Investigating the pharmacology of ladybird alkaloids on ligand-gated ion channels

Abstract

The WHO has forecasted the global population to reach 9 billion by 2050 and increased productivity of crops and livestock is crucial in order to meet the heightened demand for food. Pest organisms are a major factor in limiting this productivity and pesticides are a key tool in their control. Environmental concerns and the spread of pesticide resistance have hastened the need for new pesticides. Natural toxins targeting the nicotinic acetylcholine receptor (nAChR) have proven a valuable source of lead compounds for the development of new pesticides. Alkaloids from multiple species of ladybird beetles have been shown to target the nAChR. Recently, studies have shown that harmonine from the Harlequin ladybird Harmonia axyridis and (-)-adaline from the 2-spot ladybird Adalia bipunctata show promise as leads for new pesticides as they have demonstrated to be selective for the neuronal-type nAChRs expressed in locust neurons over mammalian muscle-type nAChRs expressed in human TE671 cells. However, mammalian neuronal-type nAChRs need to be investigated in order to confirm this selectivity. The Xenopus oocyte expression system was used to investigate the effects of (-)-adaline and whole alkaloid extract from the harlequin ladybird (HAE) on multiple mammalian neuronal-type nAChRs and the hybrid Dα2/β2 Drosophila/chick receptor using the two electrode voltage-clamp technique. This technique also allowed the expression and investigation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors. HAE was found to be selective for the insect hybrid Dα2/β2 over the other mammalian receptors investigated with an IC50 (95% CI) of 0.44 μg/ml (0.211 – 0.91). Analysis of the current-voltage (IV) relationship of HAE inhibition showed voltage-dependent block of rat α4β2 and voltage-independent block of rat α3β4 and human α7. Examination of the effect of HAE on the ACh EC50 revealed competitive block for rat α4β2 and potential non-competitive block of human α7 and rat α3β4. Selectivity for the hybrid insect/chick receptor was not demonstrated with (-)-adaline instead showing selectivity for rat α3β4 with an IC50 (95% CI) of 0.66 μM (0.24 – 1.67). (-)-adaline was found to competitively block rat α3β4 and rat α4β2 with potential non-competitive block of human α7. (-)-adaline was also found to block in all nAChRs examined in a voltage-independent manner. As HAE showed selectivity for the hybrid Dα2/β2 receptor there is promise for component(s) of HAE to serve as lead structures for the development of new pesticides. However, HAE is a mixture of various alkaloids that could have differing modes of action. This study revealed a competitive mode of action consistent with binding to the ACh binding site and voltage-dependent block consistent with open channel blocking on rat α4β2. Separation of HAE into individual components needs to be carried out for further investigation. HAE was also found to inhibit the rat GluN1-1a/2A NMDA receptor with an IC50 (95% CI) of 0.179 μg/ml (0.116 – 0.278) at Vh = -75 mV indicating a potential therapeutic use in neurodegenerative diseases associated with NMDA receptor excitotoxicity as well as pesticidal use