Characterization of the Preferred Stereochemistry for the Neuropharmacologic Actions of Antillatoxin

Abstract

Antillatoxin is a potent ichthyotoxin and cytotoxin previously discovered from the marine cyanobacterium Lyngbya majuscula. Ensuing studies of its mechanism of action showed it to activate the mammalian voltage-gated sodium channel at a pharmacological site that is distinct from any previously described. The structure of antillatoxin, initially formulated from spectroscopic information, was subsequently corrected at one stereocenter (C-4) as a result of synthesis of four different antillatoxin stereoisomers (all possible C-4 and C-5 diastereomers). In the current study these four stereoisomers, (4R,5R)-, (4S,5R)-, (4S,5S)-, and (4R,5S)-antillatoxin, were characterized in five different biological assay systems: ichthyotoxicity to goldfish, microphysiometry using cerebellar granule cells (CGCs), lactose dehydrogenase efflux from CGCs, monitoring of intracellular Ca(2+) concentrations in CGCs, and cytotoxicity to Neuro 2a cells. Across these various biological measures there was great consistency in that the natural antillatoxin (the 4R,5R-isomer) was greater than 25-fold more potent than any of the other stereoisomers. Detailed NMR studies provided a number of torsion and distance constraints that were modeled using the MM2 force field to yield predicted solution structures of the four antillatoxin stereoisomers. The macrocycle and side chain of natural (4R,5R)-antillatoxin present an overall "L-shaped" topology with an accumulation of polar substituents on the external surface of the macrocycle and a hydrogen bond between N(H)-7' and the C(O)-1 carbonyl. The decreased potency of the three non-naturally occurring antillatoxin stereoisomers is certainly a result of their dramatically altered overall molecular topologies.

Antillatoxin, a Novel Lipopeptide, Enhances Neurite Outgrowth in Immature Cerebrocortical Neurons through Activation of Voltage-Gated Sodium Channels

“…The structure of ATX includes asymmetric carbon atoms, and the (4R,5R)-isomer is the naturally occurring compound. The (4R,5R)-isomer appears in profile as an "L" shape with a hydrophobic interior and a cluster of hydrophilic groups on the exterior of the macrocycle (Li et al, 2004). Thus, the (4R,5R)-configuration is important for creating a molecular topology that is recognized by the acceptor site on the voltage-gated sodium channel ␣ subunit.…”

“…Previous work has demonstrated that ATX is a potent activator of voltagegated sodium channels (VGSCs) that elevates intracellular Na ϩ concentration ([Na ϩ ] i ) in intact neurons (Li et al, 2001;Cao et al, 2008). Moreover, ATX has been shown to be neurotoxic in cerebellar granule cells through an indirect activation of N-methyl-D-aspartate receptors (NMDARs) as a consequence of glutamate release (Li et al, 2001(Li et al, , 2004.…”

Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 alpha subunits

“…Characterization of the four ATX stereoisomers (all possible C-4 and C-5 diastereomers) has revealed that the preferred stereochemistry for the neuropharmacologic actions of ATX is the (4R, 5R)-isomer [13]. In addition to its stereochemistry, the twisted side chain of ATX has also been demonstrated to be important for its toxicity in neuro-2a mouse neuroblastoma cells.…”

Aspects of Chirality in Natural Products Drug Discovery

“…The natural 4R,5R-isomer was the most potent analog with an LD 50 = 0.18 mM against a mouse neuro 2 a cell line, 25-50 times more potent than the next most active 4S,5S-isomer. The isomers showed the same order of potency in the test systems [76].…”