Frances Emms

Regional Differences in the Inhibition of Mouse In Vivo [3H]Ro 15-1788 Binding Reflect Selectivity for α1 versus α2 and α3 Subunit-Containing GABAA Receptors

The benzodiazepines flunitrazepam, diazepam, and Ro 15-1788 and the β-carboline DMCM bind with equivalent affinity to the benzodiazepine binding site of GABAA receptors containing different α subunits (i.e., α1, α2, α3, or α5); whereas, the triazolopyridazine CL 218,872 and imidazopyridine zolpidem have higher affinity for α1 subunit-containing GABAA receptors. In the present study, the in vivo binding of [3H]Ro 15-1788 in mouse cerebellum and spinal cord was used to establish the occupancy of the benzodiazepine binding site of GABAA receptors containing primarily α1 and α2/α3 subunits, respectively. Thus, the nonselective compounds flunitrazepam, diazepam, and DMCM all produced a similar inhibition of binding in cerebellum and spinal cord (respective ID50 values of 0.2 to 0.3 mg/kg, 2 mg/kg, and 10 mg/kg IP); whereas, the α1 selective compounds CL 218,872 and zolpidem were more potent at inhibiting [3H]Ro 15-1788 binding in the cerebellum (ID50 values 4.5 mg/kg and 10 mg/kg IP) compared to the spinal cord (ID50 values 12 mg/kg and >30 mg/kg IP). Thus, the reduction of in vivo f [3H]Ro 15-1788 binding in tissues containing α1 and α2/α3 receptor populations reflects the in vitro affinities of subtype selective compounds and should help to interpret the behavioral profile of such compounds.

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, B max = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp‐dependent shifts in [3H]N‐0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1–10 μM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3, dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3‐like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Antagonism of the effects of (+)-PD 128907 on midbrain dopamine neurones in rat brain slices by a selective D2 receptor antagonist L-741,626.

1 The ability of PD 128907 to activate dopamine receptors in the ventral tegmental area, substantia nigra pars compacta, and striatum was investigated by use of in vitro electrophysiological recording and fast cyclic voltammetry. The affinity of a novel D2 selective antagonist L‐741,626 for receptors activated by this agonist was measured to determine if its effects were mediated by D2 or D3 receptors. 2 The active (+) enantiomer of PD 128907 bound with high affinity and selectivity to rat D3 dopamine receptors. The Ki values for (+)‐PD 128907 were 620 nM at D2, 1 nM at D3 and 720 nM at D4 receptors. 3 (+)‐PD 128907 inhibited cell firing in both the ventral tegmental area and substantia nigra pars compacta with EC50 values of 33 nM (pEC50 = 7.48 ± 0.10, n= 10) and 38 nM (pEC50 = 7.42 ± 0.15, n = 5), respectively. No effects of (+)‐PD 128907 (100 nM) were observed on glutamate or GABA‐mediated synaptic potentials elicited by focal bipolar stimulation. 4 L‐741,626 antagonized these effects of (+)‐PD 128907 in a concentration‐dependent and surmountable manner with an affinity, determined from Schild analysis, of 20 nM (pKB = 7.71 ± 0.14) in the ventral tegmental area and 11 nM (pKB = 7.95 ± 0.18) in the substantia nigra pars compacta. 5 (+)‐PD 128907 also inhibited dopamine release in the caudate‐putamen with an EC50 of 66 nM (n = 5). The affinity of L‐741,626 for these nerve terminal autoreceptors (pKB = 7.71 ± 0.06; =20 nM) was identical to that observed on midbrain dopamine neurones. 6 These data demonstrate that the D3 receptor ligand (+)‐PD 128907 is a potent agonist on rat midbrain dopamine neurones. However, its lack of regional selectivity, and the high affinity of the selective D2 receptor antagonist L‐741,626 for receptors activated by (+)‐PD 128907, was more consistent with an action on D2 autoreceptors rather than upon a D3 dopamine receptor subtype.

Functional Coupling of Human D2, D3, and D4 Dopamine Receptors in HEK293 Cells

The D2 dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3 and D4 dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3 and D4 receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3 or D4 receptors resulted in the inhibition of forskolin-stimulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4 receptor than the D3 or D4 receptors, although only 6-fold higher than the D2 receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4 dopamine receptor mRNA in human brain regions.

Synthesis and sar of 2- and 3-substituted 7-azaindoles as potential dopamine D4 ligands.

7-azaindole compounds bearing a cyclic amine moiety linked by a one or two carbon chain attached at the 2- or 3-position were synthesised and evaluated as potential dopamine D4 ligands. Highest affinity and selectivity for the D4 receptor resided in the 3-aminomethyl-7-azaindole series.

4-N-linked-heterocyclic piperidine derivatives with high affinity and selectivity for human dopamine D4 receptors

The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the structure 1 are described (compounds 3-12) as hD4 ligands. After further optimisation the best compound identified was 13 which has high affinity for hD4 (5.2 nM) and >300-fold selectivity for hD4 receptors over hD2 and hD3 receptors.

Binding of 2,4-disubstituted morpholines at human D4 dopamine receptors.

The synthesis of a series of 2,4-disubstituted morpholines is described and their affinities at human dopamine receptors reported. The orally bioavailable 7-azaindole compound 11 has nanomolar affinity at the hD4 receptor with > 1000-fold selectivity over the hD2 receptor.

3-(1-Piperazinyl)-4,5-dihydro-1H-benzo[g]indazoles: high affinity ligands for the human dopamine D4 receptor with improved selectivity over ion channels

3-(4-Piperidinyl)-5-arylpyrazoles, such as 1, were selective for the cloned human dopamine D4 receptor (hD4), but also showed affinity at voltage sensitive calcium, sodium and potassium ion channels. A combination of substituent changes to reduce the basicity of the piperidine nitrogen and conformational restriction to give 4,5-dihydro-1H-benzo[g]indazoles reduced this ion channel affinity at the expense of selectivity for hD4 over other dopamine receptors. Incorporation of piperazine into the 4,5-dihydro-1H-benzo[g]indazoles in place of piperidine gave a novel series of high affinity, selective, orally bioavailable hD4 ligands, such as 16, with improved selectivity over ion channels.

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.