The Effects of Conformational Constraints in the Polyamine Moiety of Philanthotoxins on AMPAR Inhibition

Abstract

[a] IntroductionPhilanthotoxin-433 (PhTX-433, 1; the numerals indicate the number of methylene groups spacing the nitrogens in the polyamine moiety; Figure 1) is a toxin found naturally in the venom of the solitary wasp, Philanthus triangulum. [1,2] PhTX-433 and many of its synthetic analogues have been shown to have non-competitive inhibitory effects at both ionotropic glutamate receptors and nicotinic acetylcholine receptors. [3][4][5][6] In that respect PhTXs are attractive molecules to investigate further given that both of these receptor types are accepted as valid drug targets for a variety of neurodegenerative and other disorders of the central nervous system. [7] The modular butyryl-tyrosylthermospermine composition of 1 has allowed for efficient generation of many synthetic analogues demonstrating the importance of all of these structure segments. [4,6,[8][9][10][11] PhTXs 1, 2 and an array of other analogues have been shown to produce powerful voltage-dependent inhibition of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) currents suggesting a binding mode with the polyamine inserted deeply within the pore region of the ion channel.[3] This hypothesis is supported by the observation that AMPARs containing the GluA2 subunit with arginine at the "Q/R" site in the selectivity filter of the pore exhibit drastically reduced inhibition by PhTX-343 and other polyamine-containing molecules. [12] In the last two decades advanced methodologies for solidphase synthesis (SPS) of polyamines have been developed, [13] however, no examples of SPS of cyclopropane-containing polyamine derivatives have been reported. The commonly used solution-phase method for obtaining polyamines displaying a cyclopropane moiety is alkylation of mesitylenesulfonamides with mesitylenesulfonates of cyclopropane diols, but this is not readily transferred into an SPS protocol due to the harsh conditions required for deprotonation of the sulfonamide and the risk of cross-linking the resin due to the bifunctional building block. [14] Cyclopropane-trans-1,2-dicarboxylic acid [15] may be readily obtained from the corresponding ethyl diester and the cisanhydride 3-oxabicyclo[3.1.0]hexane-2,4-dione was commercially available, and therefore, we chose an approach involving on-resin reduction of the diamide corresponding to the desired 4,4'-dimethoxytrityl-protected polyamine. [16] In the present work we focus on incoporating unprecedented structural variations of the polyamine moiety present in both 1 and its well-studied close structural analogue, PhTX-343 (2), and examine how these influence the inhibitory effects of the resulting PhTX analogues on a specific subunit, GluA1flop, present in members of the AMPAR subdivision of the ionotropic glutamate receptor family. This subunit is characteristic of a calcium-permeable and polyamine-sensitive subtype of AMPARs, with the flop splice variant (a 38 amino acid region upstream of the fourth transmembrane region) being upregulated in place of the flip splice variant during early...

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

“…Apart from its inhibitory action on insect nAChRs and iGluRs, PhTX-433 and its closely related synthetic analogue, PhTX-343 (Fig. 1), also exhibit potent activity at vertebrate ionotropic receptors, and their receptor interactions have been quite extensively characterized for mammalian iGluRs, including the AMPA, kainate and NMDA receptor subtypes3456, as well as for vertebrate muscle-type nAChRs78. These investigations have inferred that philanthotoxins (PhTXs) display some selectivity towards iGluRs over nAChRs.…”

Structurally Diverse Polyamines: Solid‐Phase Synthesis and Interaction with DNA

“…Partially constrained polyamines do not occur naturally, to the best of our knowledge. However, several polyamines in this class have been synthesized and show interesting activities, such as DNA triplex stabilization,20 growth‐inhibitory effects on human tumor cell lines,21 AMPA receptor inhibition,22 and stereoselective DNA binding 19. 23 Branched polyamines are found in the cells of hyperthermophiles.…”

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

“…Furthermore, compounds 2 and 7 also had subnanomolar IC 50 values, and 2 was previously found to have increased potency at torpedo electric organ nAChRs through testing in a [ 3 H]­H 12 –HTX binding assay, presumably because of an increased headgroup hydrophobicity induced by the Tyr → Phe substitution, resulting in the absence of a hydroxyl group. Compound 7 has a cyclopropane functionality inserted into the center of the spermine moiety and previous modelling studies indicate that internal hydrogen bonding between the amine closest to the headgroup and the amide oxygens will position the cyclopropane unit so that it contributes to the bulkiness of the headgroup. , …”

“…ACh was from Sigma. PhTX-343 ( 1 ) was synthesized as described by Wellendorph et al Some analogues have been synthesized previously: compounds 2 and 3 ; compound 4 , compounds 7 and 8 , as well as compounds 9 , 12 , and 18 . cDNA clones of rat neuronal nAChR subunits (α3, α4, β2 and β4) were from the Salk Institute for Biological Studies (Professor Stephen Heinemann).…”