Molecular determinants of sarco/endoplasmic reticulum calcium ATPase inhibition by hydroquinone-based compounds

The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5-di-tert-butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQ's four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5-substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzyme's E(2) conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp-59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E(1) conformation of the enzyme failed, because the conformational changes accompanying the E(2)/E(1) transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E(2) form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors.