Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.
Malaria is responsible for approximately 1 million deaths
annually; thus, continued efforts to discover new antimalarials are
required. A HTS screen was established to identify novel inhibitors
of the parasite's mitochondrial enzyme NADH:quinone oxidoreductase
(PfNDH2). On the basis of only one known inhibitor of this enzyme,
the challenge was to discover novel inhibitors of PfNDH2 with diverse
chemical scaffolds. To this end, using a range of ligand-based chemoinformatics
methods, ∼17000 compounds were selected from a commercial library
of ∼750000 compounds. Forty-eight compounds were identified
with PfNDH2 enzyme inhibition IC50 values ranging from
100 nM to 40 μM and also displayed exciting whole cell antimalarial
activity. These novel inhibitors were identified through sampling
16% of the available chemical space, while only screening 2% of the
library. This study confirms the added value of using multiple ligand-based
chemoinformatic approaches and has successfully identified novel distinct
chemotypes primed for development as new agents against malaria.
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