Search

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 non-selectively 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 seventeen analogues of PhTX-343 against ganglionic (?3?4) and brain (?4?2) nAChRs expressed in Xenopus oocytes by using two-electrode voltage-clamp at -100 mV. IC50 values for PhTX-343 inhibition of ?3?4 and ?4?2 receptors were 7.7 nM and 80 nM, respectively. All of the studied analogues had significantly higher potency at ?3?4 nAChRs with IC50 values as low as 0.16 nM and with up to a 91-fold selectivity for ?3?4 over ?4?2 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 ?3?4 nAChRs. 2

An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.

A protocol for parallel solid-phase synthesis of ω-amino acid-elongated philanthotoxins was developed for exploration of structure-activity relationships for unnatural wasp toxin analogues. Several methods for on-resin activation of N-trityl-linked ω-amino acids were investigated, and the influence of chain length upon activation as pentafluorophenyl (Pfp) esters was examined. Comparison of Pfp-ester activation with phosphonium- and aminium/ uronium-promoted coupling without pre-activation of the resin-bound carboxylic acids showed that only benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) exhibited a comparable efficiency. The present Pfp ester protocol gave high yields and purities of the resulting philanthotoxin analogues; however, in the case of γ-aminobutyric acid (GABA), use of PyBOP was required in order to avoid lactamization to 2-pyrrolidone.

The modular structure of philanthotoxins was exploited for construction of the first combinatorial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S)-3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine, and 4,9-dioxa-1,12-dodecanediamine as amine components, and butanoyl, phenylacetyl, and cyclohexylacetyl as N-acyl groups. Following automated preparative HPLC, the resulting 18 compounds were isolated as the S-forms in 40-70% yields. The purity of the products was determined by HPLC with evaporative light scattering detection and by (1)H and (13)C NMR. The thus obtained philanthotoxins were tested electrophysiologically for their antagonist properties on human muscle-type nicotinic acetylcholine receptors (nAChR) expressed in TE671 cells and on rat brain non-NMDA glutamate receptors (non-NMDAR) expressed in Xenopus oocytes. 4-Hydroxy analogues lacking the secondary amino groups (PhTX-12 and 4,9-dioxa-PhTX-12 and their analogues) were inactive on non-NMDAR, whereas the potency of the spermine derivatives (PhTX-343 and its analogues) increased with steric bulk of the N-acyl group. The analogue of PhTX-343 in which the N-butanoyl group was replaced by phenylacetyl group had IC(50) of 15 +/- 4 nM on non-NMDAR. Increasing the steric bulk of the N-acyl group was not advantageous for activity at nAChR, and a sharp decrease in potency with increased steric bulk was observed with the derivatives of PhTX-12. 3-Hydroxy analogues generally exhibited lower activity and different response to alterations of the N-acyl groups as compared to the 4-hydroxy analogues. Since the acyl group alterations in PhTX-343 and 4,9-dioxa-PhTX-12 have a similar effect on potency, which is distinctly different from that observed for PhTX-12, the two former compounds may bind to nAChR in a similar fashion but differently from that of PhTX-12. The combinatorial library approach described in this work represents a prototype methodology for future exploration of structure-activity relationships of philanthotoxins.

Philanthotoxin (PhTX-433), a potent noncompetitive inhibitor of the L-glutamate receptors and the nicotinic acetylcholine receptors of vertebrates and invertebrates, has a butyryl-tyrosyl-thermospermine structure. Several synthetic analogs of PhTX with hydrophobic alkyl branches in the polyamine chain exhibit 6- to 10-fold enhanced activities to various receptors. Because the branched analogs exhibit such unique activities and because of their importance in tertiary structural studies of ligand/receptor binding, methods for preparing branched PhTX analogs, including photolabile analogs, are presented

The synthetic methods for 27 novel and photolabile philanthotoxin analogs are described. Most analogs were synthesized by two general methods with modifications of these methods where necessary.

The synthetic methods for 27 novel and photolabile philanthotoxin analogs are described. Most analogs were synthesized by two general methods with modifications of these methods where necessary.

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-aspartate (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 Ca2+ currents in rat hippocampal CA1 neurons. At a concentration of 10 microM it reduces the Ca2+ current to 40%. Two voltage-dependent potassium currents, the A current and the delayed rectifier, were hardly affected by dideaza-PhTX-12, indicating selectivity of the drug for Ca2+ currents. As a consequence the philanthotoxins will inhibit the calcium influx via voltage dependent as well as NMDA mediaded calcium channels and thus reduce excitability in the hippocampus.

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

1. The molecular structure of β-philanthotoxin (β-PTX), a toxin from the insect paralysing venom of the wasp Philanthus triangulum, has been elucidated using NMR and mass spectrometry. β-PTX has a polyamine character: C5H11·NH·CO·(CH2)4·NH·(CH2)3·NH2. This structure has been confirmed by synthesis. 2. In the locust muscle fibre β-PTX blocks iontophoretically evoked glutamate potentials in a non-activation induced manner. β-PTX also blocks the nicotinic transmission in the insect CNS, however, at much higher toxin concentrations. 3. β-PTX reduces the frequency of postsynaptic channel opening and reduces the duration of open times. 4. These results suggest an effect of β-PTX on kinetics of glutamate activated ion channels, different from acting as an open channel blocker.

A general, improved procedure for rapid synthesis of philanthotoxin analogues, a pharmacologically important class of polyamine conjugates, is described. The solution-phase procedure is illustrated by gram-scale synthesis of philanthotoxins PhTX-343 and PhTX-12. Selectively protected polyamines are coupled to N(alpha)-Fmoc-protected amino acid pentafluorophenyl esters. After removal of the N(alpha)-Fmoc group, the amine is coupled with carboxylic acid pentafluorophenyl esters. Deprotection followed by a rapid and efficient purification by vacuum liquid chromatography on octadecylsilyl silica (RP-18 phase) gave the philanthotoxin analogues in 74-78% overall yield.

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 (IC50 = 17 microM 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 microM PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 microM 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 (IC50 = 0.77 microM 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 microM 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.

Three photosensitive, synthetic analogues of δ-philanthotoxin (PhTX-433) have been tested on the locust, excitatory nerve-muscle system. At 10 −9 M they inhibit reversibly the postjunctional currents (EPSCs) recorded from muscle fibres during motor nerve stimulation, mainly by non-competitively antagonizing postjunctional quisqualate-sensitive glutamate receptors (Quis-R), probably through open channel block. This use-dependent antagonism is characteristic of the philanthotoxins. When the preparation was irradiated with 270 nm U.V. during toxin application and nerve stimulation the EPSCs were inhibited irreversibly. Irradiation of the preparation alone or in the presence of philanthotoxins (e.g. PhTX-433) which are not photosensitive did not lead to irreversible inhibition of the EPSC. It is concluded that the three photosensitive toxins bind covalently to Quis-R in its open channel conformation during U.V. irradiation, thereby irreversibly blocking the channel gated by this receptor.

1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.

M. Bruce , R. Bukownik , A. T. Eldefrawi , M. E. Eldefrawi , R. Goodnow , Jr, T. Kallimopoulos , K. Konno , K. Nakanishi , M. Niwa and P. N. R. Usherwood . Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors. Toxicon 28, 1333–1346, 1990.—Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which ic 50 s were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

In this chapter we will briefly review some of the evidence from electrophysiological studies of mammalian neurones in vivo which relates to the subtypes of glutamate receptors. In particular we will concentrate on recent experiments elucidating the mechanism of action of HA-966 as an N-methyl-D-aspartate (NMDA) antagonist and of a new tricyclic quinoxalinedione (NBQX) and a wasp venom, philanthotoxin, as quisqualate antagonists. These studies were designed to test the hypothesis that the NMDA subtype and one other non-NMDA subtype of glutamate receptor mediate the excitation of rat spinal and brainstem neurones produced by acidic amino acids.

Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.