Article Cite This: J. Med. Chem. 2019, 62, 6214−6222
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Philanthotoxin Analogues That Selectively Inhibit Ganglionic Nicotinic Acetylcholine Receptors with Exceptional Potency Hamid S. Kachel,†,§ Henrik Franzyk,‡ and Ian R. Mellor*,† †
School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K. Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, Copenhagen DK-2100, Denmark
‡
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ABSTRACT: 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.
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INTRODUCTION A constituent of the venom of the Egyptian digger wasp, Philanthus triangulum, is the polyamine-containing toxin, known as philanthotoxin-433 (PhTX-433; Figure 1), which
the wasp has evolved to produce toxins targeting iGluRs and nAChRs in insects, PhTX-433 and its structurally very similar synthetic analogue, PhTX-343 (1; Figure 1), are also potent inhibitors of vertebrate ionotropic receptors. These latter interactions have been well characterized at mammalian iGluRs, including the N-methyl-D-aspartate (NMDA), kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors;3,4 as well as for vertebrate muscle-type5,6 and neuronal-type7,8 nAChRs. In our recent study on neuronal nAChRs, it was found that PhTX-343 is a very potent inhibitor of heteromeric receptors with selectivity for the ganglionic α3β4 subtype.7 Inhibition of both nAChRs and iGluRs by PhTX-343 has been shown to be use- and voltage-dependent (with increased inhibition occurring at more negative membrane potentials), and this has led to the hypothesis that open-channel blocking is the dominant mode of action, with the polyamine tail penetrating deep into the channel pore and interacting with polar amino acids, while the headgroup interacts with rings of hydrophobic amino acids at a more extracellular location in the pore.9,10 Further evidence for this mechanism resulted from experiments showing that AMPA receptors lacking the GluA2 subunit are highly sensitive to PhTX-343, whereas those containing GluA2 are almost insensitive.11 This can be explained by RNA editing of GluA2, resulting in single amino acid substitution at the so-called “Q/R site” that forms
Figure 1. Structures of naturally occurring PhTX-433 and its synthetic analogue PhTX-343 (1), used as a template for the analogues studied in the present work.
enables paralysis of its insect prey through inhibition of ionotropic glutamate receptors (iGluRs) and nicotinic acetylcholine receptors (nAChRs).1,2 The structure of PhTX433 consists of a central tyrosine residue amide-linked to a thermospermine moiety on one side and to an n-butanoyl chain on the other (Figure 1). The resulting molecule thus has a relatively bulky and hydrophobic “headgroup” and a positively charged (+3) “tail” at physiological pH. Although © 2019 American Chemical Society
Received: March 25, 2019 Published: June 5, 2019 6214
DOI: 10.1021/acs.jmedchem.9b00519 J. Med. Chem. 2019, 62, 6214−6222
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Table 1. IC50 Values for Inhibition of α3β4 or α4β2 Receptors by Compounds 1−18a
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DOI: 10.1021/acs.jmedchem.9b00519 J. Med. Chem. 2019, 62, 6214−6222
Journal of Medicinal Chemistry
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Table 1. continued
a RP = relative potency as compared to that of 1 for the receptor subtype; Sel = selectivity of the compound for α3β4 over α4β2; 95% CI = 95% confidence interval; n = 4−20 oocytes.
Scheme 1. SPS of Novel PhTX Analoguesa
Reagents: (i) iPr2EtN (3 equiv), CH2Cl2, 3 days at room temperature; (ii) iPr2EtN−MeOH−CH2Cl2 (5:15:80, 2 × 15 min); (iii) Bu4NF (4 equiv), DMF, 50 °C, 1 h, then 1 h at room temperature; (iv) Fmoc-Cha-OH or Fmoc-Tyr(tBu)-OH (3 equiv), DIC (3 equiv), HOBt (3 equiv), DMF, pre-activation for 20 min then 20 h after addition to resin; (v) 20% piperidine−DMF, 2 × 10 min; (vi) R′-COOH (5 equiv), DIC (5 equiv), HOBt (5 equiv), DMF, 16 h; (vii) 50% TFA−CH2Cl2, 2 h.
a
the selectivity filter within the pore.9 Likewise, mutations in the outer pore of nAChR affect the affinity for PhTX-343.7 Studies into the importance of the amine functionalities resulted in the first breakthrough in receptor selectivity. Thus, the analogue PhTX-12, in which the two secondary amines in PhTX-343 were replaced with methylene groups, had an expected reduced potency at AMPA receptors and NMDA receptors, but unexpectedly, inhibition of muscle-type nAChRs was significantly increased.4 However, the latter was accompanied by a change in the mode of action because inhibition
remained strong at positive membrane potentials and voltage dependence was weak.6,10 In another study, it was found that exchange of the tyrosine moiety in PhTX-343 for a cyclohexylalanine (Cha) unit generated a toxin with considerably enhanced inhibitory activity at muscle-type nAChR.12 In the present work, we investigated PhTX-343 (1) and its headgroup-modified analogues (i.e., compounds 2−18; Table 1) with single or combined structural alterations in the nbutanoyl, tyrosine, and polyamine moieties for their inhibitory actions against the α4β2 and α3β4 subtypes of neuronal 6216
DOI: 10.1021/acs.jmedchem.9b00519 J. Med. Chem. 2019, 62, 6214−6222
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nAChRs. We aimed to develop analogues capable of distinguishing between neuronal heteromeric nAChR subtypes. Recently, there has been considerable interest in compounds that selectively inhibit the α3β4 subtype of nAChRs as a potential treatment for nicotine addiction because of their significant expression in the medial habenula that modulates the mesolimbic dopaminergic response to nicotine.13 Hence, potent and selective α3β4 nAChR antagonists have the potential to aid smoking cessation.14,15
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RESULTS Synthesis of PhTX-343 Analogues. The novel PhTX analogues 5, 6, 10, 11, and 13−17 were prepared by using a solid-phase synthesis (SPS) protocol similar to that reported previously for the known analogues 9, 12, and 18 displaying a Cha residue.16 In brief, a large batch of trityl chloride polystyrene resin was loaded with an equimolar amount of mono-[2-(trimethylsilyl)ethyloxycarbonyl] (Teoc)-protected di-Boc-spermine (Scheme 1) in order to avoid the issue with cross-linking of the trityl linker moieties observed when employing di-Boc-spermine (i.e., H 2 N(CH 2 ) 3 NHBoc(CH2)4NHBoc(CH2)3NH2).17 Next, unreacted trityl groups were capped with methanol in the presence of base (i.e., iPr2EtN in CH2Cl2), and subsequent removal of the Teoc Nprotecting group by treatment with tetrabutylammonium fluoride afforded resin 19. The prepared large batch of resin 19 was divided into several reactors to complete the last steps in the SPS of the individual analogues in parallel. This comprised acylation with the appropriate fluoren-9-yl-oxycarbonyl (Fmoc)-protected amino acid building blocks (i.e., Fmoc-Tyr(tBu)-OH or Fmoc-Cha-OH) by standard diisopropylcarbodiimide (DIC)−1-hydroxybenzotriazol (HOBt) amide coupling to provide resins 20a and 20b, which then were Fmoc-deprotected and acylated with the selected different carboxylic acids under standard DIC−HOBt conditions. Subsequent cleavage (from resins 21a−21i) and purification furnished PhTX analogues 5, 6, 10, 11, and 13− 17 in 16−73% yield. Our reference compound, PhTX-343 (1), had IC50 values of 7.7 and 80 nM at α3β4 and α4β2 receptors, respectively, thus displaying a ∼10-fold selectivity for the α3β4 subtype over the α4β2 subtype (Figure 2; Table 1). Of the 17 analogues (i.e., 2−18) only one compound (i.e., 9) was a less potent antagonist of the α4β2 subtype, while all analogues were more efficient antagonists of the α3β4 subtype (Figure 3A,B). With respect to high selectivity for α3β4 over α4β2 receptors, two compounds (i.e., 5 and 16) were identified as being particularly promising (Figure 3C). The results obtained for each subset of analogues, characterized by the number and type of structural modifications, are presented in more detail below. Potency of Analogues with Substitution of Tyrosine Only. Three of the investigated analogues (2−4) contained alternative central amino acids instead of tyrosine (Tyr), thereby conferring increased hydrophobicity to the side chain (Table 1). Compounds 2 and 3 with a phenylalanine (Phe) moiety or its homologue (hPhe) instead of Tyr exhibited increased potency at both nAChR subtypes, which was more pronounced for α3β4, thus leading to increased selectivities of 30-fold and 12-fold, respectively. Compound 4, having a Cha residue in place of Tyr, proved more potent than 1 at both nAChR subtypes, but noticeably, all selectivity for α3β4 over α4β2 was abrogated (Figure 2; Table 1). Interestingly,
Figure 2. Inhibition of nAChR responses by PhTX-343 analogues. (A,B) Two-electrode voltage clamp (TEVC) current responses of α3β4 (A) or α4β2 (B) nAChRs to 100 or 10 μM acetylcholine (ACh), respectively, in the absence and co-applied with compounds 1, 4 or 16. (C,D) Concentration-inhibition curves for inhibition of α3β4 (C) or α4β2 (D) nAChRs by compounds 1, 4 or 16. Data points are represented as mean ± SEM (n = 3−9), and curves were obtained via fitting to eq 1 (see the Experimental Section on data analysis). IC50 values are given in Table 1.
analogue 4 was the only compound tested in this study that showed this nonselective behavior, while it was the most potent inhibitor of α4β2 receptors (Figure 2B). Potency of Analogues with Alternative N-Acyl Moieties. In analogues 5−6, the N-acyl moieties comprise cyclohexanoyl or cyclopentanoyl instead of the original Nbutanoyl group, which confer increased bulk and hydrophobicity to their headgroups (Table 1). Analogues 5 and 6 showed a strong increase in potency at α3β4 receptors, but less so at α4β2 receptors, and hence selectivity for α3β4 was increased up to 80-fold for compound 5. Potency of Analogues with Modifications in the Polyamine Tail Moiety. Analogues 7 and 8 contain a cyclopropane ring in the center of the polyamine moiety in a trans or cis conformation, respectively (Table 1). Both compounds exhibited moderately increased potency at α3β4 receptors as compared to that of 1, whereas they only displayed weakly improved inhibition of α4β2 receptors. This gave rise to increased selectivity of 7 and 8 for α3β4 receptors to 48-fold and 33-fold, respectively. 6217
DOI: 10.1021/acs.jmedchem.9b00519 J. Med. Chem. 2019, 62, 6214−6222
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selectivity for α3β4 over α4β2, and based on our recent observations that PhTX-343 selectively inhibits β4-containing nAChRs (especially α3β4) over all of the other major nAChR subtypes (i.e., α4β2, α3β2, α7 and α1β1γδ),7 it is expected that this will be the case also for 16. Many of the compounds described in the present work also compare favorably to a recently identified synthetic α-conotoxin, TP-2212-59, that selectively targets α3β4 nAChRs with a low nanomolar IC50 value (2.3 nM).18 However, a direct comparison between compounds belonging to these two distinct classes of antagonists are not straightforward because of their different binding sites and modes of action. The proposed binding site for PhTX-343 and its analogues, having similar highly charged polyamine tail regions, is located within the nAChR pore, whereby the polyamine moiety inserts deeply into the pore in order to interact with the threonine and serine rings beyond the equatorial leucine gate, while the bulky and hydrophobic headgroup interacts with hydrophobic residues of the valine and leucine rings that line the outer part of the pore.10 Compound 16 possesses modifications in both the N-acyl and the central amino acid residue, and it may thus be considered to be a further modification of PhTX(Cha)-343 (4), which previously was identified as an antagonist of human muscle-type nAChRs in TE671 cells, where it proved 277-fold more potent than 1 at VH = −100 mV.12 Nevertheless, in the present study, by using the same holding potential, only a 2.9fold increase in relative potency of 4 (vs that of 1) at α3β4 was found, most likely arising from the fact that 1 is much more potent (317-fold) at α3β4 than at the α1β1γδ subtype.7 Interestingly, the four most potent analogues at α3β4 receptors, having IC50 values
