ABSTRACT:The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.
The synthesis of pamidronic acid and sodium pamidronate dihydrate from β‐alanine and P‐reagents (phosphorus trichloride and phosphorous acid) was investigated at 75°C in different solvents, and the preparation was optimized. In sulfolane, the use of 2 equiv of phosphorus trichloride and phosphorous acid was found the optimum to lead to pamidronic acid in a yield of 63%. In methanesulfonic acid, 3.2 equiv of phosphorus trichloride was necessary without any phosphorous acid to give pamidronate dihydrate in the best yield (57%) after hydrolysis and pH adjustment. In the first case, the P‐nucleophile may be (HO)2P–O–PCl–O–P(OH)2 or (HO)2P–O–PCl2, whereas in the second case, the P‐reactant is probable Cl2P–O–S(O)2Me. It can be said that the mechanism proposed for the formation of pamidronic acid is highly influenced by the solvent used, as it determines the necessary P‐reagent(s). Our results promote the “on purpose” planning of the synthesis of dronates.
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