C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors

Abstract

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a, with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.

Strychnine and its mono- and dimeric analogues: a pharmaco-chemical perspective

“…127 Inspired by the observation that the GlyR activity of strychnine is almost retained in 2-propionamidostrychnine (39b), we recently developed three series of strychnine dimers with C-2 linkers of lengths varying from 6-16 atoms, 15-24 atoms, and of 57 and 69 atoms, respectively. 107 All of these dimeric analogues displayed signicant GlyR inhibition with IC 50 values in the 0.43-10 mM range, and thus they were all signicantly weaker antagonists than strychnine. 107 Even though the bivalent analogues 81a and 81b (in which the two strychnine pharmacophores are separated by 57 and 69 atoms, respectively) both displayed higher antagonist potencies at GlyRs than their respective monomeric controls, it still remains a question whether any of the dimers are true bivalent ligands capable of bridging between two orthosteric sites in the GlyR and facilitate simultaneous occupation of two of these binding sites, or whether one of the pharmacophores binds to another receptor region or just protrudes from and do not form any interactions with the receptor.…”

“…Pharmacological prole. Strychnine displays low-tomid-nanomolar binding affinities (K i /IC 50 ) to native and recombinant GlyRs, 59,107,121 and the alkaloid displays IC 50 values in the 10-200 nM range at the recombinant receptors expressed in mammalian cells 59,107,122,123 and Xenopus oocytes 124 in electrophysiological recordings and somewhat higher IC 50 values at the receptors expressed in mammalian cells in a uorescencebased membrane FLIPR potential (FMP) assay. [125][126][127] In light of the highly conserved binding sites and the very similar binding modes of strychnine to the orthosteric sites in a and ab GlyRs (outlined above), it is hardly surprising that strychnine exhibits comparable binding affinities to and mediates equipotent inhibition of homomeric and heteromeric GlyRs.…”

“…While in the bis-quaternary analogues ( 80a–c ), two molecules of strychnine or brucine are connected via tertiary nitrogens using polymethylene spacers, 49 the attachment point for the amide spacer in the bis-tertiary series ( 81a,b ) is the aromatic carbon C2. 107…”

“…107 Even though the bivalent analogues 81a and 81b (in which the two strychnine pharmacophores are separated by 57 and 69 atoms, respectively) both displayed higher antagonist potencies at GlyRs than their respective monomeric controls, it still remains a question whether any of the dimers are true bivalent ligands capable of bridging between two orthosteric sites in the GlyR and facilitate simultaneous occupation of two of these binding sites, or whether one of the pharmacophores binds to another receptor region or just protrudes from and do not form any interactions with the receptor. Interestingly, however, in patch-clamp recordings 81a and 81b exhibited substantially slower GlyR binding kinetics than strychnine and bivalent analogues with shorter linkers, 107 which seems to indicate some involvement or impact of either the linker or the “second” strychnine pharmacophore in the binding of the dimeric analogue.…”