The effects of two pyrethroids on recombinant wild-type and mutant (pyrethroid-resistant) Na + channels of Drosophila melanogaster have been studied. Three mutations that confer resistance ( ...kdr/superkdr ) to pyrethroids were inserted, either individually or in combination, into the para Na + channel of D. melanogaster : L1014F in domain IIS6, M918T in the IIS4-S5 linker, and T929I in domain IIS5. Channels were expressed in Xenopus laevis oocytes and the effects of the pyrethroids permethrin (type I) and deltamethrin (type II) on Na + currents were investigated using voltage clamp. The Na + channels deactivated slowly after deltamethrin treatment, the resultant âtailâ currents being used to quantify the effects
of this pyrethroid. The Hill slope of 2 for deltamethrin action on the wild-type channel and the mutant L1014F channel is
indicative of cooperative binding at two or more sites on these channels. In contrast, binding to the mutants M918T and T929I
is noncooperative. Tail currents for the wild-type channel and L1014F channel decayed biphasically, whereas those for M918T
and T929I mutants decayed monophasically. The L1014F mutant was â¼20-fold less sensitive than the wild-type to deltamethrin.
Surprisingly, the sensitivity of the double mutant M918T+L1014F to deltamethrin was similar to that of M918T alone, whereas
the sensitivity of T929I+L1014F was >30,000-fold lower than that of T929I. Permethrin was less potent than deltamethrin, and
its binding to all channel types was noncooperative. The decays of permethrin-induced tail currents were exclusively monophasic.
These findings are discussed in terms of the properties and possible locations of pyrethroid binding sites on the D. melanogaster Na + channel.
Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle
nicotinic acetylcholine receptors (nAChR) by the philanthotoxins, PhTX-343 ...and PhTX-(12). When coapplied with acetylcholine
(ACh), PhTX-343 caused activation-dependent, noncompetitive inhibition (IC 50 = 17 μM at -100 mV) of whole-cell currents that was strongly voltage-dependent. However, preapplication of PhTX-343 unveiled
a voltage-independent antagonism that also required receptor activation, which is suggestive of desensitization enhancement.
In single-channel studies, 10 μM PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 μM ACh
from 4.42 ± 0.44 to 1.58 ± 0.10 ms with a minor increase (1.26-fold) in mean closed time. These data indicate that PhTX-343
predominantly blocks the open channel gated by ACh. In contrast, PhTX-(12) caused potent (IC 50 = 0.77 μM at-100 mV), activation-dependent, noncompetitive inhibition of ACh-induced whole-cell currents that was only weakly
voltage-dependent and suggestive of desensitization enhancement. It caused only a small decrease (7.5%) in the mean open time
of channel openings induced by 1 μM ACh, whereas the mean closed time was significantly increased from 200 ± 45 ms to 586
± 145 ms. The different voltage-dependencies of the two modes of action of these philanthotoxins suggest two binding sites,
one deep in the nAChR pore, the other near the extracellular entrance to the pore.
Philanthotoxin-343 (PhTX-343), a synthetic analogue of wasp toxin PhTX-433, is a noncompetitive antagonist at ionotropic receptors (e.g., AChR or iGluR). To determine possible effects of variations ...of the amino acid side chain, a library consisting of seventeen PhTX-343 analogues was prepared. Thus, tyrosine was replaced by either apolar, conformationally constrained, or bulky amino acids, whereas the acyl unit and the polyamine moiety were kept unchanged. Analogues with tertiary amide groups were prepared for the first time. Pentafluorophenyl esters were employed for amide bond formation, establishing general protocols for philanthotoxin solution- and solid-phase synthesis (39−90% and 42−54% overall yields, respectively). The analogues were tested for their ability to antagonize kainate-induced currents of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazoyl)propanoic acid receptors (AMPAR) expressed in Xenopus oocytes from rat brain mRNA. This showed that steric bulk in the amino acid moiety is well tolerated and suggests that binding to AMPAR does not involve the α-NHCO group as a donor in hydrogen bonding.
Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in ...which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.
The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and ...philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3−25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M2) and ileum longitudinal muscle (M3) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M2 and M3 receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7−25 μM range while not showing any antagonism for muscarinic receptors up to 10 μM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9−11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M2 receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M2 receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the 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 13C 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 IC50 of 0.3 μM (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.
Receptor antagonists with sting: The natural wasp toxin PhTX‐433, a noncompetitive antagonist of ionotropic receptors, is relatively nonselective in its action. Solid‐phase synthetic methods gave ...rise to analogues (shown) with nanomolar potency toward the nicotine acetylcholine receptor. The inhibition was voltage‐dependent, which suggests that the analogues bind deep inside the transmembrane pore.
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.
Black widow spider venom (BWSV) kills Caenorhabditis elegans after injection owing to the presence of heat- and detergent-sensitive components, which are high-molecular-mass latrotoxins. A C. elegans ...homologue of latrophilin/CIRL (calcium-independent receptor for latrotoxin), B0457.1, was identified and shown to have five conserved domains. RNAi (RNA interference) of this gene rendered C. elegans resistant to BWSV, whereas RNAi for CYP37A1 or a neurexin I homologue, and a deletion mutant of the related B0286.2 gene, had no effect on BWSV toxicity. The latrophilin RNAi mutants exhibit changes in defaecation cycle and alterations in drug sensitivity. These results demonstrate that latrophilin mediates the toxicity of BWSV and provide evidence for a physiological function of this receptor.
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