Hugh J. Herdon

Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor

This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB‐674042 (1‐(5‐(2‐fluoro‐phenyl)‐2‐methyl‐thiazol‐4‐yl)‐1‐((S)‐2‐(5‐phenyl‐(1,3,4)oxadiazol‐2‐ylmethyl)‐pyrrolidin‐1‐yl)‐methanone), to the human orexin‐1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane‐based scintillation proximity assay (SPA) format. Specific binding of [3H]SB‐674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high‐affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein−1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin‐A displaying a ∼five‐fold higher affinity than orexin‐B (Ki values of 318±158 and 1516±597 nM, respectively). SB‐334867, SB‐408124 (1‐(6,8‐difluoro‐2‐methyl‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) and SB‐410220 (1‐(5,8‐difluoro‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB‐334867, SB‐408124 and SB‐410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50‐fold selectivity over the orexin‐2 receptor. These studies indicate that [3H]SB‐674042 is a specific, high‐affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors.

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172EDGE175, Tyr177, and Thr423. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr177 and Thr423, rather than the 172EDGE175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1

M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimers disease and schizophrenia. However, the discovery of subtype‐selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh‐binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl]‐piperidine), which bind to an allosteric site and selectively activate the M1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs.

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1 Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M1 receptor previously suggested to interact with an “ectopic” site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M1 receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [3H]N-methylscopolamine ([3H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [3H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [3H]NMS dissociation from CHO-hM1 cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M1 receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M1 receptors in the absence of the orthosteric agonist.

Characterization of the binding of [125I]‐human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor

GPR10 is a novel G‐protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor‐1 (UHR‐1). Human prolactin‐releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [125I]‐PrRP‐20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [125I]‐PrRP‐20 was saturable, and analysis suggested evidence of both high and low affinity sites, with KD values of 0.026±0.006 and 0.57±0.14 nM respectively, and Bmax values of 3010±400 and 8570±2240 fmol mg protein−1 respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a KD of 0.012 nM. Competition studies revealed that human and rat PrRP‐20 and PrRP‐31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF‐amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP‐20 failed to alter basal or forskolin‐stimulated levels of intracellular cyclic AMP in HEK293‐GPR10 cells, suggesting that GPR10 does not couple via either Gs or Gi. Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP‐20 and PrRP‐31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca2+ stores. These studies indicate that [125I]‐PrRP‐20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor

Recent years have witnessed the discovery of novel selective agonists of the M1 muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M1 mAChR and three mutant M1 mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [3H]N-methyl scopolamine ([3H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [3H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y381A (transmembrane helix 6) and W101A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F77I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca2+ elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible “bitopic” binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.

Identification of (-)-cis-6-acetyl-4S-(3-chloro-4-fluoro-benzoylamino)- 3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol as a potential antimigraine agent.

Optimisation of novel cis- and trans-4-(substituted-amido)benzopyran-3-ol derivatives has led to the identification of SB-220453 20 with an in vivo pre-clinical CNS profile predictive of potential antimigraine activity.

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures

1 Earlier optimization of structure‐activity relationships in a novel series of 4‐(benzoylamino)‐benzopyrans, led to the discovery of SB‐204269 (trans‐(+)‐6‐acetyl‐4S‐(4‐fluorobenzoylamino)‐3,4‐dihydro‐2,2‐dimethyl‐2H‐benzo[b]pyran‐3R‐ol, hemihydrate), a potent orally‐active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2 Studies have now been undertaken to determine the effects of SB‐204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3 SB‐204269 proved to be an orally‐effective anticonvulsant agent, at doses (0.1–30 mg kg−1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MES)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)‐evoked tonic extension seizures. However, the compound did not inhibit PTZ‐induced myoclonic seizures at doses up to 30 mg kg−1, p.o. 4 SB‐204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1–10 μM) that had no effect on normal synaptic activity and neuronal excitability. 5 In all of these seizure models, SB‐204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6 Unlike SB‐204269, the corresponding trans 3S,4R enantiomer, SB‐204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7 In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB‐204269 was inactive even at doses as high as 200 mg kg−1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB‐204269 of >31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8 At concentrations (10 μM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 μM in brain), SB‐204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB‐204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9 The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB‐204269 for the treatment of refractory partial and generalized tonic‐clonic seizures.

Characterization of the binding of [3H]‐SB‐204269, a radiolabelled form of the new anticonvulsant SB‐204269, to a novel binding site in rat brain membranes

1 SB‐204269 (trans‐(+)‐6‐acetyl‐4S‐(4‐fluorobenzoylamino)‐3,4‐dihydro‐2,2‐dimethyl‐2H‐benzol[b]pyran‐3R‐ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side‐effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]‐SB‐204269 in rat forebrain membranes. 2 Specific [3H]‐SB‐204269 binding was saturable and analysis indicated binding to a homogenoeous population of non‐interacting binding sites with a dissociation constant (KD) of 32±1 nM and a maximum binding capacity (Bmax) of 253±18 fmol mg−1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris‐HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. 3 Specific [3H]‐SB‐204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB‐204269 and its enantiomer SB‐204268 for the binding site. The affinities of analogues of SB‐204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. 4 None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]‐SB‐204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. 5 These studies indicate that SB‐204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.

Glycine transporter 1 (GlyT1) inhibitors exhibit anticonvulsant properties in the rat maximal electroshock threshold (MEST) test

Glycine can act as either an inhibitory neurotransmitter or as a potentiator of NMDA-dependent excitatory neurotransmission. There is some evidence that glycine can have both pro- and anticonvulsant properties in various rodent models of epilepsy. In the present study we tested several glycine transporter 1 (GlyT1) inhibitors including NFPS, SSR 504734, Lu AA21279, Org 25935, SB-710622, GSK931145, as well as the glycine agonist d-serine, in the maximal electroshock threshold (MEST) test in the rat. In a series of experiments, male Sprague-Dawley rats (n=12/group) were pre-treated with a compound of interest and then received an electric shock delivered via corneal electrodes. A cohort of satellite animals (n=3/group) was also used to measure blood and brain levels of Org 25935. All GlyT1 inhibitors increased seizure thresholds dose-dependently, indicative of anticonvulsant activity. SB-710622 and GSK931145 had lower minimum effective doses (MEDs) in the MEST test than other GlyT1 inhibitors. At estimated t(max), increases in dose administered were paralleled by increases in blood and brain concentrations of Org 25935. Thus, increasing extracellular concentration of glycine via inhibition of its uptake protects from electroshock-induced seizures in the rat. Whether strychnine-sensitive or strychnine-insensitive glycine binding sites are involved in this effect remains to be determined.