A novel class of insecticidal alkylsulfones are potent inhibitors of vesicular acetylcholine transport

Pyridine alkylsulfone derivatives typified by oxazosulfyl (Sumitomo Chemical Company Ltd.) and compound A2 (Syngenta) represent a new class of insecticides, with potent activity against several insect orders. Whilst the MOA of this class has been attributed to interaction with the voltage-gated sodium channel (VGSC), here we present strong evidence that their toxicity to insects is mediated primarily through inhibition of the vesicular acetylcholine transporter (VAChT). Alkylsulfone intoxication in insects is characterised by (i) a reduction in cholinergic synaptic transmission efficiency demonstrated by a depression of cercal afferent activity in giant-interneurone preparations of American cockroach (Periplaneta americana), (ii) selective block of cholinergic-transmission dependent post-synaptic potentials in the Drosophila giant-fibre pathway and (iii) abolition of miniature excitatory post-synaptic currents (mEPSCs) in an identified synapse in Drosophila larvae.Ligand-binding studies using a tritiated example compound ([3H]-A1) revealed a single saturable binding-site, with low nanomolar Kd value, in membrane fractions of green bottle fly (Lucilia sericata). Binding is inhibited by vesamicol and by several examples of a previously identified class of insecticidal compounds known to target VAChT, the spiroindolines. Displacement of this binding by analogues of the radioligand reveals a strong correlation with insecticidal potency. No specific binding was detected in untransformed PC12 cells but a PC12 line stably expressing Drosophila VAChT showed similar affinity for [3H]-A1 as that seen in fly head membrane preparations. Previously identified VAChT point mutations confer resistance to the spiroindoline class of insecticides in Drosophila by Gal-4/UAS directed expression in cholinergic neurones and by CRISPR gene-editing of VAChT, but none of these flies show detectable cross-resistance to this new chemical class.Oxazosulfyl was previously shown to stabilise voltage-gated sodium channels in their slow-inactivated conformation with an IC50 value of 12.3μM but inhibits binding of [3H]-A1 with approximately 5000 times greater potency. We believe this chemistry class represents a novel mode-of-action with high potential for invertebrate selectivity.