Development of Tetramethylenedisulfotetramine (TETS) Hapten Library: Synthesis, Electrophysiological Studies, and Immune Response in Rabbits.

Article abstract of DOI:10.1002/chem.201700783

There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABA_A) receptors. General synthetic approach toward two groups of analogues was dev

Full text of DOI:10.1002/chem.201700783

A Journal of
Accepted Article
Title: Development of tetramethylenedisulfotetramine (TETS) hapten
library: synthesis, electrophysiological studies and immune
response in rabbits
Authors: Bogdan Barnych, Natalia Vasylieva, Tom Joseph, Susan
Hulsizer, Hai Nguyen, Tomas Cajka, Isaac Pessah, Heike
Wulff, Shirley Gee, and Bruce Hammock
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To be cited as: Chem. Eur. J. 10.1002/chem.201700783
Link to VoR: http://dx.doi.org/10.1002/chem.201700783
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10.1002/chem.201700783
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Development of tetramethylenedisulfotetramine (TETS) hapten
library: synthesis, electrophysiological studies and immune
response in rabbits.
Bogdan Barnych,[a] Natalia Vasylieva,[a] Tom Joseph,[a] Susan Hulsizer,[b] Hai M. Nguyen,[c] Tomas
Cajka,[d] Isaac Pessah,[b] Heike Wulff,[c] Shirley J. Gee,[a] Bruce D. Hammock*[a]
Abstract: There is a need for fast detection methods for the banned
rodenticide tetramethylenedisulfotetramine (TETS), a highly potent
blocker of the γ-aminobutyric acid (GABAA) receptors. General
synthetic approach toward two groups of analogues was developed.
Screening of the resulting library of compounds by FLIPR or whole-
cell voltage-clamp revealed that despite the structural differences
some of the TETS analogues retained GABAA receptor inhibition,
however their potency was an order of magnitude lower. Antibodies
raised in rabbits against some of the TETS analogues conjugated to
protein recognized free TETS and will be used for the development
of an immunoassay for TETS.
highly toxic rodenticides such as TETS.[2] Access to TETS in
China and its illegal export to other countries necessitates better
methods for its detection.[2] Current methods for the detection of
TETS are mainly GC-MS based and thus require a laboratory
setting, are laborious and expensive limiting their use.
Immunoanalytical methods, on the other hand, are widely used
for the detection of small molecules in different matrices[4] and
have the advantage of being cheap, portable and high
throughput. However, they require conjugation of the small-
molecule analyte to a larger protein in order to generate an
appropriate immune response and raise analyte-selective IgG
antibodies.
Tetramine has a unique chemical structure including a rigid cage
and multiple heteroatoms that may provide recognition points for
antibodies. However, TETS lacks reactive functional groups that
could be easily functionalized and used as the attachment points
for the preparation of the immunizing and coating antigens.
Therefore, haptens have to be synthesized de novo, not by
modification of the target analyte (TETS) or its precursors as it is
typically done for other analytes.[5] An additional challenge is to
develop a synthetic route that would exclude production of free
TETS as a by-product. These are probably the main reasons for
the lack of an immunoassay for TETS to date. Interestingly, one
monoclonal antibody (mAb) developed against cyclodiene
pesticides, such as aldrin, was shown to cross-react with
TETS.[6] However, due to significant structural differences
between cyclodienes and TETS, the affinity of the monoclonal
antibody (mAb) to TETS was low (IC50 = 3 µM or 0.72 µg/mL)
and not suitable for analytical use. Thus, the development of
structurally close TETS analogues possessing active functional
groups will facilitate development of a cheap immunoanalytical
method for its detection, and may be useful for regulatory and
enforcement agencies charged with environmental, agricultural
and homeland security. Additionally, such analogues could be
used for the development of photoaffinity labels allowing
identification of the TETS binding site, in-depth study of the
mechanisms of its toxicity and evaluation of treatment options.
Therefore, in this work we developed a synthetic route to
generate a library of TETS-like compounds. The potency of
these compounds as excitotoxicants were assessed in
bioassays with primary cultures of mouse hippocampal neurons
and cultured cells expressing human GABAA receptors, and
compared directly to TETS. The most promising analogues were
conjugated to the carrier protein and injected in rabbits to
produce polyclonal antibodies.
Introduction
TETS (tetramethylenedisulfotetramine, tetramine) is a highly
lethal neurotoxic rodenticide. It is a non-competitive channel
blocker of the γ-aminobutyric acid (GABAA) receptors that
induces excessive excitation of the adult central nervous system
(CNS). The LD50 in laboratory animals is 0.1 mg/kg, and 7-10 mg
is considered to be a lethal dose for humans.[1] Despite being
banned worldwide it is still available on the black market in
China and other countries because of its ease of manufacture,
profitability and effectiveness as a rodenticide.[2] The illegal use
of TETS has led to multiple accidental human poisoning cases.
Additionally, being water soluble, odorless and tasteless it has
been often used in intentional poisonings and thus is considered
as
a
potential threat agent.[3] More than three thousand
poisonings between 2000 and 2012 in China have been
associated with TETS.[3b]
China implemented multiple regulatory enforcement measures
which had a positive impact on the frequency of such events but
failed to completely remove TETS-containing products from the
open market because of the lack of technical means to test for
[a]
Dr. B. Barnych, Dr. N. Vasylieva, T. Joseph, S. J. Gee, Prof. B. D.
Hammock; Department of Entomology and Nematology, and UCD
Comprehensive Cancer Center, University of California Davis,
Davis, California 95616, United States
E-mail: bdhammock@ucdavis.edu
[b]
[c]
[d]
S. Hulsizer, Prof. I. Pessah; Department of Molecular Biosciences,
School of Veterinary Medicine, University of California Davis, Davis,
California 95616, United States
Dr. H. M. Nguyen, Prof. H. Wulff; Department of Pharmacology,
School of Medicine, University of California Davis, Davis, California
95616, United States
Dr. T. Cajka; UC Davis Genome Center-Metabolomics, University of
California Davis, Davis, California 95616, United States
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201700783
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Results and Discussion
substituted analogues 4 (Me = R1 ≠ R2). Unfortunately,
condensation of equimolar amounts of N-methylsulfamide and
methyl 6-(sulfamoylamino)hexanoate with polyformaldehyde not
only gave poor yields of asymmetric analogue 4b (Table 1) but
also its separation from the symmetrical analogues was
complicated.
We designed four types of analogues with different degrees of
similarity to TETS (Scheme 1). Although overall surface
complementarity is considered to be an important determinant
for antigen recognition, specific interactions like electrostatic and
hydrogen bonding are frequently more critical determinants of
antibody affinity.[7] It is therefore generally accepted that a good
hapten will preserve the distinctive functional groups as well as
the overall antigen structure.[8] Additionally, to ensure that
distinctive functional groups remain well exposed and available
for interaction with the antibody, the spacer arm should be as
remote from them as possible.[8c] Following these considerations,
TETS analogue 1 having a linker arm attached to one of the
methylene bridges should be an ideal hapten because it
preserves all the structural features of the parent compound like
the adamantane structure and both sulfamide functions.
Theoretically, its synthesis would involve co-condensation of
formaldehyde and aldehyde with sulfamide resulting in formation
of the mixture of TETS-like compounds including analogue 1
and TETS. Clearly, this approach would suffer from drawbacks
such as poor yields and complicated chromatographic
separation of the desired product. Most importantly, the
possibility of formation of analogue 1 or its stability is doubtful. At
least under standard reaction conditions, previous publications
show that replacement of formaldehyde by more bulky
aldehydes in the condensation reaction with sulfamide precludes
the formation of the tricyclic core.[9] Thus either structural or
functional group modifications were required during the process
of hapten design.
Table 1. Synthesis of TETS analogues
2a (92 %)
2b (87 %)
2c (53 %)
2d (85 %)
2e (9.4 %)
2f (30 %)
2g (55 %)
2h (5.2 %)
2i (79 %)
3a (-)
2j (61 %)
3b (-)
Scheme 1. Design of compounds with functional determinants similar to TETS.
It was shown previously that monoalkyl or monoaryl sulfamides
could be engaged in a similar reaction with formaldehyde as a
parent nonsubstituted sulfamide giving TETS-like compounds 4
lacking one methylene bridge (Scheme 1).[9-10] Although
synthetically very attractive this approach would result in
haptens having two linker units that may cause complications
during conjugation or negatively influence TETS recognition.
This problem could be overcome by using asymmetrically
4b (<20 %, crud)
4a (73 %)
7 (-)
Next, we studied whether compounds having two sulfamide
functions connected through a variable-length linker undergo
intramolecular condensation with formaldehyde to give tricyclic
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TETS like compounds. To answer this question disulfamides 8
had to be synthesized. Among multiple synthetic procedures
available[11] we chose the method described by Masui et al.[12]
because of its reported water tolerance and high product yields.
Thus, reaction of the CSI (chlorosulfonyl isocyanate) with tert-
butanol followed by the addition of the triethylamine and primary
amine provided the Boc protected sulfamides 8 in good to
excellent yields (Supporting Information, SI). The next steps
called for cleavage of the Boc protecting group followed by
condensation with formaldehyde. Since acidic media is
necessary for both reactions these two steps were tested in a
one-pot procedure. Since the bicyclic TETS analogue 4a is
known, this condensation reaction was first tested with tert-butyl
(N-methylsulfamoyl)carbamate 8k and produced the compound
having identical physicochemical characteristics to the
previously reported 4a in 73 % yield. Next this reaction was
tested with a simple di-Boc protected disulfamide derived from
easily available ethylene diamine. Reaction of di-tert-butyl (((2-
only 5.2 % yield was the disulfamide 2h structurally related to 2e,
but missing -CH2OCH2- bridge. Its structure was confirmed by
spectroscopic methods and X-ray crystallography. Replacement
of the ethylene bridge by phenylene was well tolerated (Table 1,
2i-j).
Studies of NOE and COSY spectra of analogues 2 revealed the
following interesting features. A NOE effect was observed
between spatially close pairs of hydrogens of the methylene
bridges H1a-H2b and H1b-H3b, whereas spin-spin correlations in
the COSY spectra were observed only for distant protons of the
same methylene groups H1b-H2a and H1a-H3a (Figure 1).
Modeling of the analogues 2 revealed that the systems H-C-N-
C-H for which these spin-spin correlations were observed are
almost flat and thus they could be attributed to long-range “W”-
type correlations.[13] A NOE effect was also observed between
spatially close protons of methylene and ethylene bridges H2a-
H6a and H3a-H5b, and therefore could be used to confirm the
relative stereochemistry in these analogues.
(hydrosulfonylamino)ethyl)amino)sulfonyl)dicarbamate
with
dimethoxymethane in trifluoroacetic acid gave the TETS
analogue 2a as a racemic mixture in 92 % yield (Table 1). To
investigate the scope of this transformation and optimize
reaction conditions a variety of differently substituted 1,2-
diamines were tested. Since the choice of commercially
available functionalized diamines is limited the substrate scope
was first tested with most commonly available nonfunctionalized
diamines followed by functionalized ones, which were obtained
by multistep synthesis as described in the supporting information.
Presence of aliphatic or carboxylate substituents on the ethylene
bridge proved to be tolerable, however in this case the products
were obtained as a mixture of two diastereomers (Table 1, 2b-d).
Although, flash column chromatography has been tested as a
purification technique, due to relatively low polarity and very
poor solubility of 2 in non-polar solvents, use of this technique
for purification was complicated. The major products were
obtained in pure or almost pure form by recrystallization from
methanol. Interestingly, introduction of a benzyl group on the
ethylene bridge not only dramatically deteriorated the reaction
yield but also resulted in formation of product 2e with an
unexpected structure. Replacement of the benzyl group by p-
nitrobenzyl resulted in formation of a mixture of at least 2
products which after recrystallization gave pure 2f in 30 % yield.
Comparison of the NMR spectra of crude product, 2f and 2e
revealed that chemical shifts of the minor product from the
mixture were very similar to those of 2e and thus it likely had the
same structure. Absence of the TETS-like product in the case of
disulfamide 8e can be explained by intramolecular
sulfamidoalkylation of the aromatic ring resulting in the formation
of benzo-annelated side-products similar to a previous report.[9]
Disulfamide derived from (1R,2R)-cyclohexane-1,2-diamine also
reacted with dimethoxymethane resulting in formation of TETS
analogue 2g in 55 % yield as a single diastereoisomer. RS-
cyclohexane-1,2-diamine, on the other hand, failed to give the
desired tricyclic product probably because of considerable van
der Waals repulsive forces between the tricyclic core and the
axial hydrogens of the cyclohexane moiety which render this
compound unstable. The only product which was obtained in
Figure 1. Significant NOE and spin-spin correlations observed for 2.
Next, we tested if one of the methylene bridges in TETS could
be replaced by propylene or if it could be completely deleted.
For this purpose, we synthesized di-Boc protected disulfamides
starting from 1,3-diaminopropane, 1,8-diaminonaphthalene and
hydrazine, which were then reacted with dimethoxymethane in
trifluoroacetic acid (Table 1, 3a, 3b, 7). All three reactions
resulted in complex reaction mixtures showing no sign of the
desired product by NMR spectroscopy or mass spectrometry.
For 3a-b this result compares well with literature data showing
that unlike ethylenediamine, 1,3-diaminopropane does not give a
tricyclic condensation product with formaldehyde presumably
because of steric factors.[14] However, it is likely that faster
alternative condensation reactions compared to the formation of
the eight-membered ring, required for construction of 3a-b
skeleton, are responsible for absence of the desired product.
We also studied approaches to synthesize TETS analogues 5
and 6 which possess a lower degree of similarity to the parent
molecule, but still have an adamantane-like structure and one of
the two sulfamide functions preserved. Hapten 5 was very
attractive from the synthetic point of view since precursors –
bispidine derivatives have been described and are easily
accessible, thus leaving solely the feasibility of sulfamide bridge
formation uncertain. Thus, after the intermediates 10 and 11
were synthesized (Scheme 2) according to
a literature
procedure[15] the possibility of introduction of a sulfamide bridge
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was studied. For this transformation we first tested the reaction
conditions employing sulfuryl chloride as sulfurylation reagent
and triethylamine or pyridine as a base. In both cases, reactions
resulted in complex mixtures as judged by NMR spectra. To
overcome this problem a range of alternative sulfurylation
reagents were tested, but none were successful at introducing
the sulfamide bridge in bispidine derivatives 10, 11.
Scheme 3. First synthetic approach toward hapten 6a.
Scheme 4. Synthesis of the hapten 6a.
Elimination of the deprotection step was envisioned to avoid this
problem and to considerably shorten the synthetic pathway.
Thus, the unprotected triamino alcohol 18, an analogue of 16,
was prepared in two steps from commercial pentaerythritol
tribromide by nucleophilic substitution with azide ion followed by
palladium catalyzed hydrogenation[17] (Scheme 4). The resulting
triamine 18 was transformed into TETS analogue 6a by
treatment with catechol sulfate followed by addition of
formaldehyde. The yield of 6a (8.4 – 15 %) was considerably
lower than the yield for 17. This might be attributed to poor
solubility of the reaction intermediates obtained from 18 in
dioxane (gummy substance was observed in the reaction
mixture) leading to higher amounts of polymeric sulfamides.
Scheme 2. Synthesis of bispidine derivatives 10-11 and tentatives of their
sulfurylation.
In parallel to our studies toward the TETS analogue 5 we also
explored approaches aiming at synthesis of TETS analogues 6.
Among the multitude of possible radicals R in the TETS
analogue with general formula 6 (Scheme 1), we chose
hydroxymethylene (HOCH2-) and amino groups because of the
availability of precursors pentaerythritol and Tris base. Synthesis
of compound 6a commenced by preparation of the known
triamine 16[16] (Scheme 3). Briefly, pentaerythritol was first
converted into monobenzyl derivative 15 via orthoester
protection, alkylation and deprotection sequence. Tosylation of
derivative 15 followed by nucleophilic substitution with azide ion
and palladium catalyzed hydrogenation gave triamine 16.
Transformation of 16 into the adamantane-like sulfamide 17 was
achieved in 24 - 45 % yield by its treatment with an equimolar
amount of catechol sulfate under reflux followed by addition of
formaldehyde. Alternatively, refluxing triamine 16 with sulfamide
in pyridine followed by the addition of formaldehyde also gave
the tricyclic sulfamide 17, but in lower yield. The next step was
benzyl deprotection using hydrogen and palladium on carbon.
However, despite all efforts, the hydrogenation either did not
proceed or was not chemoselective as concomitant
hydrogenative cleavage of the C-N bond at the methylene bridge
was occurring.
Scheme 5. Synthesis of the hapten 6b.
Synthesis of TETS analogue 6b started from Tris base, which
was first transformed into trichloride followed by the azidation
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and protection of the amine function by a Boc group to give
intermediate triazide (Scheme 5).[18] Palladium catalyzed
hydrogenation of this triazide gave monoprotected tetraamine
19[19] that was reacted with catechol sulfate in refluxing dioxane
followed by reaction with formaldehyde at RT. Treatment of the
resulting intermediate 20 with trifluoroacetic acid liberated the
free TETS analogue 6b in quantitative yield. All products and
intermediates were characterized by NMR and other
spectroscopic methods. Additionally, the identities of compounds
2e, 2g - 2j, 4a, 17 and 20 were proven by X-ray crystallography
(see SI).
contrast, even though sera obtained from rabbits immunized
with haptens 6b and 6a still recognized the corresponding
immunizing haptens, their binding was not altered by TETS.
These data suggest that either haptens 6b and 6a are fairly
distinct from the TETS structure and thus sera do not recognize
the analyte or the affinity of the developed antibodies is much
greater for the haptens and thus the concentration of TETS
tested is not high enough to produce a visible inhibition effect.
Here we present promising preliminary data for immunoassay
development from rabbit sera immunized with haptens 2. Further
evaluation of developed antibodies against TETS is a subject of
a separate study.[21]
Development of immune response in rabbits. Analogues 2d,
2j, 2k (nitro group reduced to amine in 2f), 6a and 6b were
selected from our library for animal immunization. These
compounds represented the diversity of the synthesized library
and possessed functional groups that could be used for
chemical linking to the carrier protein. Thyroglobulin was chosen
as a carrier protein for immunization because of its high
immunogenicity and ease of use. Bovine serum albumin and
conalbumin were used as carrier proteins in the preparation of
coating antigens. For the conjugation 6a and 6b were first
reacted with succinic anhydride to give corresponding
monoester and monoamide respectively. The resulting
monosuccinates of 6, as well as haptens 2d, 2j and 2c (2c was
only used for making coating antigen) were then directly
conjugated to the carrier protein via carboxylic acid function
using the standard activated ester method. Amine 2k was
conjugated to the carrier protein by using either diazotization or
glutaraldehyde methods (see SI).[20]
TETS analogues as tools for biological applications. The
history of terror acts involving chemical agents[22] has raised
concern about banned substances with high toxicity, including
TETS. For instance, within the U.S. Department of Health and
Human Services, the NIH is making a significant effort to pursue
the
development
of
new
and
improved
medical
countermeasures designed to prevent, diagnose, and/or treat
the pathology caused by TETS.[23] To develop a successful
treatment approach for the poisoning it is important to
understand the mechanism of action of the agent. So far, TETS
has only been identified to block the GABA receptor chloride
channel (GABAAR), but the binding site remains unidentified.
Knowledge of structure-activity relationship of TETS and
analogues may prove to be useful in this regard, for example by
helping design photoaffinity probes.
Figure 3. Screening for neuroactive TETS analogues with the FLIPR bioassay
in cultured hippocampal neurons. The SCO amplitude after the addition of
TETS at 10 µM or TETS analogs at 30 µM, compared to the addition of 0.03%
DMSO vehicle control. The mean amplitude for each well after the addition is
compared to the mean amplitude for that well before the addition, then
normalized to the vehicle response. n = 7 or 8 for TETS and analogues; n = 12
for vehicle. ANOVA comparison with vehicle control, *** indicates p < 0.0001,
all other differences were not significant from vehicle. Bars indicate SEM.
Figure 2. Inhibition of antibody binding on homologous coating antigen in the
presence of TETS at 5 mg/L. Each bar represent serum from individual animal.
After immunization of rabbits, serum from the final bleed was
analyzed in a competitive ELISA format with a concentration of
TETS of 5 mg/L. The aim of this experiment was to study if
synthetic analogues used for immunization could elicit an
immune response and result in antibodies that recognized TETS.
Figure 2 demonstrates that sera from rabbits immunized with
haptens 2j, 2k and 2d recognized the corresponding haptens
and its binding was to some extent inhibited by TETS. By
Effects of TETS and TETS analogs on Ca2+ Oscillations in
Primary Cultured Hippocampal Neurons. For initial screening for
neuroactive analogues we used a FLIPR bioassay detecting
Ca2+ oscillations in cultured hippocampal neurons. Cultured
hippocampal neurons (13–28 days in vitro (DIV)) display a
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balance of glutamatergic (excitatory) and GABAergic (inhibitory)
signaling, which results in spontaneous synchronous Ca2+
oscillations of approximately 10 second duration. Application of
GABAA receptor (GABAAR) antagonists such as picrotoxin,
bicuculin and TETS, result in an increase in the amplitude of
intracellular calcium peaks as revealed through a Ca2+ indicator,
such as Fluo-4. Therefore, the FLIPR assay allows sensitive
detection and high throughput screening of neuroactive
compounds. Recordings of individual somas reveal that these
oscillations are synchronous across a field of view, making them
appropriate to study with lower spatial resolution, as the average
signal from a large portion of a well in a 96-well plate. As with all
primary neuronal cultures, there are culture-to-culture variations
in the precise balance of types of neurons, but with so many
wells from one culture, the complete dose-response curve for
TETS can be obtained from one plate in one run with 5 duplicate
wells with FLIPR.[24]
µM). Our study used a narrower range of TETS, and indicated a
lower EC50 of 0.5 µM, but did not include measurements at the
lower concentrations of TETS needed to state the EC50 with
confidence. Using both values, our conclusion is that TETS is at
least 4 times more potent than the most effective TETS analog
tested (4a), whereas the maximal response of the two is the
same (efficacy).
Figure 4. TETS analog 4a has lower potency but similar efficacy to TETS. n =
4 for each group, bars indicate SEM.
Figure 5. Effect of TETS and its analogs on GABA-induced currents. Top:
Dose-response association curves showing percentage of the α1β22L current
blocked by increasing concentrations of either TETS or its analog 4a. Bottom:
Percentage of current blocked by TETS and its analogs at test concentration
of 50 µM. Percentage blocked for TETS is 69.4 ± 1.0% (n = 4), 4a is 43.9 ±
2.1% (n = 3), 2a is 59.8 ± 20.5% (n = 5) and 2c is 59.4 ± 12.6% (n = 3). Error
bars indicate SD.
Addition of vehicle (0.03% dimethyl sulfoxide [DMSO]) had no
significant effect on properties of the synchronous Ca2+
oscillations (SCO). By contrast, TETS at 10 µM significantly
increased the amplitude and decreased the frequency of the
SCOs.[24] Eight analogs of TETS were tested at 30 µM, and
compared to TETS (Figure 3). Analog 4a produced a similar
effect to TETS, with an increased SCO amplitude and lower
spike frequency. The traces from this analog were
indistinguishable from TETS (Figure S1). None of the other
analogs tested had a significant difference in the SCO amplitude
Inhibition of GABAA currents by TETS and its analogs. TETS
causes neuronal hyperexcitability by competitively binding to
GABAA receptors and reducing the hyperpolarizing chloride
currents. To evaluate further neuromodulating properties and to
determine the potency of the TETS analogs in inhibiting GABA-
induced currents, the effect of the TETS analogs on currents
produced by cultured cells expressing α1β22L GABAA
receptors were measured using whole-cell voltage-clamp and
compared against current inhibition by TETS. Despite being a
low-throughput technique, manual patch-clamping directly
measures the activity of the compound on GABAAR current.
Additionally, in contrast to cultured hippocampal neurons, we
from vehicle. The TETS analog 4a was tested at
3
concentrations: 3, 10 and 30 µM and compared to TETS at
concentrations from 0.06 µM to 20 µM (Figure 4). The EC50
value for 4a was found to be 7.15 µM (95% CI, 3.13 to 16.35
µM). Our use of TETS in this study was to compare on each
plate, the amplitude increase of each analog with a maximal
TETS response. An earlier study,[24] used a wider range of TETS
concentrations on identically prepared cultures with the same
procedures as this study, in the same lab, found the EC50 of
TETS on amplitude of SCO to be 1.8 µM (95% CI: 1.12 to 2.80
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controlled the receptor subunits being expressed in our
heterologous cells and thus the exact identity of the GABAA
receptors being tested is always known. In this report, we chose
the most abundant subunit combination in the mammalian CNS
(α1β22L) to determine the potency of TETS and its analogues.
In contrast to TETS (IC50 = 7.9 ± 2.6 µM, n = 12), TETS analog
4a, identified as the most active analog in the SCO assays, only
exhibited modest activity (IC50 = 48.0 ± 13.2 µM, n = 10) (Figure
5). Two additional analogs, 2a and 2c, which also showed some
activity in the FLIPR assays, were determined to have
comparable inhibitory effect on GABA-induced currents as 4a at
the 50 µM test concentration (4a 43.9 ± 2.1%, n = 3; 2a 59.8 ±
20.5%, n = 5; 2c 59.4 ± 12.6%, n = 3). None of the tested
analogs were more potent than TETS (69.4±1.0%, n = 4) on our
receptors of choice.
antibodies recognized TETS with sensitivities higher than 5
µg/mL. Use of these sera for the development of the first
immunoassay for sensitive detection and quantification of TETS
will be published in due course.
Acknowledgements
This work was supported by the CounterACT Program, National
Institutes of Health Office of the Director, and the National
Institute of Neurological Disorders and Stroke, Grant Number
U54 NS079202; and NIEHS, Superfund Research Program, P42
ES04699.
Keywords: poly-heterocycles, tetramine, cage convulsants,
Thus, we have successfully synthesized and identified
analogues of TETS that are active on GABAA receptors. Despite
being several fold less potent then TETS in our
electrophysiological studies, these analogues retained the
functional groups required for binding and blocking α1β22L
GABAA receptors. Additionally, although TETS is a known
GABAA receptor inhibitor, its exact binding site and selectivity for
the various GABAA receptor subtypes have not been
investigated. Thus, further testing of these analogues and TETS
on additional GABAA receptor subtypes will provide the
information on their exact potency and selectivity on the GABAA
receptors. The fact that analogues and TETS exhibit a different
potency ranking in the SCO and the patch-clamp experiments is
probably caused by the fundamentally different nature of the two
assays which in one case uses Ca2+ signaling as a downstream
effect of GABAA receptor blockade while directly measuring
blockade of GABA-induced chloride currents in the other.
Another possibility could be that the TETS analogous exhibit
differential selectivity for different GABAA receptor subtypes
present in the hippocampal neurons used for the SCO
experiments.
neurotoxicity, GABA, antibody-based assay.
[1] International
Program
on
Chemical
Safety
http://www.inchem.org/documents/pims/chemical/pim982.ht
m#2.1.
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the neurotoxic TETS molecule. Bioactivity of some of the
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Mimicking neurotoxic TETS: TETS
and newly synthesized polycyclic
sulfamides were shown to have
similar GABAA receptor channel
blocking properties and were both
recognized by antibodies raised
against synthetic TETS haptens.
Author(s), Corresponding Author(s)*
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