Lisa M. Broad

Sazetidine-A Is a Potent and Selective Agonist at Native and Recombinant α4β2 Nicotinic Acetylcholine Receptors

Sazetidine-A has been recently proposed to be a “silent desensitizer” of α4β2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes α4β2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of α4β2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by α4β2* and α6β2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant α4β2 nAChRs in more detail. We expressed the two alternative stoichiometries of α4β2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both α4(2)β2(3) and α4(3)β2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of α4β2 nAChR: it was a full agonist on α4(2)β2(3) nAChRs, whereas it had an efficacy of only 6% on α4(3)β2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant α4β2 nAChRs but shows differential efficacy on α4β2 nAChRs subtypes.

Identification and Pharmacological Profile of a New Class of Selective Nicotinic Acetylcholine Receptor Potentiators

Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (α1β1γδ, α2β4, α3β2, α3β4, α4β2, α4β4, and α7) expressed in mammalian cells or Xenopus oocytes. At α2β4, α4β2, α4β4, and α7, but not α1β1γδ, α3β2, or α3β4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABAA, and N-methyl-d-aspartate receptors or voltage-gated Na+ and Ca2+ channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at α4β2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [3H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.

Species Selectivity of a Nicotinic Acetylcholine Receptor Agonist Is Conferred by Two Adjacent Extracellular β4 Amino Acids that Are Implicated in the Coupling of Binding to Channel Gating

5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one (TMAQ) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for β4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human β4 subunit but having no detectable agonist activity on nAChRs containing the rat β4 subunit. With the aim of identifying subunit domains and individual amino acids, which contribute to the species selectivity of TMAQ, a series of chimeric and mutated β4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric, or mutated β4 subunits have been examined by radioligand binding, intracellular calcium assays, and electrophysiological recording. Two adjacent amino acids located within the extracellular loop D domain of the β4 subunit (amino acids 55 and 56) have been identified as playing a critical role in determining the agonist potency of TMAQ. Mutagenesis of these two residues within the rat β4 subunit to the corresponding amino acids in the human β4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human β4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the nonselective nicotinic agonist epibatidine. Despite acting as a potent agonist of human β4-containing nAChRs, TMAQ acts as an antagonist of rat β4-containing receptors. Our experimental data, together with homology models of the rat and human α3β4 nAChRs, suggest that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.

Synthesis and Pharmacological Characterization of C4-Disubstituted Analogs of 1S,2S,5R,6S-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: Identification of a Potent, Selective Metabotropic Glutamate Receptor Agonist and Determination of Agonist-Bound Human mGlu2 and mGlu3 Amino Terminal Domain Structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

Characterisation of inositol 1,4,5-trisphosphate binding sites in rabbit aortic smooth muscle.

The present study investigated the characteristics of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites in crude membrane preparations of rabbit aortic smooth muscle. A particular aim was to demonstrate if increases in cytoplasmic cyclic guanosine 3:5 monophosphate (cGMP), which mediates the effect of nitrovasodilators, may cause smooth muscle relaxation in part by the displacement of Ins(1,4,5)P3 binding. Negligible Ins(1,4,5)P3 binding was observed at pH < 7, while maximum binding occurred over the pH range 8-9. Saturation analysis of isotopic dilution binding data revealed an apparently homogenous population of Ins(1,4,5)P3 binding sites with a KD of 4.02 +/- 0.53 nM and a Bmax of 27.7 +/- 4.6 fmol/mg protein. Heparin, an Ins(1,4,5)P3 receptor antagonist, inhibited binding with an IC50 of 11.43 +/- 2.81 micrograms/ml. The ability of other polyphosphate compounds to inhibit Ins(1,4,5)P3 binding in this preparation was also examined. D-myo-Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), adenosine 5-triphosphate (ATP) and guanosine 5-triphosphate (GTP) inhibited Ins(1,4,5)P3 binding, although each was significantly less potent that Ins(1,4,5)P3. In contrast, cyclic guanosine 3:5 monophosphate (cGMP) did not significantly alter Ins(1,4,5)P3 binding in rabbit aortic smooth muscle. This observation suggests that competitive inhibition of Ins(1,4,5)P3 receptor binding is not an important consideration in cGMP-mediated vascular smooth muscle cell relaxation.