Philanthotoxin-433 Research Articles

Philanthotoxin Analogues That Selectively Inhibit Ganglionic Nicotinic Acetylcholine Receptors with Exceptional Potency.

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 nonselectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here, we examined the action of 17 analogues of PhTX-343 against ganglionic (α3β4) and brain (α4β2) nAChRs expressed in Xenopus oocytes by using a two-electrode voltage clamp at -100 mV. IC50 values for PhTX-343 inhibition of α3β4 and α4β2 receptors were 7.7 and 80 nM, respectively. All the studied analogues had significantly higher potency at α3β4 nAChRs with IC50 values as low as 0.16 nM and with up to 91-fold selectivity for α3β4 over α4β2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for α3β4 nAChRs.

The effects of conformational constraints in the polyamine moiety of philanthotoxins on AMPAR inhibition.

Philanthotoxin-433 (PhTX-433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype by using two-electrode voltage-clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX-343, with trans-cyclopropyl-PhTX-343 being the most potent (∼28-fold) and cis-cyclopropyl-PhTX-343 least potent (∼4-fold). Both cis- and trans-cyclopropyl-PhTX-444 had intermediate potency (both∼12-fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX-444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.

Subunit-Dependent Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Philanthotoxins

Philanthotoxin-433 (PhTX-433) is the active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. To improve selectivity two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), were developed. Previous work showed a 22-fold selectivity for PhTX-12 on embryonic muscle-type nicotinic acetylcholine receptors (M-nAChR) naturally expressed in the TE671 cell line in comparison to PhTX-343. In this study, we investigated the pharmacological action of both analogues on different mammalian hetero-oligomeric neuronal nicotinic acetylcholine receptor (N-nAChR) subunit combinations expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX-analogue were studied electrophysiologically using two-electrode voltage-clamp at three different membrane holding potentials (VH = −60 mV, −80 mV and −100 mV). Concentration-inhibition curves were constructed and IC50 values estimated for each holding potential. The IC50 values for PhTX-343 inhibition of α3β4, α3β2, α4β2 and α4β4 peak currents at −100 mV were 0.077 µM (n=9), 3.20 µM (n=8), 0.170 µM (n=7) and 0.28 µM (n=6) respectively; for PhTX-12 they were 2.03 µM (n=8), 36.0 µM (n=10), 0.430 µM (n=7) and 1.82 µM (n=9) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. The potency of PhTX-343 was strongly augmented by holding the cell at more negative VH while this was not the case for PhTX-12 where weak voltage-dependence was observed. The variation in potency is most likely due to a single amino acid change in the β2/4 subunit pore lining region. Therefore, we conclude that PhTX-343 works as a potent open channel blocker of the mammalian heteromeric N-nAChR and has binding site deep in the channel while the PhTX-12 site is near to the outside of channel.

Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

Polyamines and polyamine toxins are biologically important molecules, having modulatory effects on nucleotides and proteins. The wasp toxin, philanthotoxin-433 (PhTX-433), is a non-selective and uncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic acetylcholine receptors. Polyamine toxins are used for the characterization of subtypes of ionotropic glutamate receptors, the Ca 2+-permeable AMPA and kainate receptors. A derivative of the native polyamine toxin, philanthotoxin-56 (PhTX-56), has recently been shown to be an exceptionally potent and selective antagonist of Ca 2+-permeable AMPA receptors. PhTX-56 and its labeled derivatives are promising tools for structure–function studies of the ion channel of the AMPA receptor. We now describe the design and synthesis of 3H-, 13C-, and 15N-labeled derivatives of PhTX-56 for molecular level studies of AMPA receptors. [ 3H]PhTX-56 was prepared from a diiodo-precursor with high specific radioactivity, providing the first radiolabeled ligand binding to the pore-forming part of AMPA receptors. For advanced biological NMR studies, 13C and 15N-labeled PhTX-56 were synthesized using solid-phase synthesis. These analogs can provide detailed information on the ligand–receptor interaction. In conclusion, synthesis of labeled derivatives of PhTX-56 provides important tools for future studies of the pore-forming region of AMPA receptors.

Diols as Building Blocks in Solid-Phase Synthesis of Polyamine Toxins by Fukuyama-Mitsunobu Alkylation

Philanthotoxin-433 (PhTX-433) is a polyamine wasp toxin that antagonizes certain ionotropic receptors. In the present work three analogues of PhTX-433 were synthesized in good overall yields (27-31 %) employing a Fukuyama-Mitsunobu strategy on solid phase. Primary diols were used as building blocks in solid-phase synthesis of polyamines for the first time. Furthermore, experiments with sterically congested alcohols indicated that incorporation of such structures by the solid-phase Fukuyama-Mitsunobu approach is not possible.

Solid-phase synthesis of polyamine toxin analogues: potent and selective antagonists of Ca2+-permeable AMPA receptors.

The wasp toxin philanthotoxin-433 (PhTX-433) is a nonselective and noncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic acetylcholine receptors. Polyamine toxins are extensively used for the characterization of subtypes of ionotropic glutamate receptors, in particular Ca(2+)-permeable AMPA and kainate receptors. We have previously shown that an analogue of PhTX-433 with one of the amino groups replaced by a methylene group, philanthotoxin-83 (PhTX-83) is a selective and potent antagonist of AMPA receptors. We now describe the solid-phase synthesis of analogues of PhTX-83 and the electrophysiological characterization of these analogues on cloned AMPA and kainate receptors. The polyamine portion of PhTX-83 was modified systematically by changing the position of the secondary amino group along the polyamine chain. In another series of analogues, the acyl moiety of PhTX-83 was replaced by acids of different size and lipophilicity. Using electrophysiological techniques, PhTX-56 was shown to be a highly potent (K(i) = 3.3 +/- 0.78 nM) and voltage-dependent antagonist of homomeric GluR1 receptors and was more than 1000-fold less potent when tested on heteromeric GluR1+GluR2, as well as homomeric GluR5(Q) receptors, thus being selective for Ca(2+)-permeable AMPA receptors. Variation of the acyl group of PhTX-83 had only minor effect on antagonist potency at homomeric GluR1 receptors but led to a significant decrease in the voltage-dependence. In conclusion, PhTX-56 is a novel, very potent, and selective antagonist of Ca(2+)-permeable AMPA receptors and is a promising tool for structure/function studies of the ion channel of the AMPA receptor.

Effect of Two Polyamine Toxins on the Bacterial Porin OmpF

Spermine, a polyamine based on a 12-carbon motif, is an effective inhibitor of E. coli OmpF porin. Here we study the inhibition of porin by two polyamine toxins commonly used as modulators of polyamine-sensitive channels: Philanthotoxin-433 (PhTX) from wasp venom and Joro spider toxin (JSTX). Both are highly asymmetric molecules, with at one end a 12-carbon chain polyamine targeting the molecule to the porin constriction zone, and at the other end large aromatic groups conferring to this extremity a size in the order of the OmpF constriction zone. Here we report that PhTX, but not Joro toxin, induces a high degree of flickering in the OmpF-mediated current. The effect is concentration and voltage-dependent, and greatly diminished in a mutant lacking D113 on the constriction loop, a residue previously shown to be required for spermine sensitivity. Possible reasons for the distinct sensitivity of OmpF to PhTX and Joro toxin are discussed.

Analogues of neuroactive polyamine wasp toxins that lack inner basic sites exhibit enhanced antagonism toward a muscle-type mammalian nicotinic acetylcholine receptor.

Philanthotoxin-433 (PhTX-433), a natural polyamine wasp toxin, is a noncompetitive antagonist of certain ionotropic receptors. Six analogues of PhTX-343 (a synthetic analogue of the natural product), in which the secondary amino groups are systematically replaced by oxygen or methylene groups, have been synthesized by coupling of N-(1-oxobutyl)tyrosine with 1,12-dodecanediamine, 4,9-dioxa-1, 12-dodecanediamine, or appropriately protected di- and triamines, the latter being obtained by multistep syntheses. The resulting PhTX-343 analogues were purified and characterized, and their protolytic properties (stepwise macroscopic pK(a) values) were determined by (13)C NMR titrations. All analogues are fully protonated at physiological pH. The effects of these compounds on acetylcholine-induced currents in TE671 cells clamped at various holding potentials were determined. All of the analogues noncompetitively antagonized the nicotinic acetylcholine receptor (nAChR) in a concentration-, time-, and voltage-dependent manner. The amplitudes of acetylcholine-induced currents were compared at their peaks and at the end of a 1 s application in the presence or absence of the analogues. Most of the analogues were equipotent with or more potent than PhTX-343. The dideaza analogue PhTX-12 [IC(50) of 0.3 microM (final current value)] was the most potent, representing the highest potency improvement (about 50-fold) yet achieved by modification of the parent compound (PhTX-343). Thus, the presence of multiple positive charges in the PhTX-343 molecule is not necessary for antagonism of nAChR. In contrast, the compounds were much less potent than PhTX-343 at locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The results demonstrate that the selectivity for different types of ionotropic receptors can be achieved by manipulating the polyamine moiety of PhTX-343.

Labeling studies of photolabile philanthotoxins with nicotinic acetylcholine receptors: mode of interaction between toxin and receptor

Background: The nicotinic acetylcholine receptors (nAChRs) and glutamate receptors are ligand-gated cation channels composed of five separate polypeptide chains. A 43 kDa protein of unknown function is noncovalently associated with the cytoplasmic side of nAChR in vivo. The -venoms of many wasps and spiders ccntain toxins that block the activity of these channels. Philanthotoxin-433 (PhTX-433) is a non-competitive channel blocker found in the venom of the wasp Philanthus. We have used a photolabile derivative to investigate how PhTX-433 interacts with nAChRs. Results: A radiolabeled PhTX analog, containing a photolabile group substituted on one of its aromatic rings, photocrosslinked to all five subunits (α, α′, β, γ, δ) of purified nAChR in the absence of the 43 kDa protein. In the presence of the 43 kDa protein, the a subunit was preferentially labeled. Proteolysis of the receptor after crosslinking indicated that the hydrophobic end (head) of the PhTx-433 analog bound to the cytoplasmic loop(s) of the a-subunit. Binding is inhibited by other non-competitive channel blockers such as the related polyamine-amide toxins from spiders and chlorpromazine. Conclusions: These results, coupled with previous structure/ activity studies, lead to a putative model of the binding of PhTx and related polyamine toxins to nAChRs in vitro. The 43 kDa protein appears to influence the orientation of toxin binding. Further binding studies are necessary to confirm the model and to define how toxins enter the receptor and how they are oriented within it. A precise understanding of ligand/receptor interaction is crucial for the design of drugs specific for a particular subtype of receptor.

Philanthotoxin inhibits Ca 2+ currents in rat hippocampal CA1 neurons

The wasp venom philanthotoxin-4.3.3 (PhTX-4.3.3) is an antagonist of glutamate transmission in the insect as well as in the mammalian brain. It was recently shown that PhTX-4.3.3 inhibits the N- methyl- D-asparttate (NMDA) transmission in rat hippocampus. In this study we show that dideaza-philanthotoxin-12 (dideaza-PhTX-12), an analogue of PhTX-4.3.3, is a potent antagonist of voltage-dependent Ca 2+ currents in rat hippocampal CA1 neurons. At a concentration of 10 μM it reduces the Ca 2+ current to 40%. Two voltage-dependent potassium currents, the A current and the delayed rectifier, were hardly affected calcium influx via voltage dependent as well as NMDA mediaded calcium channels and thus reduce excitability in the hippocampus.

Synthesis of glutamate receptor antagonist philanthotoxin-433 (PhTX-433) and its analogs

The synthesis of the non-competative glutamate receptor inhibitor, philanthotoxin 433 (PhTX-433) is described. Furthermore, two synthetic routes are described for the preparation of a variety of structural analogs.

Structure and synthesis of a potent glutamate receptor antagonist in wasp venom.

A low molecular weight toxin isolated from the venom of the digger wasp Philanthus triangulum, first noted by T. Piek, is a potent antagonist of transmission at quisqualate-sensitive glutamate synapses of locust leg muscle. This philanthotoxin 433 (PTX-433) has been purified, chemically characterized, and subsequently synthesized along with two closely related analogues. It has a butyryl/tyrosyl/spermine sequence and a molecular weight of 435. Its two analogues, PTX-343 and PTX-334 (the numerals denoting the number of methylenes between the amino groups of the spermine moiety), are also active on the glutamate synapse of the locust leg muscle; PTX-334 was more potent and PTX-343 was less potent than the natural toxin. Such chemicals are useful for studying, labeling, and purifying glutamate receptors and may become models for an additional class of therapeutic drugs and possibly insecticides.