Jeannette M. Watson

SB-224289–a novel selective (human) 5-HT1B receptor antagonist with negative intrinsic activity

1 Human 5‐HT1B (h5‐HT1B) and human 5‐HT1D (h5‐HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB‐224289 (1′‐Methyl‐5‐[[2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)biphenyl‐4‐yl]carbonyl]‐2,3,6,7‐tetrahydrospiro [furo [2,3‐f]indole‐3,4′‐piperidine] oxalate), which has high affinity for h5‐HT1B receptors (pK1=8.16±0.06) and displays over 75 fold selectivity for the h5‐HT1B receptor over all other 5‐HT receptors including the h5‐HT1D receptor and all other receptors tested thus far. 2 Functional activity of SB‐224289 was measured in a [35S]GTPγS binding assay on recombinant h5‐HT1B and h5‐HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB‐224289 displayed negative intrinsic activity at both receptors with higher potency at h5‐HT1B receptors. SB‐224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3 SB‐224289 potentiated [3H]5‐HT release from electrically stimulated guinea‐pig cerebral cortical slices to the same extent as as the non‐selective 5‐HT1 antagonist methiothepin. SB‐224289 also fully reversed the inhibitory effect of exogenously superfused 5‐HT on electrically stimulated release. 4 Using SB‐224289 as a tool compound, we confirm that in guinea‐pig cerebral cortex the terminal 5‐HT autoreceptor is of the 5‐HT1B subtype.

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.

Serotonergic targets in depression

Serotonin reuptake inhibitors have proved to be a very effective treatment for depression and have strengthened the hypothesis that impaired 5-hydroxytryptamine (5-HT) neurotransmission may contribute to the underlying cause of depressive disorders. Extensive research has been carried out to investigate other 5-HT targets associated with the disease and studies involving combination treatments with selective serotonin reuptake inhibitors and 5-HT(1A) receptor ligands are currently being carried out in the clinic. Whether other 5-HT receptor subtypes are involved in the aetiology of depression remains to be seen.

Vilazodone: a 5-HT1A receptor agonist/serotonin transporter inhibitor for the treatment of affective disorders.

Vilazodone (EMD 68843; 5‐{4‐[4‐(5‐cyano‐3‐indolyl)‐butyl]‐1‐piperazinyl}‐benzofuran‐2‐carboxamide hydrochloride) is a combined serotonin specific reuptake inhibitor (SSRI) and 5‐HT1A receptor partial agonist currently under clinical evaluation for the treatment of major depression. This molecule was designed based on the premise that negative feedback circuitry, mediated via 5‐HT1 receptors, limits the acute SSRI‐induced enhancements in serotonergic neurotransmission. If the hypothesis is correct, combination of SSRI with 5‐HT1A partial agonism should temporally enhance the neuroplastic adaptation and subsequently hasten therapeutic efficacy compared to current treatments. Preclinical in vitro evaluation has confirmed vilazodones primary pharmacological profile both in clonal and native systems, that is, serotonin reuptake blockade and 5‐HT1A partial agonism. However, in vivo and in contrast to combination of 8‐OH‐DPAT and paroxetine, vilazodone selectively enhanced serotonergic output in the prefrontal cortex of rats. Behavioral evaluations, in the ultrasonic vocalization model of anxiety in rats, demonstrated anxiolytic efficacy. In the forced swim test (a putative model of depression), vilazodone also showed efficacy but at a single dose only. In man, vilazodone abolished REM sleep and demonstrated clinical antidepressant efficacy equivalent to an SSRI. Ongoing clinical evaluations will hopefully reveal whether the founding hypothesis was valid and if vilazodone will produce a more rapid onset of antidepressant efficacy.

GPR105, a novel Gi/o-coupled UDP-glucose receptor expressed on brain glia and peripheral immune cells, is regulated by immunologic challenge: possible role in neuroimmune function

We have recently shown that UDP-glucose, and some related UDP-sugars, are potent agonists of the novel G protein-coupled receptor GPR105 (recently re-named P2Y(14)). GPR105 is widely expressed throughout many brain regions and peripheral tissues of human and rodents, and couples to a pertussis toxin-sensitive G protein. To further characterise the role of GPR105, we demonstrate by immunohistochemistry with receptor-specific antiserum that GPR105 protein is widely distributed throughout the post mortem human brain where it is localised to glial cells, and specifically co-localises with astrocytes. Using quantitative RT-PCR we also show that GPR105 mRNA exhibits a restricted expression profile in an array of human cell lines and primary cells, with prominent expression detected in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. To investigate the G protein selectivity of GPR105, we used chimeric Galpha subunits (Galpha(qi5), Galpha(qo5), and Galpha(qs5)) and an intracellular Ca(2+) mobilisation assay to demonstrate that GPR105 couples to Galpha subunits of the G(i/o) family but not to G(s) family proteins or to endogenous G(q/11) proteins in HEK-293 cells. Finally, we show that expression of GPR105 mRNA in the rat brain is up-regulated by immunologic challenge with lipopolysaccharide. Based on these observations, we propose that G(i/o)-coupled GPR105 might play an important role in peripheral and neuroimmune function in response to extracellular UDP-sugars.

5-HT1A receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5‐HT1A receptors. Saturation studies using [3H]‐8‐OH‐DPAT and [3H]‐MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5‐HT1A receptors and rat cortex. In recombinant cells, [3H]‐MPPF labelled 3–4 fold more sites than [3H]‐8‐OH‐DPAT suggesting the presence of more than one affinity state of the receptor. [3H]‐Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5‐HT1A receptors but did not bind to native tissue 5‐HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5‐HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. Receptor agonists inhibited [3H]‐MPPF binding from recombinant 5‐HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]‐8‐OH‐DPAT and [3H]‐spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]‐MPPF and [3H]‐8‐OH‐DPAT in a monophasic manner. Functional evaluation of compounds, using [35S]‐GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5‐HT and 5‐CT to inverse agonism displayed by spiperone. [3H]‐8‐OH‐DPAT : [3H]‐MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]‐8‐OH‐DPAT : [3H]‐spiperone pKi difference with functional intrinsic activity (r=0.96). Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5‐HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5‐HT1A receptors in which only the high agonist affinity state was detectable.

GR127935 acts as a partial agonist at recombinant human 5-HT1Dα and 5-HT1Dβ receptors

In this study we have investigated the functional activity of GR127935 (2-methyl-4-(5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4-carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]-amide) at human 5-HT1Dα and 5-HT1Dβ receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [35S]GTPγS binding to cell membranes as a measure of receptor-G protein coupling, GR127935 showed partial agonist activity in both 5-HT1Dα and 5-HT1Dβ receptor expressing cells (Emax: 29 and 31% above basal control; pEC50: 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT1Dα (app. pA2 8.5) and 5-HT1Dβ (app. pA2 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT1Dα receptors which stimulate cAMP levels and 5-HT1Dβ receptors which inhibit cAMP levels (app. pA2 8.6 and 9.7, respectively). The 5HT1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [35S]GTPγS binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-methoxy-1,2-dihydroquinoline) the 5-HT1Dα cell line displayed a lack of receptor reserve but it was evident in the 5-HT1Dβ cell line. In previous studies we have also shown that agonist stimulation of 5-HT1Dα receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 μM EEDQ to reduce 5-HT1Dα receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, aswell as a potent antagonist, at the human 5-HT1Dα and 5-HT1Dβ receptors when expressed in CHO cells.

Functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM1, hM2, hM3, hM4 and hM5 using microphysiometry

This study describes the pharmacological comparison of the muscarinic partial agonists sabcomeline, xanomeline and milameline at human cloned muscarinic receptor subtypes (hM1–5). Radioligand binding studies at the hM1–5 muscarinic receptor subtypes were compared with functional studies using microphysiometry using carbachol as the standard full agonist. In binding assays none of the compounds studied displayed preferential affinity for the M1,3,4 or M5 subtypes although carbachol was less potent at hM1 than hM3,4,5. In functional studies, all of the compounds studied displayed similar levels of efficacy across the muscarinic receptors with the exception of M3, where there was a large apparent receptor reserve and the compounds behaved essentially as full agonists. Sabcomeline was the most potent agonist in functional studies but also showed the lowest efficacy. In terms of potency, xanomeline showed some selectivity for M1 over M2 receptors and milameline showed some selectivity for M2 over M1 receptors. These results show the value of microphysiometry in being able to compare receptor pharmacology across subtypes irrespective of the signal transduction pathway. None of the partial agonists showed functional selectivity for M1 receptors, or indeed any muscarinic receptor, in the present study.

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.

SB-649915-B, a Novel 5-HT 1A/B Autoreceptor Antagonist and Serotonin Reuptake Inhibitor, is Anxiolytic and Displays Fast Onset Activity in the Rat High Light Social Interaction Test

Preclinically, the combination of an SSRI and 5-HT autoreceptor antagonist has been shown to reduce the time to onset of anxiolytic activity compared to an SSRI alone. In accordance with this, clinical data suggest the coadministration of an SSRI and (±) pindolol can decrease the time to onset of anxiolytic/antidepressant activity. Thus, the dual-acting novel SSRI and 5-HT1A/B receptor antagonist, SB-649915-B, has been assessed in acute and chronic preclinical models of anxiolysis. SB-649915-B (0.1–1.0 mg/kg, i.p.) significantly reduced ultrasonic vocalization in male rat pups separated from their mothers (ED50 of 0.17 mg/kg). In the marmoset human threat test SB-649915-B (3.0 and 10 mg/kg, s.c.) significantly reduced the number of postures with no effect on locomotion. In the rat high light social interaction (SI), SB-649915-B (1.0–7.5 mg/kg, t.i.d.) and paroxetine (3.0 mg/kg, once daily) were orally administered for 4, 7, and 21 days. Ex vivo inhibition of [3H]5-HT uptake was also measured following SI. SB-649915-B and paroxetine had no effect on SI after 4 days. In contrast to paroxetine, SB-649915-B (1.0 and 3.0 mg/kg, p.o., t.i.d.) significantly (p<0.05) increased SI time with no effect on locomotion, indicative of an anxiolytic-like profile on day 7. Anxiolysis was maintained after chronic (21 days) administration by which time paroxetine also increased SI significantly. 5-HT uptake was inhibited by SB-649915-B at all time points to a similar magnitude as that seen with paroxetine. In conclusion, SB-649915-B is acutely anxiolytic and reduces the latency to onset of anxiolytic behavior compared to paroxetine in the SI model.