Kathy M. O’ Boyle

Hybrid angiogenesis inhibitors : Synthesis and biological evaluation of bifunctional compounds based on 1-deoxynojirimycin and aryl-1,2,3-triazoles

Synthesis of hybrids of 1-deoxynojirimycin (DNJ) and 5-aryl-1,2,3-triazole as potential bifunctional inhibitors of angiogenesis is described. The DNJ component inhibits the biosynthesis of cell surface oligosaccharides necessary for angiogenesis, whereas the aryl-1,2,3-triazole inhibits methionine aminopeptidase II, a target in angiogenesis therapy. One bifunctional compound was a more potent inhibitor of angiogenesis in vitro than DNJ alone or the 5-aryl-1,2,3-triazole alone.

Hybrids of 1-deoxynojirimycin and aryl-1,2,3-triazoles and biological studies related to angiogenesis.

Hybrids of 1-deoxynojirimycin (DNJ) and aryl-1,2,3-triazole have been synthesized with a view to identifying an inhibitor of both alpha-glucosidase and methionine aminopeptidase 2 (MetAP2). One compound was a potent inhibitor of alpha-glucosidase at both the enzyme and cellular level, and this agent also inhibited bovine aortic endothelial cell (BAEC) growth and tube formation. The anti-proliferative activity of this hybrid is due to its ability to induce cell-cycle arrest in the G(1) phase. The novel agent caused a reduction in the expression of cyclin D1 but did not promote apoptosis or inhibit the phosphorylation of ERK1/2. These observations indicate that its mechanism of action is distinct from fumagillin and its analogues, which inhibit MetAP2. Stress-fibre assembly in BAECs was abolished by the novel agent indicating that the inhibition of BAEC tube formation observed is partially a result of a reduction in cell motility.

D2 receptor‐mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB1 receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D2 receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D2 receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC50: 3.25 × 10−8 M). The CB1 receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB1 receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB1 receptors and D2 autoreceptors in regulating rat striatal dopamine release.

Pharmacologically distinct binding sites in rat brain for [3H]thyrotropin-releasing hormone (TRH) and [3H][3-methyl-histidine2]TRH

We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [3H][3-Me-His(2)]TRH or [3H]TRH binding to rat cortical homogenates. [3H][3-Me-His(2)]TRH was observed to bind to a single population of high-affinity, low-capacity sites (K(d): 4.54+/-0.62 nM, N=5; B(max): 4.38+/-0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K(i) of 22 nM. K(i) values for the library peptides at [3H][3-Me-His(2)]TRH-labelled sites varied from 10(-3) to 10(-9)M; the potency order was: [3-Me-His(2)]>His>Thi>Leu,Phe,Asn>Gln, Arg, Thr, Ala, HomoPhe. All other replacements had K(i) values >10(-4)M. [3H]TRH was observed to label a single population of low-affinity, high-capacity sites (K(d): 7.55+/-1.23 microM, N=6; B(max): 3.40+/-0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [3H]TRH-labelled sites did not correlate with their affinities for [3H][3-Me-His(2)]TRH-labelled sites (r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme (r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [3H]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.