A Taxon-Specific and High-Throughput Method for Measuring Ligand Binding to Nicotinic Acetylcholine Receptors

Abstract

The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids

“…The diet of E. boulengeri remains elusive within the literature, and, thus, it is difficult to make any assumptions between diet and neurotoxic selectivity toward the lizard mimotope. Snakes are well documented as being the preferred prey for O. hannah [32,38,48,49], to the extent that the genus name means snake ('Ophio') eater ('phagus'). All O. hannah localities studied here ( Figure 4A,B) showed a high selectivity and potency for the snake mimotope ( Figure 4A).…”

“…In order to help our understanding of prey-specific binding of toxins and how these shape overall venom evolution, this study utilised a validated biolayer interferometry (BLI) assay [32] to test a large diversity of 3FTx-rich venoms from 29 African and Asian elapids (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) and a 3FTx representative (alpha-cobratoxin) against the alpha-1 nAChR orthosteric sites from some of the major groups of potential prey types: amphibian, lizard, snake, bird, and rodent. Our results provide evidence that prey-specific binding does occur within some of the species tested at both the toxin and crude-venom level.…”

Novelty in three-finger toxin evolution

“…We use a recently described bio-layer interferometry technique to assess the the α-neurotoxicity of Micrurus venoms [229]. This technique uses mimotopes-small peptides designed to mimic the region of interest of a larger protein-attached to optical biosensors to measure the binding of ligands [230].…”

“…This makes these venoms an ideal test of whether short-chain neurotoxins bind to mimotopes and how they behave on this mimotope assay. No other research has examined the binding of short-chain neurotoxins to α 1 subunit mimotopes specifically and the study demonstrating this method only used venoms that contain both short and long-chain neurotoxins [229].…”

“…One of the interesting results of the previous study using this methodology was that venom from the two species studied with specialized diets (Aipysurus laevis and Ophiophagus hannah) showed much greater effects on the mimotopes designed after the receptors of their preferred prey type (fish and snakes, respectively) than those from other taxa [229]. Prey specificity is a widespread phenomenon in snake venoms and these results suggest that our assay should be able to detect if Micrurus venoms have undergone similar adaptation [20,47,75,76].…”