A series of substituted omega-[2-(1H-imidazol-1-yl)ethoxy]alkanoic acid derivatives were synthesized and evaluated for their ability to inhibit thromboxane synthase both in vitro and in vivo. Compound 13 was identified as a potent and selective competitive inhibitor of human platelet thromboxane synthase having a Ki value of 9.6 X 10(-8) M. In collagen-treated human whole blood, 13 potentiated levels of 6-keto PGF1 alpha. Enantiospecific syntheses afforded the R and S enantiomers of 13, of which the S enantiomer 13b was the more potent. Compounds 13 and 13b were potent in vivo inhibitors of thromboxane synthase with good oral activity and duration of action.
Analogues of 4-[[2-(1H-imidazol-1-yl)-1-[[(4-methoxyphenyl)methoxy]methyl] ethoxy]methyl]benzoic acid (5m) were prepared and evaluated as thromboxane synthase inhibitors. A series of esters of 5m showed a parabolic relationship between lipophilicity and inhibition of TxB2 generation in intact platelets, with activities up to 50 times greater than that of dazoxiben. However, on administration to rabbits the ethyl ester 5d had a short duration of action, due to rapid metabolism and excretion via deesterification and beta-glucuronidation. Attempts at replacing the carboxylate group with other potential pharmacophores were unsuccessful.
We have recently described in detail the structure of an epitope defined by a monoclonal antibody which enhances in vivo the biological activity of bovine GH @GH) [ 11. Although several theories have been advanced in relation to the mechanism associated with this enhancement phenomenon, there is little conclusive data supporting any hypothesis [2]. To examine the molecular and cellular aspects of this phenomenon more closely, we have undertaken studies using the well characterised GH-responsive 3T3-F442A preadipocyte cell line and the FDCP-I cell line transfected with full length mouse growth hormone receptor (GHR). As a prelude to this, we examined the binding properties of OA15 in relation to the subsequent interaction between bGH and its receptor. Initial examination of the epitope defined on bGH by OA15 suggested that it was removed from binding sites 1 and 2 on GH required for interaction with the GH receptor [3]. Using the recombinant bGH binding protein (bGHBP), which represents the extracellular domain of the receptor, we have demonstrated, using optical evanescent wave biosensor technology, that engagement of bGH by OA15 still allows for the interaction between bGH and bGHBP. Although further experiments are underway to investigate the effects on the affinity of bGH-bGHBP interaction following bGH binding to OA15, at this stage we can tentatively conclude that these real time binding studies agree with our initial epitope mapping work ie that the epitope defined by OA15 is removed from receptor interaction sites on the hormone. The results of these experiments suggested that in cell culture studies tnpartite complexes of antibody-hormone and receptor would exist. In the context of mechanistic studies, this in turn would suggest that OA15 would not inhibit GH signalling events through disruption of GH-GHR interactions. To examine this hypothesis in more detail we used two GH-responsive cell culture systems -the 3T3-F442A preadipocyte cell line and IL-3 dependant FDCP-I cell line which has been transfected with the full length mouse GHR [4]. In F442A cells, treatment with GH leads to the rapid tyrosine phosphorylation of proteins of the JAK-STAT-MAP pathway(s). Figure 1 is an antiphosphotyrosine (py) blot of lysates from F442A cells treated with bGH alone or hormone pre-complexed with OA15. As can be clearly seen, GH treatment stimulates the level of a 9lkDa tyrosine phosphorylated protein. This is most likely to be a member of the Stat (signal transducers and activators of transcription) family of transcription factors. The level of tyrosine phoshporylated 9 lkDa 9lkL)a GH + + + OA15 -+ -+ CMAb ---+ Fig. 1. The effect ofbGH (50ng/ml) f OA15 Mab (1:lOO dilution of hybndoma supernatant containing 0.6mg/ml total protein) on tyrosine phosphorylation of 9lkDa Stat protein. The location of 9lkDa Stat is arrowed. 160 1 8 6 0 1 Fig. 2 The effect of varying doses of Mab OA15 on bGH (4.5ng/ml) stimulation of mGHR-transfected FDCP-1 cells. Bioactivity of hormone and hormone-antibody complexes was determined colourim...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.