NITROMETHYLENEIMIDAZOLIDINE RADIOLIGAND ([H-3]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 [H-3]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-chioro-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. [H-3]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 [H-3]NMI and [H-3]IMI, each with a single saturable specific binding site of K-d = 1.2 nM and B-max = 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 [H-3]IMI but only a low affinity one for [H-3]NMI. Competing ligands are less effective at displacing [H-3]NMI than [H-3]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. (C) 1994 Academic Press, Inc.