Phenolphthalein as a Prototype Drug for a Group of Structurally Related Calcium Channel Blockers in Human Platelets

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Thrombin increases intracellular free Ca2+ ([Ca2+]i) in human platelets by 2 mechanisms: internal mobilization and the influx of Ca2+ via store-operated Ca2+ entry (SOCE). 2-Aminoethoxydiphenyl borate (2-APB) is an inhibitor of SOCE. In search for nonboron analogues of 2-APB, we identified a well-known compound, phenolphthalein, structurally related to 2-APB. Many phenolphthalein analogues inhibited the ability of thrombin and thapsigargin (a specific activator of SOCE) to increase [Ca2+]i. Phenolphthalein has an IC50 ≈10 μM to inhibit thrombin-induced [Ca2+]i elevation, its active analogues have a similar potency. Several phenolphthalein analogues also inhibited thrombin-induced intracellular Ca2+ mobilization, which indicates action on inositol 1,4,5-trisphosphate receptors. We identified structural features among active and inactive phenolphthalein analogues that are responsible for the activity. Opening of the 5-membered lactone ring of phenolphthalein resulted in a total loss of activity. If the diphenyl rings possessed primary amine, dimethyl amine, or a cyano group, there was no activity. Modifications to the diphenyl groups that were tolerated include phosphate, sulfate, iodine, bromine, methyl, nitrite, and methoxy. Inhibition of thrombin-induced [Ca2+]i increase by phenolphthalein was not mediated by an increase in cyclic adenosine monophosphate because the inhibitor of cyclic adenosine monophosphate-dependent protein kinase A, 4-cyano-3-methylisoquinoline, did not affect the inhibitory action of phenolphthalein. The inhibitory effect of phenolphthalein was not mediated by an increase in NO/cyclic guanosine monophosphate (cGMP) because the inhibitors of NO-sensitive soluble guanylyl cyclase, methylene blue, and ODQ did not affect the inhibition. Phytohemagglutinin and thapsigargin-induced SOCE in Jurkat cells was also inhibited by phenolphthalein and 2-APB to the same extent as seen in platelets. Therefore, phenolphthalein and its derivatives structurally similar to 2-APB inhibit SOCE in platelets and other cells.

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