Siegfried Wurster

Fipamezole (JP-1730) is a potent alpha2 adrenergic receptor antagonist that reduces levodopa-induced dyskinesia in the MPTP-lesioned primate model of Parkinson's disease.

Previous studies in the MPTP‐lesioned primate model of Parkinsons disease have demonstrated that α2 adrenergic receptor antagonists such as idazoxan, rauwolscine, and yohimbine can alleviate L‐dopa–induced dyskinesia and, in the case of idazoxan, enhance the duration of anti‐parkinsonian action of L‐dopa. Here we describe a novel α2 antagonist, fipamezole (JP‐1730), which has high affinity at human α2A (Ki, 9.2 nM), α2B (17 nM), and α2C (55 nM) receptors. In functional assays, the potent antagonist properties of JP‐1730 were demonstrated by its ability to reduce adrenaline‐induced 35S‐GTPγS binding with KB values of 8.4 nM, 16 nM, 4.7 nM at human α2A, α2B, and α2C receptors, respectively. Assessment of the ability of JP‐1730 to bind to a range of 30 other binding sites showed that JP‐1730 also had moderate affinity at histamine H1 and H3 receptors and the serotonin (5‐HT) transporter (IC50 100 nM to 1 μM). In the MPTP‐lesioned marmoset, JP‐1730 (10 mg/kg) significantly reduced L‐dopa–induced dyskinesia without compromising the anti‐parkinsonian action of L‐dopa. The duration of action of the combination of L‐dopa and JP‐1730 (10 mg/kg) was 66% greater than that of L‐dopa alone. These data suggest that JP‐1730 is a potent α2 adrenergic receptor antagonist with potential as an anti‐dyskinetic agent in the treatment of Parkinsons disease.

Conserved structural, pharmacological and functional properties among the three human and five zebrafish α2‐adrenoceptors

1 Zebrafish has five distinct α2‐adrenoceptors. Two of these, α2Da and α2Db, represent a duplicated, fourth α2‐adrenoceptor subtype, while the others are orthologue of the human α2A‐, α2B‐ and α2C‐adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish α2‐adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist‐stimulated [35S]GTPγS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3 The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G‐protein coupling of the zebrafish and human α2‐adrenoceptors, separated by ∼350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish α2‐adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40‐fold affinity differences. 4 The α2A orthologues and the zebrafish α2D duplicates clustered as close pairs, but the relationships between the orthologues of α2B and α2C were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the α2‐adrenoceptors, we suggest that the second extracellular loop – where significant sequence variability is located – might contribute significantly to the observed affinity differences.

Molecular mechanisms of ligand–receptor interactions in transmembrane domain V of the α2A-adrenoceptor

The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 α2‐adrenergic agonists and a panel of mutated human α2A‐adrenoceptors. The α2ASer201 mutant had a Cys → Ser201 (position 5.43) amino‐acid substitution, and α2ASer201Cys200 and α2ASer201Cys204 had Ser → Cys200 (5.42) and Ser → Cys204 (5.46) substitutions, respectively, in addition to the Cys → Ser201 substitution. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand‐binding assays and functional [35S]GTPγS‐binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild‐type α2A‐adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the α2A‐adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta‐hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para‐hydroxyl group of the catecholic ring.

α2-adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner

Transfected Chinese hamster ovary cells expressing human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes were used to monitor alpha2-adrenoceptor-stimulated GTP hydrolysis. Incubation with 100 microM (-)-adrenaline resulted in stimulation of pertussis toxin-sensitive GTPase by 380% after activation of the alpha2A-subtype, by 320% after activation of the alpha2B-subtype and by 110% after activation of the alpha2C-subtype. The agonists dexmedetomidine, UK14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxaline) and oxymetazoline showed subtype-dependent efficacy. Dexmedetomidine was a full agonist at the alpha2B-subtype and a partial agonist at the alpha2A- and the alpha2C-subtypes. UK14,304 was a full agonist at the alpha2A-subtype and a partial agonist at the other two. Oxymetazoline showed strong partial agonism at the alpha2B-subtype (63% of adrenaline), but did not significantly activate the alpha2A- and the alpha2C-subtypes. These results agreed with cAMP accumulation experiments carried out with cell lines endogenously expressing the alpha2A-subtype (human erythroleukemia, HEL) or the alpha2B-subtype (neuroblastoma-glioma, NG108-15). The GTPase assay may thus provide a valuable tool for the identification of subtype-selective alpha2-adrenoceptor agonists.

Differential Efficacies of Somatostatin Receptor Agonists for G-Protein Activation and Desensitization of Somatostatin Receptor Subtype 4-Mediated Responses

Although desensitization represents an important physiological feedback mechanism that protects against overstimulation, it can significantly limit the therapeutic usefulness of drugs. In the current investigation, we have employed Cytosensor microphysiometry for the purpose of determining the propensity of somatostatin receptor agonists to induce desensitization of the human somatostatin receptor subtype 4 (h sst4)-mediated extracellular acidification rate (EAR) response in intact Chinese hamster ovary (CHO) cells. We have compared this propensity with the efficacies of the agonists as measured in a [35S]guanosine-5′-O-(3-thio)triphosphate binding assay with membranes of the same CHO-h sst4 cell line. We observed that (1′S,2S)-4-amino-N-(1′-carbamoyl-2′-phenylethyl)-2-(4′′-methyl-1′′-naphthalenesulfonylamino)butanamide (J-2156), a superagonist at the h sst4 with higher efficacy than somatostatin-14 itself (Engström et al., 2005), was considerably less prone to cause desensitization of the EAR response than somatostatin-14, somatostatin-28, and cortistatin-17. In contrast, compound A (methyl (2S)-5-{[amino(imino)methyl]amino}-2-{[4-[5–7-difluoro-2-phenyl-1H-indol-3-yl)butanoyl]amino}-pentanoate), which we also found to be an h sst4 superagonist, albeit to a lesser degree than J-2156, demonstrated a high propensity to cause desensitization. Our results indicate that there is no relationship between the efficacy of the agonists to cause G-protein activation and their ability to induce desensitization of the h sst4-mediated EAR responses. The finding that on the h sst4, J-2156 is not only a superagonist but also shows a low propensity to cause desensitization, might offer therapeutic advantages. At a minimum, the compound will be a powerful tool to study the mechanisms connected to efficacy and desensitization of h sst4-mediated responses.

Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay.

We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [35S]guanosine-5-O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH2 exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.

Different apparent modes of inhibition of alpha2A-adrenoceptor by alpha2-adrenoceptor antagonists

The inhibition of alpha2A-adrenoceptor-mediated Ca2+ elevation by alpha2-adrenoceptor antagonists was measured in HEL human erythroleukemia cells. The antagonists could be divided in two classes: those that displayed surmountable inhibition (right-shift of the agonist dose-response curve), and those that displayed different degrees of insurmountable inhibition (depression of the maximum signal and a possible right-shift of the agonist dose-response curve). The degree of surmountability of the inhibition correlated well with the measured antagonist dissociation rates, suggesting that the hypothesis of the antagonist dissociation rate governing the mode of inhibition of fast responses, holds true. HEL cells thus provide a useful model system for the investigation of physiological consequences of different dissociation rates. Also, the dissociation rates of antagonists not available in radiolabelled form can be predicted from the functional data. The data stresses the importance of measurement of kinetic parameters of the drug-receptor interaction in addition to the equilibrium binding constants.

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors.

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [35S]guanosine-5-O-(3-thio)triphosphate ([35S]GTPgammaS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a super-agonist. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [35S]GTP-gammaS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.

Interaction of folk medicinal plant extracts with human α2-Adrenoceptor subtypes

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human α2-adrenoceptor subtypes α2A, α2B and α2C. Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of α2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the α2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the α2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPγS binding, thus indicating a predominantly antagonistic nature of the compounds with α2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 ± 40 nᴍ for α2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating α2-adrenoceptor related diseases