alpha/beta hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase alpha/beta hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tu4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.
A diversity of nicotinic acetylcholine receptor (nAChR) subtypes has been identified in mammalian brain using recombinant DNA technology. Alterations in the activity of these acetylcholinegated ion channels have been implicated in a number of central nervous system disorders including Alzheimer's disease (AD). The potential therapeutic usefulness of (-)-nicotine [(S)-3-(1-methyl-2-pyrrolidinyl) pyridine], the prototypic agonist at nAChRs, is severely limited by side effects that are the result of activation of both cholinergic and noncholinergic pathways in the central and peripheral nervous systems. This study sought to determine the in vitro selectivity of (S)-3-methyl-5-(1methyl-2-pyrrolidinyl)isoxazole (ABT 418), a novel analog of (-)-nicotine in which the pyridine ring was replaced with an isoxazole bioisotere, to activate nAChRs. ABT 418 was a potent inhibitor of [3H]-cytisine binding to nAChR in rat brain (Ki = 3 nM) but was inactive (Ki > 10,000 nM) in 37 other receptor/neurotransmitter-uptake/enzyme/transduction system binding assays, including those for alpha-bungarotoxin, muscarinic and 5-hydroxytryptamine3 receptors. In PC12 cells, patch-clamp studies indicated that ABT 418 was an agonist with an EC50 value of 209 microM, a potency to activate cholinergic channel currents some 4-fold less than that of (-)-nicotine (52 microM). Channel current responses elicited by ABT 418 were prevented by the cholinergic channel blocker, mecamylamine. ABT 418 was also approximately 10-fold less potent (EC50 value = 380 nM) than (-)-nicotine (40 nM) in increasing [3H]-dopamine release from rat striatal slices, an effect that was blocked by the nAChR antagonist, dihydro-beta-erythroidine (10 microM).2+ In contrast, ABT 418 appeared equipotent with (-)-nicotine in enhancing 86Rb+ flux from mouse thalamic synaptosomes. ABT 418 demonstrated an in vitro pharmacological profile of cholinergic channel activation that was robust at some nAChR, but not others. The reasons for this are unclear. However, a nAChR subtype selectivity may account for the in vitro potency differences of ABT 418 on various neurotransmitter systems, and the substantial separation between the cognitive enhancement/anxiolytic benefits, and the reduced central nervous system side-effect liabilities seen in vivo. ABT 418 represents the first neuronal nAChR ligand that differentiates the toxicities/liabilities and other negative aspects normally associated with liabilities and other negative aspects normally associated with (-)-nicotine from the potential pharmacological benefits of selective cholinergic channel activation.
(+/-)-Epibatidine, exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1] heptane, is a novel, potent analgesic agent that acts through nicotinic acetylcholine receptor (nAChR) mechanisms. This study sought to establish whether (+/-)-epibatidine, like (-)-nicotine, also displays a wide diversity of behavioral responses that are known to be elicited by nAChR activation or whether it demonstrates subtype selectivity for its interactions with nAChRs.(+/-)-Epibatidine displaced [3H](-)-cytisine binding to the alpha 4 beta 2 nAChR subtype in rat brain membranes with high affinity (Ki, 43 pM). The compound was approximately 5000-fold less potent (Ki = 230nM) in the displacement of [125I] alpha-bungarotoxin binding from the alpha-bungarotoxin-sensitive nAChR subtype present in rat brain but was a potent inhibitor (Ki, 2.7 nM) of [125I] alpha-bungarotoxin binding to the nAChR subtype in Torpedo electroplax, which is similar to that present in the neuromuscular junction. Functionally, (+/-)-epibatidine enhanced 86Rb+ flux in IMR 32 cells with an EC50 value of 7 nM. It was some 3000-fold more potent than (-)-nicotine (EC50 value, 21,000 nM) and was approximately 150-fold more potent (EC50 value, 0.4 nM) than (-)-nicotine (EC50 value = 60 nM) in increasing [3H]dopamine release from rat striatal slices. Remarkably, (+/-)-epibatidine was 40% to 50% more efficacious than (-)-nicotine in both functional assays. Both functional effects were blocked by the nAChR channel blocker, mecamylamine (100 microM). (+/-)-Epibatidine was 300 to 1000 times more potent than (-)-nicotine in the reduction of body temperature and locomotor activity in mice.(ABSTRACT TRUNCATED AT 250 WORDS)
