Kerry L. Chapman

Evidence from gene knockout studies implicates Asc-1 as the primary transporter mediating d-serine reuptake in the mouse CNS.

In the mammalian central nervous system, transporter‐mediated reuptake may be critical for terminating the neurotransmitter action of d‐serine at the strychnine insensitive glycine site of the NMDA receptor. The Na+ independent amino acid transporter alanine–serine–cysteine transporter 1 (Asc‐1) has been proposed to account for synaptosomal d‐serine uptake by virtue of its high affinity for d‐serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc‐1 to d‐serine uptake in mouse brain synaptosomes using Asc‐1 gene knockout (KO) mice. Total [3H]d‐serine uptake in forebrain and cerebellar synaptosomes from Asc‐1 knockout mice was reduced to 34 ± 5% and 22 ± 3% of that observed in wildtype (WT) mice, respectively. When the Na+ dependent transport components were removed by omission of Na+ ions in the assay buffer, d‐serine uptake in knockout mice was reduced to 8 ± 1% and 3 ± 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc‐1 plays a major role in the Na+ independent transport of d‐serine. Potency determination of d‐serine uptake showed that Asc‐1 mediated rapid high affinity Na+ independent uptake with an IC50 of 19 ± 1 µm. The remaining uptake was mediated predominantly via a low affinity Na+ dependent transporter with an IC50 of 670 ± 300 µm that we propose is the glial alanine–serine–cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc‐1 is the only high affinity d‐serine transporter in the mouse CNS and is the predominant mechanism for d‐serine reuptake.

Identification of signal transduction pathways used by orphan g protein-coupled receptors.

The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.

Measurement of central nervous system activity of systemically administered CCKB receptor antagonists by ex vivo binding

In the present study we have described an ex vivo binding assay in mice to measure the central nervous system (CNS) activity of systemically administered CCKB receptor antagonists. This assay incorporated a transcardiac perfusion step to remove the residual blood from the brain, which otherwise may result in an overestimation of CNS activity. The benzodiazepine CCKB receptor antagonist L-365,260 had marked CNS activity in this assay following i.v. (ED50 12.0 mg/kg) and p.o. (ED50 20.0 mg/kg) administration, whereas the dipeptoid CCKB receptor antagonist, CI988 exhibited relatively weak CNS activity following i.v. injection (ED50 > 30.0 mg/kg). In contrast, following i.c.v. administration, CI988 potently inhibited ex vivo binding of [125I]Bolton Hunter-CCK-8S to mouse brain. The recently described acidic tetrazole CCKB receptor antagonist, L-368,935 had potent CNS activity with an ED50 of 5.6 mg/kg i.v. and an ED50 of 1.9 micrograms/kg i.c.v. These studies suggest that the weak CNS activity of CI988 following systemic injection may, in part, be due to poor brain penetration and that the ex vivo binding assay is a useful way of assessing the brain penetration of CCKB receptor antagonists.

Pharmacological and functional characterisation of dopamine D4 receptors in the rat retina

In the retina, activation of dopamine receptors, particularly the D2-like family (D2, D3, D4 receptor subtypes), with quinpirole suppresses the light sensitive cAMP pool and inhibits melatonin synthesis in photoreceptor cells. We have characterised rat retinal D4 receptors using the D4 selective radioligand [(125)I] L-750667 which bound specifically and saturably to rat retinal membranes with high affinity (K(d) 0.06+/-0.02 nM) and exhibited a D4 receptor pharmacology. Comparison of the binding kinetics of [(125)I] L-750667 and [(3)H] spiperone revealed B(max) values of 134+/-27 fmol/mg and 219+/-47 fmol/mg respectively, indicating that the dopamine D4 receptor is a major component of D2-like dopamine receptors in the rat retina. Modulation of retinal cAMP levels by quinpirole was used to evaluate the functional relevance of rat retinal dopamine D4 receptors. Quinpirole (0.03-3 micro ) produced a dose-related decrease of the light sensitive cAMP pool which was reversed by haloperidol, clozapine and the D4 selective antagonist, L-745870 with a rank order of potency suggesting that the quinpirole effect is due to activation of the dopamine D4 receptors. The D2 selective ligand L-741626 had no effect on the quinpirole response confirming that the D4 receptor is the major receptor subtype mediating dopamine induced suppression of adenylate cyclase in the retina.

Substituted pyrazoles as novel selective ligands for the human dopamine D4 receptor

Two novel series of 3-(heterocyclylmethyl)pyrazoles have been synthesised and evaluated as ligands for the human dopamine D4 receptor. Compounds in series I (exemplified by 8k) have a phenyl ring joined to the 4-position of the pyrazole while those in series II (exemplified by 15j) have a 5-phenyl ring linked by a saturated chain to the 4-position of the pyrazole. Both series supplied compounds with excellent affinity for the human D4 and good selectivity over other dopamine receptors. Excellent selectivity over calcium, sodium, and potassium ion channels was also achieved.

Are radioligand antagonist/agonist binding ratios in rat pancreas predictive of functional efficacy of cholecystokinin receptor agonists and antagonists?

Radioligand binding assays have been previously used to predict the relative efficacy of novel ligands. In the present study we have investigated whether for the cholecystokinin CCK-A receptors in the rat pancreas, the ratio of binding affinities for compounds for antagonist and agonist radioligands are predictive of functional activity. A number of classical cholecystokinin agonists, such as CCK-8S, caerulein, CCK-8DS, pentagastrin and CCK-4 had antagonist/agonist binding ratios of 4-fold or greater. All compounds behaved as full agonists in the stimulation of phosphatidylinositol (PI) turnover and increase in amylase secretion in rat pancreas. In contrast, compounds such as the benzodiazepine derivatives devazepide and L-365,260 had binding ratios of less than one and lacked agonist activity in either functional assay. Interestingly, the dipeptide derivative CI-988, which has been described as a selective CCK-B antagonist, was found to have an antagonist/agonist binding ratio of 1.5 for the CCK-A receptors in rat pancreas which was sufficiently high for this compound to behave as a full agonist in the amylase assay, although CI-988 did not exhibit agonist activity in the PI assay. These results suggest that the effective receptor reserve in the amylase assay is greater than that required to stimulate PI turnover, and that the selective peptoid CCK-B antagonist CI-988 has weak agonist activity at CCK-A receptors.

1,2,4-Triacylpiperidine substance p antagonists: Separation of affinities for the NK-1 receptor and the L-type calcium channel

Abstract A series of 1,2,4-triacylpiperidines are shown to be potent antagonists of the NK-1 (Substance P) receptor with significant affinity for the L-type calcium channel as well. The latter property can be diminished by suitable substitution on the terminal nitrogen while maintaining good NK-1 receptor binding.

Mitigation of Acetylcholine Esterase Activity in the 1,7-Diazacarbazole Series of Inhibitors of Checkpoint Kinase 1

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.