Izumi Ikeda

Picrodendrin and related terpenoid antagonists reveal structural differences between ionotropic GABA receptors of mammals and insects.

Twenty-eight picrotoxane terpenoids, including picrodendrins isolated from the Euphorbiaceae plant, Picrodendron baccatum (L.) Krug and Urban, have been evaluated for their ability to inhibit the specific binding of [3H]EBOB, the noncompetitive antagonist of ionotropic GABA receptors, to rat-brain and housefly (Musca domestica L.)-head membranes. Picrodendrin Q was the most potent competitive inhibitor of [3H]EBOB binding, with IC50 values of 16 nM (rat) and 22 nM (Musca). We find that the spiro gamma-butyrolactone moiety at the 13-position, which contains a carbonyl group conjugated with an unsaturated bond, and the substituents at the 4-position play important roles in the interaction of picrodendrins with their binding site in rat receptors. In contrast, such structural features are not strictly required in the case of the interaction with Musca receptors; the spiro saturated gamma-butyrolactone moiety at the 13-position, which bears the 16-sp3 carbon atom, and the hydroxyl groups at various positions are somewhat tolerated. Quantitative structure-activity studies have clearly shown that the electronegativity of the 16-carbon atom and the presence or absence of the 4- and 8-hydroxyl groups are important determinants of the potency of nor-diterpenes in Musca receptors, while the negative charge on the 17-carbonyl oxygen atom is likely important in the case of rat receptors. These findings indicate that there are significant differences between the structures of the complementary binding sites in rat GABA receptors and Musca GABA receptors. We also infer differences between native Musca GABA receptors and the Drosophila Rdl subunit-containing homo-oligomeric GABA receptors in the structures of their binding sites.

Actions of cyclic esters, S-esters, and amides of phenyl- and phenylthiophosphonic acids on mammalian and insect GABA-gated chloride channels.

Cyclic esters, S-esters, and amides of phenyl(thio)phosphonic acid were synthesized to probe the interaction between noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors and their binding site. Some of these compounds competitively inhibited the specific binding of [3H]EBOB, a noncompetitive GABA antagonist, to rat-brain and housefly-head membranes. The trans isomer of the ester bearing a tert-butyl group at the 5-position and a bromine atom at the p-position (5t) was most potent in rat receptors with an IC50 value of 40 nM, while the trans isomer of the S-ester bearing the same substituents (10t) was most potent in housefly receptors with an IC50 value of 55 nM. In both cases, the corresponding amide analogue (12t) was less potent. The potencies of 5t and 12t tended to decrease in the presence of GABA, particularly in housefly receptors, while that of 10t remained unchanged. The rank order of activity in inhibiting [3H]EBOB binding to housefly-head membranes in the presence of GABA (10t > 5t > 12t) was in accord with that of insecticidal activity. S-Ester 10t depressed 10 microM and 300 microM GABA-induced 36Cl- influx into mouse cerebral synaptoneurosomes, whereas ester 5t depressed 10 microM GABA-induced 36Cl- influx but not 300 microM GABA-induced flux. Amide 12t was inactive at both GABA concentrations. These findings indicate that six-membered cyclic phenylthiophosphonic acid derivatives act as noncompetitive antagonists of GABA receptors and suggest that 10t is able to bind to the receptor in the open, desensitized, and closed states, whereas the affinity of 5t and 12t is lower in the open and desensitized states than in the closed state. The derivatives have similar structures except for the heteroatoms at the 1- and 3-positions, so that the heteroatoms may play a unique role when antagonists bring the open state of the GABA-gated channel to the desensitized or closed state.

Competitive antagonism of insect GABA receptors by iminopyridazine derivatives of GABA

A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100 μM in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100 μM, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10 μM in these receptors, although the inhibition at 100 μM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500 μM using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site.

Synthesis of photoreactive ivermectin B1a derivatives and their actions on Haemonchus and Bombyx glutamate-gated chloride channels.

Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls. .

QSAR Studies of Picrodendrins and Related Terpenoids — Structural Differences Between Antagonist Binding Sites on GABA Receptors of Insects and Mammals

γ -Aminobutyric acid (GABA), an inhibitory neurotransmitter, binds to the GABAA (ionotropic) receptor, to regulate the central nervous system of vertebrates. Insects have similar ionotropic receptors with different pharmacological properties, and, as a result, their GABA receptors represent promising targets for insecticides. Recently, 3D-QSAR analyses for insecticidal activity (against houseflies) and competitive activity against the specific [35S]tert-butylbicyclophosphorothionate (TBPS) binding (to rat brain membranes) of some picrotoxinin-type GABA antagonists, including γ -BHC, endosulfan, bicyclophophates, dioxatricyclododecenes (DTD) and related compounds, were carried out1 using comparative molecular field analysis (CoMFA). The CoMFA results showed that similarities and dissimilarities in sterically and electrostatically favourable and forbidden regions on the molecule were reflected in the insecticidal and rat-receptor binding activities.

Interactions of picrodendrins and related terpenoids with ionotropic GABA receptors of mammals and insects

Different structural features govern the interaction of picrodendrins and related terpenoids with rat and with housefly GABA receptors. This supports previous studies which suggest that there are significant differences between the structures of the binding sites in these two receptors.