Nitromethyleneimidazolidine radioligand ([3H]NMI): high affinity and cooperative binding for house fly acetylcholine receptor

1-[N-(2-Chloro-5-thiazolylmethyl)]-2 nitromethylene-imidazolidine (NMI) is a very potent insecticide and is 6-fold more effective than imidacloprid (IMI) in displacing [3H]IMI from its binding site in the house fly acetylcholine (ACh) receptor (AChR). NMI differs from IMI in two isosteric replacements, i.e., 2-chloro-5-thiazolyl (CT) for 6-chloro-3-pyridinyl (CP) and nitromethylene for nitroimine. The CP and CT moieties in this series confer almost equivalent potency and binding properties allowing intercomparisons based on the nitromethylene and nitroimine substituents. [3H]NMI (55 Ci/mmol) was prepared from 2-chloro-5-(carbethoxy)thiazole by reducing with lithium aluminum tritide to the alcohol which was converted to the chloromethyl derivative and then coupled with ethylenediamine followed by reaction with 1,1-bis(methylthio)-2-nitroethylene. Binding parameters in house fly head membranes treated with Triton X-100 are very similar for [3H]NMI and [3H]IMI, each with a single saturable specific binding site of Kd = 1.2 nM and Bmax = 853-897 fmol/mg protein, and there are also similar initial rates of association and dissociation for the two radioligands. However, there is a significant difference in the Hill coefficient with 1.4 +/- 0.06 for NMI and 1.0 +/- 0.1 for IMI. Without Triton X-100 treatment, there are both low and high affinity binding components for [3H]IMI but only a low affinity one for [3H]NMI. Competing ligands are less effective at displacing [3H]NMI than [3H]IMI, e.g., 9-fold for ACh (with paraoxon to inhibit acetylcholinesterase), 40-fold for carbachol, and 2- to 6-fold for the nicotinic agents (-)-nicotine and alpha-bungarotoxin. The enhanced insecticidal activity and receptor potency of NMI compared with IMI may be associated with its higher apparent cooperativity facilitating disruption of the AChR.