SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma 1 receptor and emopamilbinding protein, have been characterised molecularly. Only the σ 1 receptor has been shown to bind (Z)Ncyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)propen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma 1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a ∆8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the ∆8-∆7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma 1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.
The DLS-VS strategy was developed as an integrated method for identifying chemical modulators for orphan GPCRs. It combines differential low-throughput screening (DLS) and virtual screening (VS). The two cascaded techniques offer complementary advantages and allow the experimental testing of a minimal number of compounds. First, DLS identifies modulators specific for the considered receptor among a set of receptors, through the screening of a small library with diverse chemical compounds. Then, an active molecular model of the receptor is built by homology to a validated template, and it is progressively refined by rotamers modification for key side-chains, by VS of the already screened library, and by iterative selection of the model generating the best enrichment. The refined active model is finally used for the VS of a large chemical library and the selection of a small set of compounds for experimental testing. Applied to the orphan receptor GPR34, the DLS-VS strategy combined the experimental screening of 20 000 compounds and the virtual screening of 1 250 000 compounds. It identified one agonist and eight inverse agonists, showing a high chemical diversity. We describe the method. The strategy can be applied to other GPCRs.
The differential pulse polarography has been used on samples taken from the medium during the culture of chinese hamster ovary cells with a view to producing the active principle of an anti-cancer drug. An electrochemical signal at a potential of -1.8 V with respect to the saturated calomel electrode was correlated with the lactic acid concentration, the pH variation and the number of cells. The peaks observed in oxidation at potentials -0.35 V and -0.05 V were associated with the transformation of the cystine present in the nutrient medium into cysteine and provide an indication of the modification of the reducing power of the medium during culture. No correlation was found between the electrochemical behaviour and cell production.
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.