title: Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.
abstract: γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan. Electronic address: ozoe-y@life.shimane-u.ac.jp.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Ozoe
authors.forename: Tomo
authors.initials: T
authors.lastname: Kita
authors.forename: Fumiyo
authors.initials: F
authors.lastname: Ozoe
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Nakao
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sato
authors.forename: Kangetsu
authors.initials: K
authors.lastname: Hirase
mesh_terms: D000495:Allosteric Site; D000818:Animals; D018756:GABA Antagonists / Q000737:chemistry / Q000378:metabolism; D006793:Houseflies; D007306:Insecticides / Q000737:chemistry* / Q000378:metabolism*; D007559:Ivermectin / Q000031:analogs & derivatives / Q000378:metabolism; D018079:Receptors, GABA / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D018756:GABA Antagonists; D007306:Insecticides; D018079:Receptors, GABA; D007559:Ivermectin; C052102:22,23-dihydroavermectin B(1)a
keywords: 3-Substituted benzamides; 3-benzamido-N-phenylbenzamide; 4′-ethynyl-4-n-propylbicycloorthobenzoate; Antagonist; BPB; EBOB; GABA; GABA receptor; GABAR; IGluR; Insecticide; SOS; TEVC; inhibitory l-glutamate receptor; standard oocyte solution; two-electrode voltage clamp; γ-Aminobutyric acid; γ-aminobutyric acid