further investigation. We elucidated and confirmed the structure of the active acylpolyamine in Pwx10. Both fraction Pwx10 and the synthesized component enhance the activity of transporters EAAT1 and EAAT2, and, importantly, offer in vitro neuroprotection against excitotoxicity in primary cultures. These data suggest that compounds with this mechanism could be developed into therapies for disorders in which l-glutamate excitotoxicity is involved.

Full text of DOI:10.1021/acschemneuro.0c00007

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Article
Elucidation of the structure and synthesis of neuroprotective low
molecular mass components of the Parawixia bistriata spider venom
Yvonne M. Forster, Jennifer Leigh Green, Apeksha Khatiwada, José Luiz
Liberato, Poli Adi Narayana Reddy, Joseph M. Salvino, Stefan Bienz, Laurent
Bigler, Wagner Ferreira dos Santos, and Andreia Cristina Karklin Fontana
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00007 • Publication Date (Web): 28 Apr 2020
Downloaded from pubs.acs.org on April 29, 2020
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Elucidation of the structure and synthesis of neuroprotective low
molecular mass components of the Parawixia bistriata spider venom
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a
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Yvonne M. Forster , Jennifer Leigh Green , Apeksha Khatiwada , José Luiz Liberato ,
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a
Poli Adi Narayana Reddy , Joseph M. Salvino , Stefan Bienz , Laurent Bigler ,
c*
b*
Wagner Ferreira dos Santos , Andréia Cristina Karklin Fontana
aDepartment of Chemistry, University of Zurich, Zurich, CH 8057, Switzerland
bDepartment of Pharmacology and Physiology, Drexel University College of Medicine,
Philadelphia, PA, 19102, United States
cDepartment of Biology, University of São Paulo, Ribeirão Preto, SP, 14040-900,
Brazil
dThe Wistar Institute, Philadelphia, PA, 19104, United States
*
Co-corresponding authors:
*Andréia C. K. Fontana
Department of Pharmacology and Physiology, Drexel University College of Medicine,
Philadelphia, PA, 19102, United States
Tel: +1 215-762-4399. E-mail: acm83@drexel.edu
*
Wagner F. dos Santos
Department of Biology, University of São Paulo
Ribeirão Preto, São Paulo, SP, 14040-900, Brazil
Tel: +55 16-3315-3657. E-mail: wagnerf@usp.br
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Abstract
The South-American social spider Parawixia bistriata produces a venom containing
complex organic compounds with intriguing biological activities. The crude venom
leads to paralysis in termites and stimulates L-glutamate uptake and inhibits GABA
uptake in rat brain synaptosomes. Glutamate is the major neurotransmitter at the
insect neuromuscular junction and at the mammalian central nervous system,
suggesting a modulation of the glutamatergic system by the venom. Parawixin1, 2,
and 10 (Pwx1, 2 and 10) are HPLC fractions that demonstrate this bioactivity. Pwx1
stimulates L-glutamate uptake through the main transporter in the brain, EAAT2, and
is neuroprotective in in vivo glaucoma models. Pxw2 inhibits GABA and glycine uptake
in synaptosomes and inhibits seizures and neurodegeneration, and Pwx10 increases
L-glutamate uptake in synaptosomes and is neuroprotective and anticonvulsant,
shown in in vivo epilepsy models. Herein, we investigated the low molecular mass
compounds in this venom and have found over 20 small compounds and 36 unique
acylpolyamines with and without amino acid linkers. The active substances in fractions
Pwx1 and Pwx2 require further investigation. We elucidated and confirmed the
structure of the active acylpolyamine in Pwx10. Both, fraction Pwx10 and the
synthesized component enhance the activity of transporters EAAT1 and EAAT2, and
importantly, offer in vitro neuroprotection against excitotoxicity in primary cultures. This
data suggests that compounds with this mechanism could be developed into therapies
for disorders in which L-glutamate excitotoxicity is involved.
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Keywords: acylpolyamine, Parawixin10 (Pwx10), UHPLC, high-resolution MS, MS/MS,
neurotransmitter, GABA, EAAT1, EAAT2, neuroprotection.
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Introduction
The spider Parawixia bistriata (Araneae; Araneidae) is a colonial orb-weaver
species, found in the Southeast of Brazil, the North of Argentina, and Paraguay. The
bioactivity of the P. bistriata venom was intensively studied by the group of Ferreira
dos Santos. In 2000, it was shown that the venom, injected into termites, leads to
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irreversible and dose-dependent paralysis . As L-glutamate serves as the major
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-3
neurotransmitter at the insect neuromuscular junction , these results indicated that
the venom might mediate its action through the effects on the glutamatergic system.
Additional in vitro studies showed that the crude venom stimulates L-glutamate uptake
and inhibits γ-amino butyric acid (GABA) uptake in rat brain cortical synaptosomes in
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a concentration-dependent manner , suggesting that the effects of the venom might
also involve modulation of the GABAergic system.
Compounds which increase L-glutamate uptake are of interest as the over-
stimulation of glutamatergic postsynaptic receptors due to sustained elevation of
extracellular L-glutamate levels in the synaptic cleft leads to excitotoxicity, which
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-6
results in neuronal death through apoptosis/necrosis . Glutamate excitotoxicity plays
a key role in secondary damage following acute pathologies, including traumatic brain
injury (TBI)7-14, hyper-excitability and seizures15-16, stroke17-21, epilepsy6, 16, 22-23, and
cerebral and retinal ischemia8-9, 24-29. Excitotoxicity also produces secondary damage
in chronic pathologies, including Amyotrophic lateral sclerosis (ALS)30-31, Alzheimer’s
disease , Huntington’s disease33-34, neuropathic pain35 and HIV-associated
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neurocognitive disorders (HAND)36-39
.
+
There are five structurally distinct subtypes of Na -dependent EAAT transporters,
including (rat/human homolog): GLAST/EAAT1/, GLT-1/EAAT2, EAAC1/EAAT3,
EAAT4 and EAAT5, which allows for precise spatial and temporal regulation of L-
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glutamate neurotransmission . Transporters EAAT1 and EAAT2, expressed
predominantly in glia cells of the central nervous system, are the major transporters
which take up synaptic glutamate to maintain optimal extracellular glutamic levels,
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thus preventing accumulation in the synaptic cleft and ensuing excitotoxicity 21, 41-44
.
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Although expressed within the same astrocytic plasma membrane , EAAT1 and
EAAT2 display functional differences, for example, cell-surface protein expression of
EAAT1 is affected by exogenous glutamate levels, but not EAAT2; and EAAT2 is
regulated by neuronal soluble factors, unlike EAAT146-47. EAAT1 is highly expressed
in the glial cells of the cerebellar Purkinje cell layer 48-49 and generally is expressed at
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higher levels in astrocytes and oligodendrocytes compared to microglia . EAAT2 is
primarily expressed in astrocytes and select neurons and oligodendrocytes of the
brain and spinal cord44, 51, and accounts for approximately 95% of the total L-glutamate
transport activity and 1% of total brain protein in the Central Nervous System (CNS)27,
45, 52-54.
Growing evidence shows that dysregulation of these transporters plays a
significant role in excitotoxicity and associated neuropathogenesis. The expression
and function of these astrocytic transporters may be dysregulated at the genetic,
epigenetic, transcriptional or translational levels, leading to high levels of extracellular
glutamate and excitotoxicity39, 55-59. On the other hand, the neuronal EAAT3 is
ubiquitously expressed in the brain, it is important in maintaining low local
concentrations of glutamate, where its predominant post-synaptic localization can
buffer nearby glutamate receptors and modulate excitatory neurotransmission and
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synaptic plasticity . Several diseases implicate in dysfunction of EAAT3, such as
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epilepsy, Obsessive-Compulsion disorder and schizophrenia . The neuronal EAAT4
and glial EAAT1 are the two predominant EAATs responsible for maintaining low
extracellular glutamate levels and preventing neurotoxicity in the cerebellum, the brain
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region essential for motor control, by limiting mGluR1 signalling . EAAT5 is important
for control of glutamate release mediates light responses in depolarizing bipolar cells
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in retina .
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Termination of GABA-mediated neurotransmission is achieved by high-affinity
transporters, named GATs, located at both GABAergic neurons and the surrounding
astrocytes. GABA transporter subtype 1 (GAT-1, mainly presynaptic) and GABA
transporter subtype 3 (GAT-3, mainly astrocytic) are the main transporters that
regulate inhibitory GABAergic transmission in the mammalian brain through GABA
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reuptake . The role of the neurotransmitter GABA in seizure disorders is not fully
understood, but several studies indicate that the inhibition of GABA uptake could lead
to suppression of seizures64-66. This is validated by studies using GABA analogues
that were shown to exhibit pronounced anticonvulsant activity in a variety of animal
seizure models, including tiagabine, a GAT inhibitor that has been developed as a
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clinically active antiepileptic drug . Moreover, glycine transporters-1 and -2 (GlyT1,
and GlyT2), regulate extracellular glycine concentrations within the CNS and as such,
play critical roles in maintaining a balance between inhibitory and excitatory
neurotransmission. The GlyT1 subtype is expressed by astrocytes at both inhibitory
and excitatory synapses as well as by a subset of glutamatergic neurons, whereas the
expression of GlyT2 is predominantly restricted to presynaptic terminals of inhibitory
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glycinergic neurons . GlyT-1 inhibition has been extensively examined as a potential
means to treat several CNS disorders that include schizophrenia, depression, anxiety,
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addiction, pain, Parkinson's disease, and epilepsy . Modulation of GlyT2 transporters
could provide therapeutic avenues for chronic pain, as exemplified by N-arachidonyl-
glycine (NAGly), an endogenous lipid that inhibits glycine transport by GlyT2 and
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shows potential as an analgesic , and novel allosteric modulators of GlyT2 with
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translational potential for pain analgesia .
The neuroactive properties of the venom of P. bistriata appeared to be related to
three HPLC fractions previously isolated from the spider venom, namely Parawixin1
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(
Pwx1)4, 71, Parawixin2 (Pwx2)72-77, and Parawixin10 (Pwx10)78-79, containing strongly
polar, low molecular mass compounds.
The most polar fraction, called Pwx1, was shown to increase the rate of L-
glutamate uptake in vitro selectively through L-glutamate transporter EAAT2.
Importantly, the Pwx1 fraction also had neuroprotective properties in an in vivo model
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of glaucoma . Additionally, the structural region of the transporter in which Pwx1
interacts sits at the interface of the rigid trimerization domain and of the central
substrate-binding transport domain, allowing for the identification of an allosteric site
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that is important for transport stimulation . Further studies used an in silico approach
to screen for novel compounds that interact with this allosteric site and were shown to
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be positive allosteric modulators of EAAT2 . Importantly, additional studies revealed
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that this class of compounds had also in vitro neuroprotective properties , validating
the premise that increasing L-glutamate clearance by activation of EAAT2 transport
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could serve as means of achieving neuroprotection .
The fraction Pwx2 inhibited the uptake of GABA and glycine in synaptosomes
and exhibited anticonvulsant effects in chemically-induced seizures models in a dose-
dependent manner73-74, 77. Furthermore, this fraction exhibited anxiolytic,
neuroprotective and anti-epileptic effects in in vivo models of Temporal Lobe
Epilepsy72, 76. The chemical structure of the active component in the fraction Pwx2,
also called FrPbAII, was proposed as 2-amino-5-ureidopentanamide73.
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Fraction Pwx10 was shown to increase L-glutamate uptake in rat brain
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synaptosomes and to have neuroprotective and anticonvulsant activities in in vivo
epilepsy models of intrahippocampal N-methyl-D-aspartate microinjection, and
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pentylenetetrazole injection in the lateral ventricle79.
With all these promising biological activities found, we decided to investigate the
chemical structures of the low molecular mass components of the P. bistriata venom
by UHPLC-HR-ESI-MS and MS/MS in an untargeted approach to get a better
understanding of the complex venom composition. We were especially interested in
the structure elucidation and the synthesis of the compounds that caused the observed
activities in the Pwx1, Pwx2, and Pwx10 fractions in order to confirm structure and
biological activity.
In this work, we have elucidated the structure of 36 acylpolyamines, with and
without amino acid linkers, in the venom of the P. bistriata spider. One of them is the
structure
of
(S)-N1-(3-(2-amino-5-guanidinopentanamido)propyl)-N4-(3-(2-(4-
hydroxy-1H-indol-3-yl)acetamido)propyl)-N1,N1,N4,N4-tetramethylbutane-1,4-
2+
diaminium iodide (4-OH-IndAc3(Me )4(Me )3Arg ), that had equivalent biological
2
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activity to Pwx10. This compound was revealed to be a doubly charged acylpolyamine
derivative showing a non-selective allosteric modulation of astrocytic L-glutamate
transporters. Importantly, this compound was neuroprotective in in vitro models of
excitotoxicity, suggesting that this class of compounds could be further developed into
therapies for neurological disorders that involved L-glutamate excitotoxicity.
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Results and discussion
Structure elucidation of the low molecular mass compounds in P.
bistriata venom
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The P. bistriata venom was extracted from the spider glands with H O/MeCN
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9:1 (v/v) and then analyzed by UHPLC-HR-ESI-MS and MS/MS. The venom was
chromatographed on a RP C18+ core shell column and a mobile phase composed of
H O/MeCN + 0.1% TFA, conditions particularly suited for the separation of polar basic
2
compounds. The MS/MS spectra were recorded in an untargeted approach with the
selection of maximum five different precursor ions after one survey scan. In addition,
the number of exchangeable protons of the spider venom metabolites was determined
in an on-line H/D exchange experiment.
The low molecular mass compounds (< 1000 Da) in spider venom can be
classified into acylpolyamine derivatives and other small molecules. The structure of
these polyamine derivatives was elucidated based on the MS/MS fragmentation rules
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defined by Tzouros et al. . These rules allow the unambiguous identification of
acylpolyamines from their MS/MS fragment ions without the need of synthesized
reference material. All polyamine derivatives were named according to the generic
acylpolyamine nomenclature84
More details about acylpolyamine nomenclature can
.
also be found in the supplementary material (including Figure 1 and tables 1-3). The
structures of the other small compounds, which are not polyamine derivatives and,
thus, do not follow the fragmentation rules of acylpolyamines, were confirmed by the
analysis of reference material.
The base peak chromatogram (BPC) of a sample of crude P. bistriata venom
measured by UHPLC-HR-MS is shown in Figure 1. With the untargeted approach, the
MS and MS/MS spectra of more than 75 features were recorded. Of these, 58
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structures could be resolved. The signals related to the proposed biologically active
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candidates found in the peak corresponding to the Pwx2 (P = m/z 175.12) and
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Pwx10 (m/z 294.72) fractions were detected with a retention time of 1.13 min and
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.84 min, respectively. The molecular mass (437 Da) characterizing the main
compound described in the Pwx1 fraction, however, could not be related to any signal
observed from the crude spider venom. Since there is no other structure-related
4
information about the active substance in Pwx1 fraction and the originally isolated
fraction was no longer accessible, the structure of the active substance in Pwx1
fraction was not further investigated in this study.
Structure elucidation of the active compound in the Pwx2 fraction
Previous studies proposed 2-amino-5-ureidopentanamide (FrPbAII) as the
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active structure in the Pwx2 fraction . In our studies, the structure of the active
compound in the Pwx2 fraction was re-examined.
In the UHPLC-MS chromatogram of the crude P. bistriata venom, the peak
+
related to m/z 175.11909 ([M+H] ) was detected with a retention time of 1.13 min.
Although retention time and accurate masses were in good agreement with
commercially available FrPbAll (1.15 min and C6H15N4O2, ∆m/z = 0.78 ppm), the
MS/MS spectra are clearly proving the presence of arginine (∆m/z = 3.63 ppm), a
structural isomer of FrPbAll (Figure 2). Our bioactivity tests with commercially available
FrPbAII did not show any effect on the inhibition of GABA (Figure 3B). Additionally,
the activity assays of arginine (Figure 3C) did not match to what was observed for the
isolated fraction Pwx2, as none of the concentrations of arginine tested elicited
inhibition of GABA uptake. This suggests that another co-eluting small molecule from
the spider venom may trigger the action of Pwx2.
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In total, 22 small compounds (mainly nucleosides, amino acids and quaternary
amines) were identified within the venom of P. bistriata (see supplementary material
for comprehensive data including all MS/MS spectra obtained from the spider venom
as well as the corresponding reference compounds) whereby only one of the identified
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small compounds, namely inosine, was previously found in the P. bistriata venom .
Inosine induced convulsive seizures in rats after intracerebroventricular injection,
whereas an analogue, inosine 5'-monophosphate, induced a delayed paralysis in
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termites . In addition, adenosine and guanosine as well as the related free bases,
adenine and guanine, were detected86-89. Nucleosides have already been found in the
venom of several spider families90-91
.
In addition to arginine, which has been found in the fraction of Pwx2, the amino
acids leucine, isoleucine, glutamic acid, glutamine, phenylalanine, tyrosine, and
tryptophan were detected. Of all these compounds, only glutamic acid has been
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reported several times as component of spider venoms . In insects, L-glutamate is
2-3
an excitatory neurotransmitter at the neuromuscular junction . The ion channel of the
activated postsynaptic L-glutamate receptor can be inhibited by acylpolyamine
compounds, which cause reversible paralysis of the spider's prey92-94. It seems to be
quite reasonable that the L-glutamate and acylpolyamines together make the venom
highly efficient. However, until now the combination of L-glutamate and
acylpolyamines have only been found in venoms of spiders from the Araneidae
91
family , in which P. bistriata belongs.
The venom further contains a number of quaternary amines, including choline,
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which was previously detected in various spider venoms , betaine, carnitine,
acetylcarnitine, propionyl carnitine, and homarine found for the first time in the spider
venom. The presence of homarine uniquely stands out since it is a compound that to
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the best of our knowledge is only known from marine species. Lastly pantothenic acid
as well as the polyamines spermine and spermidine were found. However, none of
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the three β-carboline toxins, PwTx-I , PwTx-II and 1-guanidino-6-hydroxy-3,5-
dihydro-β-carboline97 described by the group of Palma, nor structural related
compounds, were detected.
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Activity of constituents of the fraction Pwx2
We have examined and compared the effects of the natural Pwx2 fraction with
the commercially available FrPbAII on both GAT-1 and GAT-3 mediated GABA
transport, as well as GlyT1 and GlyT2 mediated glycine transport. Experiments were
performed in COS-7 transiently transfected with the appropriate DNA and empty
vector for background. Two days after transfection, a dose response assay of Pwx2
was conducted, in presence of 50 nM of the appropriated radiolabelled isotope (figure
3
). The Pwx2 fraction inhibited GABA uptake mediated by GAT-1 with an IC of 117
50
± 6 µM, while it had no effect on GABA uptake mediated by GAT-3, and glycine uptake
mediated by GlyT1 and GlyT2 (Figure 3A). In a previous study, natural Pwx2 fraction
was shown to inhibit GABA uptake in cortical synaptosomes with an IC of 24 μg/ml,
50
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which is equivalent to 114 µM , a comparable value for the inhibition of GAT-1
observed herein. We also examined Pwx2 concentrations higher than 500 mM, and
they did not inhibit GAT-1 mediated uptake more than ~52% (not shown in the graph
for simplicity). Suggesting that the natural Pwx2 fraction, even though seemingly
selective for GAT-1 rather than GAT-3 and glycine transporters, is a weak and poorly
efficacious GAT-1 inhibitor, compared to other specific inhibitors, such as SKF 89976A
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hydrochloride (IC of 0.13 μM for GAT-1 inhibition ) and tiagabine, an anticonvulsant
50
with an IC of 67 nM in vivo67, 99. Also, we did not observe any effect of the fraction
50
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Pwx2 on glycine uptake mediated by GlyT1 or GlyT2 (figure 3), even though in the
previous work it was reported to inhibit glycine uptake in synaptosomes and retinal
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preparations . Further experiments to examine whether Pwx2 modulates other
transporters, or has indirect effects on other targets, are needed to understand this
discrepancy.
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The commercial compound FrPbAII did not affect either GAT-1, GAT-3, GlyT1
or GlyT2 mediated GABA or glycine transport, as shown in Figure 3B, confirming that
the active constituent of the Pwx2 fraction is not the originally proposed FrPbAII. The
revised structure containing arginine was also tested against these 4 subtypes of
transporters. However, a dose response of arginine shows that there is no effect on
GAT-1, GAT-3, GlyT1 and GlyT2, suggesting that the active compound in the fraction
Pwx2 is not arginine either (figure 3C). Since arginine, the main compound of the Pwx2
fraction, is not active, we speculate that the inhibiting effect of the isolated fraction
Pwx2 on GAT-1 activity is either due to a minor compound or to unknown synergistic
effects among different molecules within the venom. Figure 3D shows the background
of GABA uptake assays obtained in presence of 1 mM nipecotic acid, resulting in
inhibition of 98 ± 1.5% by GAT-1, 75 ± 4% by GAT-3. Specific GlyT1 inhibitor sarcosine
results in inhibition of 90 ± 8% and specific GlyT2 inhibitor N-arachidonylglycine results
in inhibition of 95 ± 3 %, meaning that our uptake approach was fully functional.
Other potential candidates that could be causing the effect of the Pwx2 fraction
were explored, in addition to several small compounds which have been detected in
the crude P. bistriata venom (supplementary figure 2), on GABA uptake mediated by
GAT-1 and GAT-3 transporters, and glycine uptake mediated by GlyT1 transporter.
Betaine, homarine, L-carnitine hydrochloride, spermidine trihydrochloride and
spermine were examined, as they similar short retention times in the chromatographic
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separation when the crude venom was analyzed and therefore might be part of the
originally isolated fraction Pwx2. Homarine, carnitine and betaine, however, did not
affect the activities of the transporters examined (supplementary figure 2A-C). On the
other hand, spermidine and spermine selectivity increased the activity of GAT-1
mediated GABA uptake, without affecting the activities of GAT-3 or GlyT1
(supplementary figures 2D and E show the effects of spermidine trihydrochloride and
spermine on GAT-1, respectively, with variable EC50s and efficacies). Therefore,
spermidine and spermine increase GAT-1 activity, an effect that seems counter-
intuitive, as increased GABA transport results in less inhibitory neurotransmitter at the
neuromuscular junction of insects, resulting in more excitatory inputs. However, we
speculate that these effects would be annulled by the inhibiting effect of the Pwx2
fraction in GABA transport, resulting in paralysis of insects, as previously shown1.
These findings suggest that the active constituent in the fraction Pwx2 is neither of
these compounds.
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Selective modulation of the GABA uptake process through pharmacological
agents has been explored over several decades. These studies have demonstrated
that inhibition of astroglial, but not neuronal, GATs may be preferred for anticonvulsant
action. Only recently a class of specific GAT-3 inhibitors, which act by allosteric
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modulation, were elucidated . However, it remains to be investigated whether this
class of compounds has efficacy for conditions such as epilepsy in preclinical studies.
So far, the only compound with notable inhibitory effects at GATs, tiagabine, has
demonstrated clinical anticonvulsant efficacy, and is, to date, the only approved GAT
inhibitor for clinical use. Thus, efforts to identify and develop GAT subtype-specific
compounds continue to be an area of active investigation for the management of
epilepsy and other CNS disorders101.
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Structure elucidation of the acylpolyamine derivatives
Earlier studies suggested that the neuroprotective effect of the fraction Pwx10
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is probably due to an acylpolyamine associated with the doubly charged ion m/z
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2
94.7 . The corresponding peak in the UHPLC-MS chromatogram of the P. bistriata
crude venom was found at 6.84 min (Figure 1). Figure 4 displays the full scan (FS)-
HR-MS spectrum related to the chromatographic peak. The molecular formula derived
2+
from the doubly charged ion m/z 294.72047 is C H N O
(–1.00 ppm). The
30
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3
associated singly charged ion (m/z 588.4) was not detected. Instead, signals of TFA
2+
- +
+
adducts at m/z 702.42615 ([M +TFA ] , C H N O F , –1.60 ppm), m/z 351.71694
32 55
9
5 3
2+
-
+ 2+
2+
2+
-
+ +
([M +TFA +H ] , C H N O F , –0.98 ppm) and m/z 816.41902 ([M +2TFA +H ] ,
32
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5 3
+
C H N O F , –1.37 ppm) and even the signal of the triply charged ion at
34
56
9
7 6
m/z 196.81624 ([M +H ] C H N O33+, –0.42 ppm) were detected.
2
+
+ 3+
30
56
9
The structure of the acylpolyamine related to the main component of the Pwx10
fraction was elucidated by the MS/MS spectrum obtained from the precursor m/z
8
16.42 which was the only precursor that resulted in signals above m/z 231 (see
supplementary material). The compound was identified as 4-OH-
IndAc3(Me )4(Me )3Arg (compound 12; Figure 5). The base peak ion of the MS/MS
2+
2
2
+
spectrum m/z 231.11269 (a , C H N O , –0.49 ppm) together with the ion m/z
1
13 15
2
2
+
1
46.06007 (a’, C H NO , –0.20 ppm) indicated that the acylpolyamine has a 4-
9 8
hydroxy-indol acetyl head moiety connected to a C amine unit. Further, the spectrum
3
+
showed the fragments m/z 214.16636 (z , C H N O , 0.58 ppm), m/z 197.13970 (y ,
1
9
20
5
1
+
+
C H N O , 0.06 ppm), m/z 157.10854 (C H N O , 0.97 ppm), m/z 155.11782 (z -
9
17
4
6
13
4
1
+
+
Gu, C H N O , –0.45 ppm) and m/z 139.09792 (C H N , 0.70 ppm), distinctive for
8
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6
11
4
an arginine tail linked to a C unit. The identified head and tail units had to be linked
3
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by a fragment related to the formula C H N . The large number of hydrogen atoms
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2
indicated that the head and tail were not simply connected by a linear C -unit. In fact,
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only a C -unit framed by two dimethylated nitrogen atoms matched the molecular
4
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formula of the acylpolyamine. This structure was in good agreement with the remaining
+
major signals at m/z 100.11254 (dimethyl-pyrrolidin [C H N] , 4.64 ppm), m/z
6
14
+
+
259.22392 (t , C H N O , –0.64 ppm), m/z 313.27065 (z , C H N O , -1.23 ppm)
z1
11 27
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15 33
6
+
and m/z 330.21728 (a , C H N O , –0.98 ppm).
2
19 28
3
2
The presence of tetrasubstituted N-atoms in the acylpolyamine structure was
2+
confirmed in an H/D exchange experiment. The shift from m/z 294.72047 ([M] ,
C H N O , –1.00 ppm) to m/z 299.75276 ([d -M] , C H D N O32+, 1.77 ppm)
2+
2+
30
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3
10
30 45 10
9
confirmed the presence of 10 exchangeable hydrogen atoms. This finding supports
2+
the proposed structure 4-OH-IndAc3(Me )4(Me )3Arg .
2
2
2+
The structure of 4-OH-IndAc3(Me )4(Me )3Arg (12) was confirmed by the
2
2
synthesis of reference material (compound 12, see below). This acylpolyamine
derivative has never been described before. However, acylpolyamines with quaternary
amines in the polyamine backbone were previously reported by Grishin et al.102 in
Argiope lobata, Araneidae and by Yamaji et al.103 in Macrothele gigas, Hexathelidae.
Besides the acylpolyamine related to the Pwx10 fraction, the structures of 35
additional acylpolyamines were elucidated from the venom of the spider P. bistriata.
All elucidated structures are summarized in the matrix shown in Figure 6 and their
respective backbone, tail, and head highlighted as green squares. The rows represent
the head moiety and the columns a polyamine backbone of the corresponding
structure (refer to supplementary material for the drawn structures and the MS/MS
spectra of all the identified polyamine compounds). To our knowledge, only two of the
identified acylpolyamines have been described so far. The structure of N-(5-
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aminopentyl)-2,5-dihydroxy-benzamide (2,5-(OH) -Bz5) was reported in the venom of
2
1
04
1
a
Drassodes
species
(Gnaphosidae) .
N -5-aminopentyl)-2-[(1H-indol-3-
ylacetyl)amino]-butanediamide (IndAcAsn5, Pseudoargiopinin3) was earlier found in
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102
the spider venom of Argiope lobata , a species that belongs, as P. bistriata, to the
family of Araneidae.
Surprisingly, the venom of P. bistriata contained both types of acylpolyamines
with and without amino acid linkers. They are both well known, but to the best of our
knowledge they were never found coexisting in a spider venom until now. P. bistriata
spider belongs to a more recent evolved group which also has characteristics of
91
ancestral groups . We speculate the presence of both acylpolyamines types could be
related to the evolution process of this group; however, detailed studies on spider
venom content attempting to correlate the phylogenetic relationships between various
species of the Araneidae family are needed.
Acylpolyamines with the three amino acids asparagine (Asn), lysine (Lys), and
arginine (Arg) in their backbone were already described by Grishin et al. (A. lobata,
Araneidae)102 and Wakamiya et al. (Nephila species, Nephilidae)105-106. They were
isolated and their structures elucidated by NMR.
1
Somehow unusual seems the presence of the acylpolyamines N -(4-(2-amino-
5-(dimethylamino)pentanamido)butyl)-2-(2-(2,4-
dihydroxyphenyl)acetamido)succinamide (2,4-(OH) -PhAcAsn4Orn(NMe ),
see
2
2
1
Supplementary Material p. 27), and 2-(2-(1H-indol-3-yl)acetamido)-N -(4-
aminobutyl)pentanediamide (IndAcGln4, see Supplementary Material p. 20) as they
are, so far, the only derivatives in the venom that possess an ornithine or a glutamine
unit in their backbones. However, as for all other elucidated structures, the MS/MS
and the determined number of exchangeable H-atoms support the proposed
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structures. While glutamine was not found in a polyamine backbone before, ornithine
was earlier found in nephilatoxins (Nephila clavata), where the presence of ornithine
107
was confirmed by amino acid analysis .
Finally, the two bis-acetylated
azanediylbis(propane-3,1-diyl))bis(2,5-dihydroxybenzamide) (2,5-(OH) -Bz33-(2,5-
1
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polyamine
derivatives
N,N'-
(
2
(OH) -Bz))
see
Supplementary
Material
p.
23)
and
N-(3-((4-(2,5-
2
dihydroxybenzamido)butyl)amino)propyl)-2,5-dihydroxybenzamide (2,5-(OH) -Bz34-
2
(
2,5-(OH) -Bz)), mygalin, see Supplementary Material p. 25) were detected. Their
2
experimental data are in good agreement with the reported ESI-MS/MS spectrum of
mygalin that was isolated from the hemocytes of the spider Acanthoscurria gomesiana
Theraphosidae)108.
(
2+
Synthesis of 4-OH-IndAc3(Me )4(Me )3Arg (12)
2
2
2+
The synthesis of 4-OH-IndAc3(Me )4(Me )3Arg (12) was developed in order
2
2
to unequivocally confirm the structure of the active compound in the Pwx10 fraction
as compound 12 by comparison of structure and biological activity. The synthesis of
the N-Boc protected, methoxy-indole intermediate, 10, is shown in Scheme 1. In
reaction (1) N,N-dimethylpropane-1,3-diamine (1) was coupled to (S,Z)-5-(2,3-bis(tert-
butoxycarbonyl)guanidino)-2-((tert-butoxycarbonyl) amino) pentanoic acid (2) by the
action of the coupling reagent T3P to provide the key N,N-dimethylpropane tri-N-Boc
protected arginine amide (3), which will become the right hand side motif of 4-OH-
2+
IndAc3(Me )4(Me )3Arg (12). In reaction (2) 4-methoxy-1H-indole, 4, was reacted
2
2
with oxalyl chloride followed by quenching with methanol to produce methyl 2-(4-
methoxy-1H-indol-3-yl)-2-oxoacetate, 5. The reactive carbonyl in 5 is reductively
removed under palladium hydrogenation conditions to yield methyl 2-(4-methoxy-1H-
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indol-3-yl)acetate, 6, which is then hydrolysed to provide 2-(4-methoxy-1H-indol-3-
yl)acetic acid, 7. Compound 7 is then used to build the left-hand side motif of 4-OH-
2+
IndAc3(Me )4(Me )3Arg (12), first by amide coupling with diamine 1, by the action of
2
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T3P in Hunigs base and dichloromethane, followed by alkylation with 1,4-diiodobutane
to provide 4-iodo-N-(3-(2-(4-methoxy-1H-indol-3-yl)acetamido)propyl)-N,N-
dimethylbutan-1-aminium iodide, 9. Alkylation of 9 by the N,N-dimethyl amine of
intermediate 3 shown in reaction (4) provides the protected precursor, (S,Z)-N1-(6,11-
bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3-oxa-5,7,13-triazahexadec-
5-en-16-yl)-N4-(3-(2-(4-methoxy-1H-indol-3-yl)acetamido)propyl)-N1,N1,N4,N4-
tetramethylbutane-1,4-diaminium iodide, 10.
Scheme 2 shows the first approach at a stepwise removal of the protecting
groups by first removing the N-Boc protecting groups in reaction (5) by the action of
trifluoroacetic acid in dichloromethane to provide the methoxyindole 11. However, due
to the polarity of 11, we were unsuccessful in being able to further remove the indole
methoxy group for this compound. Therefore, as shown in reaction (6) 10 was treated
o
with boron tribromide at -78 C to remove the indole methoxy group under mild
conditions. Then by allowing the reaction mixture to slowly warm to room temperature
the reaction mixture became acidic enough to remove the N-Boc protection to provide
2+
4
-OH-IndAc3(Me )4(Me )3Arg (12), in a 45% yield after reverse phase HPLC
2 2
purification. Please see supplementary material for NMR and MS spectra for all
compounds.
Compound 12 was then fully characterized for comparison against the isolated
compound from the spider venom, and further evaluated for biological activity in
neurotransmitter transport and neuroprotection assays, as described below.
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Effects of Pwx10 fraction, and compounds 11 and 12 on neurotransmitter
transporter studies
Our results indicate that the natural Pwx10 fraction stimulated L-glutamate
uptake through EAAT1 and EAAT2 with no effects on EAAT3-mediated uptake. Figure
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7A shows dose response curves for the effect of the Pwx10 fraction, with the potencies
and efficacies are expressed as mean ± SD of three independent experiments. The
compound was found to have similar potencies for EAAT1 and EAAT2- mediated L-
glutamate uptake, with EC of 5.8 ± 2.9 nM for EAAT1 and 5.2 ± 2.4 nM for EAAT2.
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Efficacies of uptake augmentation were also similar, with 162 ± 11% for EAAT1 and
68 ± 9 for EAAT2. To validate the effect of natural Pwx10 fraction in an endogenous
system, we performed dose response assays for the Pwx10 fraction in cultured glia
Figure 7B), and demonstrated that the compound has a potency of 22 ± 7 nM and
1
(
efficacy of 314 ± 16% for L-glutamate uptake augmentation.
L-glutamate uptake kinetic analysis of the effect of different concentrations of
the Pwx10 fraction (10, 100 and 500 nM) in EAAT1 and EAAT2 transfected cells,
respectively, are shown in Figure 7C and D. For EAAT1-mediated uptake, Vmax values
were significantly increased, from 127 ± 15 pmol/well/min for control (vehicle)
conditions, to 168 ± 7, 313 ± 33 and 783 ± 23 pmol/well/min in presence of increasing
concentrations of the Pwx10 fraction. K values were not significantly different among
m
conditions. For EAAT2-mediated uptake, Vmax values were significantly increased,
from 133 ± 14 pmol/well/min for control (vehicle) conditions, to 170 ± 9, 309 ± 19 and
797 ± 26 pmol/well/min in presence of increasing concentrations of the Pwx10 fraction.
K values were not significantly different among conditions. These results that the
m
active compound in the Pwx10 fraction acts as a non-selective enhancer of the activity
of both subtypes of astrocytic L-glutamate transporters EAAT1 and EAAT2. Since the
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affinity for the substrate (K ) values were not significantly different among conditions
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(One-way ANOVA followed by Dunnett's post-hoc test comparing to vehicle), with an
average ± SD of 53 ± 4 M for EAAT1 and 52 ± 6 M for EAAT2, we propose that the
mechanism of stimulation to be through positive allosteric modulation of the
transporters. Figure 7E shows a kinetic experiment performed in cultured glia in
presence of different concentrations of the Pwx10 fraction, showing that the Vmax
values were significantly increased, from 19.7 ± 1 pmol/well/min for control (vehicle)
conditions, to 38.2 ± 1.3 and 59.4 ± 3.3 pmol/well/min in presence of 100 and 500 nM
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Pwx10 fraction. Similarly to the results in transfected cells, the K values were not
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significantly different among conditions (One-way ANOVA followed by Dunnett's post-
hoc test comparing to vehicle), with an average ± SD of 44 ± 3 M, suggesting once
again that the active compound in the Pwx10 fraction might be an allosteric modulator
of the activity of the transporters.
We proceed with our studies to examine the actions of synthesized compounds
11 and 12 (shown in Scheme 2). Compound 12 corresponds to the structure of the
potential active substance in the isolated Pwx10 fraction and compound 11 is a
structural analogue that has a methoxy group on the left-hand side indole ring instead
of a hydroxyl group. Dose response curves for the effects of 12 and 11 in transfected
COS-7 cells are shown in Figure 8A and B, respectively. Acylpolyamine 12 increases
EAAT1-mediated L-glutamate uptake with an EC of 0.4 ± 0.2 nM and efficacy of 171
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±
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15%, and for EAAT2-mediated uptake with an EC of 0.8 ± 0.1 nM and potency of
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44 ± 7%, with no effect on EAAT3-mediated L-glutamate uptake. Figure 8C shows
dose response assays for 12 and 11 in cultured glia, demonstrating that the
compounds increase L-glutamate uptake in this approach, however, the potency of 12
is higher (EC of 21 ± 0.6 nM) as compared to 11 (EC of 690 ± 50 nM), whereas
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efficacies for augmentation were similar (178 ± 9% for 12 and 155 ± 9% for 11),
indicating that the methoxy group in 11 results in lower potency for L-glutamate
augmentation.
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Kinetic analysis of L-glutamate uptake in presence of different concentrations
of 12 and 11 are shown in Figures 8D-F. For EAAT1-mediated uptake (figure 8D), Vmax
values were significantly increased, from 185 ± 15 pmol/well/min for control (vehicle)
conditions, to 303 ± 26 and 723 ± 44 pmol/well/min in presence of 100 and 500 nM of
12, and to 241 ± 22 and 475 ± 27 pmol/well/min in presence of 100 and 500 nM of 11.
For EAAT2-mediated uptake (Figure 8E), Vmax values were significantly increased,
from 127 ± 12 pmol/well/min for control (vehicle) conditions, to 262 ± 22 and 613 ± 55
in presence of 100 and 500 nM 12, and to 271 ± 19 and 586 ± 22 pmol/well/min in
presence of 100 and 500 nM 11, respectively. K values were not significantly different
m
among conditions (One-way ANOVA followed by Dunnett's post-hoc test comparing
to vehicle), with an average ± SD of 62 ± 22 M for EAAT1 and 57 ± 7 M for EAAT2.
These results demonstrate that 12 and 11 act as a non-selective enhancer of the
activity of both subtypes of astrocytic L-glutamate transporters EAAT1 and EAAT2,
with potentially an allosteric mechanism, since the affinity for the substrate (Km) value
was not different. Figure 8F shows kinetic experiments performed in cultured glia
showing that the Vmax values were significantly increased, from 18.1 ± 1 pmol/well/min
for control (vehicle) conditions, to 33 ± 2 and 55 ± 2 pmol/well/min in presence of 100
and 500 nM 12 and 28 ±1 and 37 ±2 pmol/well/min in presence of 100 and 500 nM
1
1. Similarly to the results in transfected cells, the K values were not significantly
m
different among conditions (One-way ANOVA followed by Dunnett's post-hoc test
comparing to vehicle), suggesting once again that these compounds enhance the
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glutamate translocation rate, with no effect on substrate interaction, suggesting an
allosteric mechanism.
Collectively, results in Figure 8 indicate that the bioactivity of synthetic compounds
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12 and 11 match the activity of the natural Pwx10 fraction, with 11 being slightly less
potent in cultured glia cells. This data suggests that the 4-methoxy substituent on the
indole ring of 11 does not negatively impact the biological activity of this component
and inspires the synthesis of additional substituted indole analogs in the future to
elaborate on the structure activity relationships for this motif. Notably other polyamine
spider toxins containing a 4-hydroxy-indole acetic acid moiety, such as in 12, are found
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to be less toxic than those with a hydroxyphenyl-based aromatic head group . With
these results of the activity study and the confirmation that the structure of compound
12 corresponds to the acylpolyamine in the P. bistriata venom, we conclude that the
2+
acylpolyamine 4-OH-IndAc3(Me )4(Me )3Arg (12) is the active compound in the
2
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fraction Pwx10.
Neither the natural Pwx10 fraction nor the synthetic compounds 12 and 11 have
any effect on GABA uptake mediated by GAT-1 and GAT-3, glycine uptake mediated
by GlyT1 and GlyT2, and on monoamine transporters (human serotonin (hSERT),
noradrenaline (hNET) and dopamine (hDAT), demonstrating these compounds are
selective for L-glutamate transporters (supplementary Figure 3).
Previous studies of the three-dimensional structure of the EAAT reveal that the
transporter is divided into two domains: a transport domain containing the substrate
binding sites, and surrounding it a peripheral rigid scaffold, designated the
trimerization domain110-111. The trimerization domain provides the interaction interface
between the three subunits of the trimer and is believed to facilitate the elevator-like
movement of the inner transport domain112-113. Our previous work suggests that
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residues at the interface between the transport and the trimerization domains are
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critical for transport stimulation . Intriguingly, preliminary modeling analysis of the
three-dimensional structure of the EAAT, searching for residues that are similar in
EAAT1 and EAAT2 (but different in EAAT3), revealed multiple molecular determinants
that could be involved in the interaction of compound 12 with transporters EAAT1 and
EAAT2 (not shown). Therefore, at this point, we are unable to propose a mechanism
of interaction between compound 12 and EAATs. However, we speculate that the
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compound might be binding to the similar previously identified allosteric site , and
possibly accelerating the rate of the transport by facilitating the movement of the
transport domain, like the recently identified positive allosteric modulators (PAMs) of
EAAT281-82. Therefore, future studies including additional molecular modeling and
mutagenesis of key residues are needed to investigate mechanisms of interaction of
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4-OH-IndAc3(Me )4(Me )3Arg (12) with the glial transporters.
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Regrettably, other PAMs and negative allosteric modulators (NAMs) of
transporters with similar structures of 12 are not available to date. However, several
allosteric modulators of glutamate receptors, such as AMPA, NMDA and metabotropic
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glutamate receptors, have been identified . For instance, polyamines spermine and
spermidine act as PAMs of the NMDA receptor to potentiate channel activity. It is
thought that they act by binding to the amino-terminal domain, resulting in the
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separation of the GluN1R and GluN2R domains and opening of the ion channel .
Due to remarkably different functions and mechanisms between these receptors and
the EAATs, we refrain to suggest any mechanistic similarities of these PAMs and
compound 12. However, polyamines from spider venoms have been studied for a long
time, and it is well stablished that many of them act as ionotropic glutamate receptor
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antagonists of the vertebrate and invertebrate central nervous systems . Some
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examples include the polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila
polyamine toxins-1 and -8 (NPTX-1 and NPTX-8), isolated from the venom of the orb-
weaver spider Nephila clavata (Joro spider). These are very potent open-channel
blockers of ionotropic glutamate receptors, providing pharmacological tools for studies
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of these receptors . Likewise, compounds 11 and 12 could be used in the future as
pharmacological tools to study the molecular scaffolds in the glial transporters that
they modulate.
Effects of Pwx10 fraction and compound 12 on excitotoxic insults in mixed
neuron-glia cultures
We evaluated Pwx10 fraction and the acylpolyamine 4-OH-
2+
IndAc3(Me )4(Me )3Arg (12) for neuroprotective effects after prolonged (24 h)
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excitotoxic insults with application of L-glutamate or acute (20 min) insults with oxygen-
glucose deprivation (OGD), in mixed neuron-glia cultures. Our cultures had optimized
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growth conditions to result in ~15% of glia growth in the cultures after 14 DIV . As
positive control for neuroprotection, we used APV (DL-2-Amino-5-
phosphonopentanoic acid), an NMDA receptor blocker. We examined the
neuroprotective potential of these compounds by immunocytochemical analysis,
comparing MAP-2 expression among different experimental groups, as previously
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2
described .
Neuronal survival after L-glutamate insult and treatments with Pwx10 fraction
and compound 12
Cultures were grown for 14 DIV before treatments, then subjected to 24h insult
in presence of vehicle (control), or natural Pwx10 fraction or synthetic compound 12.
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Figure 9A show representative images of cultures on different conditions. At
baseline conditions (no L-glutamate insult or compound treatment - vehicle), neurons
did not display pyknotic nuclei or neurite damage, which are indicators of neuronal
death117-118. Neurons treated with Pwx10 fraction alone (100 M; 24 h) also resemble
the baseline vehicle conditions, suggesting that the components in the Pwx10 fraction
are not neurotoxic. After L-glutamate insult, we observed reduced MAP-2 staining and
DAPI positive, MAP-2 negative cells, indicators of cell death. On the other hand,
neurons subjected to acute L-glutamate insult in presence of Pwx10 fraction (100 nM;
24 h) displayed morphological characteristics similar to baseline vehicle conditions,
suggesting that this compound is neuroprotective in an excitotoxic environment.
Total neuronal survival levels from different experimental conditions were
quantified. Figure 9 B illustrates the neuroprotective effects of both Pwx10 fraction and
compound 12, following the application of 100 µM L-glutamate insult in 14 DIV mixed
neuron-glia cultures. Application of 100 µM L-glutamate significantly decreased
neuronal survival to 25.9 ± 15.4% of baseline vehicle levels (red bar). Only application
of 100 nM Pwx10 fraction or 12 after L-glutamate insult resulted in a significant
neuroprotective effect, increasing survival levels to 70.5 ± 13.4 (Pwx10 fraction) and
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64.4 ± 0.5% (12) of baseline whereas 1 nM and 10 nM of the Pwx10 fraction did not
increase neuronal survival compared to the insult condition. To determine if the
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glutamate transporter, we subjected mixed cultures to prolonged L-glutamate insult,
followed by co-treatment with 100 nM Pwx10 fraction and the L-glutamate transport
inhibitor TBOA (100 µM), that inhibits all transporter subtypes at this concentration,
and with the selective EAAT2 inhibitor WAY 213613 (1 µM). Co-treatment of 100nM
of Pwx10 fraction with both L-glutamate transport blocker TBOA and EAAT2 specific
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inhibitor WAY 213613 abolish the neuroprotective effect of the compound. Co-
treatment with TBOA results in a more significant knockdown of the neuroprotective
effects of Pwx10 fraction compared to co-treatment with WAY 213613, which is
consistent with our findings that the Pwx10 fraction increases L-glutamate uptake
through both EAAT1 and EAAT2. Finally, treatment with positive control APV results
in significant neuroprotection, with survival at 80.7 ± 2.7% of baseline, confirming the
viability of our cultures, and suggesting that excessive L-glutamate transmission
through the NMDA receptor plays a crucial role in L-glutamate excitotoxicity and
neuronal death119-120, thus providing a positive control to measure the effect of the
active compound in the Pwx10 fraction.
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To better understand the contribution of the different subtypes of EAATs in the
mixed neuron-glia system, L-glutamate uptake assays in presence of several inhibitors
were performed. Supplementary Figure 3 indicates that L-glutamate uptake in
presence of 1 M WAY 213613 inhibits 50 ± 5% of L-glutamate uptake. At this
concentration, this inhibitor is selective for EAAT2 inhibition over EAAT1 and EAAT3,
indicating that ~ 50% of the transporters in this approach are EAAT2. On the other
hand, UCPH-101, at 1 M, a concentration that displays a selective inhibition of
EAAT1 over the other subtypes, inhibits 23 ± 8% of L-glutamate uptake, suggesting
that ~25% of the transporters in this system are comprised of EAAT1. When co-
incubated with both inhibitors, the transport is inhibited to a level of 6.5 ± 0.5%,
indicating the L-glutamate uptake in these cultures is mediated by EAAT1 and EAAT2,
with a small percentage of uptake probably due to EAAT3.
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Neuronal survival after Oxygen Glucose Deprivation (OGD) insult and Pwx10
fraction and compound 12 treatments
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(12) in an in vitro stroke model, by subjecting the cultures to OGD insults. Figure 10 A
shows representative images of vehicle (control) cultures, under OGD conditions, and
under OGD followed by treatment with 100 nM Pwx10 fraction. As seen above, vehicle
(control) cultures do not display pyknotic nuclei or neurite damage, as opposed to
OGD-treated cultures, in which there is a reduction in MAP-2 staining and DAPI
positive, MAP-2 negative cells, indicators of cell death. On the other hand, neurons
subjected to OGD insult followed by incubation of Pwx10 fraction (100 nM; 24 h)
displayed morphological characteristics like baseline vehicle conditions, suggesting
that this compound is neuroprotective in an excitotoxic environment. The
quantification, showed in Figure 10 B, illustrates that the Pwx10 fraction and
compound 12 displayed neuroprotective effects following 20 minutes of OGD.
Application of 10nM and 100 nM Pwx10 fraction and acylpolyamine 12 following the
OGD insult significantly increasing neuronal survival from 29.5 ± 9.6% of baseline to
70.4 ± 9.6% and 78.7 ± 4.2%, respectively. Intriguingly, the concentration of 10 nM
Pwx10 fraction was able to confer neuroprotection in this OGD model, unlike following
L-glutamate insults, where only 100 nM was effective. Future experiments determining
extracellular concentration of L-glutamate in both models could help examine the
discrepancy in effectiveness between the models. Pwx10 fraction at 1nM does not
display significant neuroprotective effects, only increasing neuronal survival to 49.7 ±
21.6% compared to the control condition. Synthetic compound 12 at 100 nM displays
similar neuroprotective effects compared to the natural compound, increasing
neuronal survival to 89.3 ± 23.9%. Co-administration with TBOA and WAY 213613
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both result in no increases in neuronal survival, and this indicates again that the
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neuroprotective effects of 4-OH-IndAc3(Me )4(Me )3Arg (12) from OGD-induced
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injury is due to modulation of L-glutamate transporters.
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Previous studies evidence that L-glutamate excitotoxicity plays an essential role
in the neurodegeneration observed following ischemic stroke and key elements
121
involved in excitotoxicity , like L-glutamate transport, may serve as potential targets
for neuroprotection after ischemic stroke. In this study, we have shown that 4-OH-
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IndAc3(Me )4(Me )3Arg (12) was neuroprotective in this model of in vitro insult. It is
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important to note that under ischemic conditions, an increase in extracellular L-
glutamate can happen by reversal of uptake122-123. However, upon cessation of the
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insult, it has been shown that L-glutamate transport activity recovers . We speculate
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that 4-OH-IndAc3(Me )4(Me )3Arg (12) is still able to accelerate the removal of L-
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glutamate to restore homeostasis during the 24h recovery time period following the
insult. This concept is supported by previous work with EAAT2 expression enhancers,
including ceftriaxone and riluzole, that offer significant protection of neuron tissues
against ischemic excitotoxicity19, 125-128. In future studies, we plan to determine the L-
glutamate concentration in culture media after insult and compound treatments to
complement and strengthen the notion that the mechanism of 4-OH-
2+
IndAc3(Me )4(Me )3Arg (12) is through increased EAAT1/EAAT2 activity resulting
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in increased L-glutamate clearance. Additionally, future studies will determine whether
NMDA, AMPA and kainate glutamate receptors modulation is involved, as inhibition of
these could result in neuroprotection (as seen in our assays with APV, an NMDA
receptor blocker). We will also examine whether metabotropic L-glutamate receptors
modulation is involved in the action of compound 12 on transporters, as these
receptors can exert different actions (toxic or compensatory) in response to excitotoxic
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stimuli . Future studies will also investigate whether apoptosis is occurring, and
which intracellular signalling pathways are involved in the insult models.
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Conclusions
In this work, we attempted to elucidate the chemical structures of compounds with
intriguing and promising biological activities that are part of three HPLC fractions that
were collected from the venom of P. bistriata spiders, namely Pwx1, Pwx2 and Pwx10.
Furthermore, we have shown that the P. bistriata venom contains 36 acylpolyamines
with and without amino acid linkers in their structures, a unique finding as both types
of substances were never found coexisting in a spider venom until now. In addition,
the presence of nucleosides and other small compounds such as amino acids free
polyamines and quaternary amines was shown. Regrettably, our approach did not
yield the identification of the chemical structure of the active substance in the Pwx1
fraction. Nonetheless, previous studies used this fraction to identify the region of L-
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glutamate transporter EAAT2 responsible for transport stimulation . This important
knowledge was used to identify novel compounds that were shown to be positive
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allosteric modulators of EAAT2 . We speculate that one or several unknown minor
active components, have, at least in part, a similar structure of these allosteric
modulators. We consider that the identification of novel positive allosteric modulators
(
PAMs) of EAAT2 holds promise to further drug development and to further our
understanding of allosteric modulation of L-glutamate transporters.
Regarding the compound found at m/z 175.2 in the Pwx2 fraction, we could not
confirm the presence of the previously proposed structure 2-amino-5-
ureidopentanamide (FrPbAII) in P. bistriata venom, but the amino acid arginine.
Unfortunately, neither arginine nor any of the additional constituents (betaine,
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