Uncompetitive Antagonism of AMPA Receptors:  Mechanistic Insights from Studies of Polyamine Toxin Derivatives

Abstract

Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. The biological activities were investigated at cloned and "native" AMPA receptors using electrophysiological techniques. A distinct relationship between length of the polyamine moiety and the location of a secondary amino group was observed. Fitting the data to the Woodhull equation allowed the first experimental demonstration of the relative location and orientation of the philanthotoxin molecule in the receptor. These results were corroborated by in silico studies using a homology model of the AMPA receptor ion channel. Together these studies provide strong evidence for a molecular mechanism by which polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors.

Evaluation of PhTX-74 as Subtype-Selective Inhibitor of GluA2-Containing AMPA Receptors

“…Binding of polyamine toxins is dependent on channel activation and membrane potential, and they are therefore classified as use-and voltage-dependent blockers. Structure-activity relationship and molecular modeling studies (Andersen et al, 2006;Tikhonov, 2007;Nelson et al, 2009;Barygin et al, 2011) have suggested a binding mode in which the polyamine toxin head group moiety is positioned in the channel vestibule with the polyamine tail permeating the Q/R site (Fig. 1A).…”

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

“…[4,10] The subunit combination of the latter is not known, but presumably it contains other AMPAR subunits that may be more sensitive to PhTX 2. All of the cyclopropane-containing PhTX analogues (3)(4)(5)(6) were significantly more potent inhibitors than 2, with the transconfigured 3 being the most potent; nearly 30-fold more so than the corresponding straight-chain PhTX 2. Table 1.…”

“…[3,4,19,20] Figure 3. Structures of philanthotoxin analogues 2-6 visualizing the three general conformations that can be adopted: "head and tail" for 2 (A) and 3 (C), "semi-folded" for 4, 5 and 6 (D-F) and "folded" for 2 (B).…”

“…[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.…”

“…[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.…”