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

Abstract

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.

Effects of High-Affinity Inhibitors on Partial Reactions, Charge Movements, and Conformational States of the Ca2+Transport ATPase (Sarco-Endoplasmic Reticulum Ca2+ATPase)

“…The discovery of SERCA inhibitors has opened favorable avenues for mechanistic studies on Ca 2ϩ homeostasis , stereochemistry of drug interactions with proteins (Logan-Smith et al, 2002;Wootton and Michelangeli, 2006;Lape et al, 2008), and possible therapeutic applications (Eckstein-Ludwig et al, 2003;Denmeade and Isaacs, 2005;Søhoel et al, 2006). With the experiments reported here, we endeavored to obtain a comparative characterization of the interactions of four inhibitors with SERCA, their interference with partial reactions of the catalytic and transport cycle, and binding to the transport sites in the absence of ATP and vectorial translocation of bound Ca 2ϩ upon addition of ATP, as sequential electrogenic steps within a single ATPase cycle.…”

Hydroquinones with Conformationally Constrained Substituents: Synthesis, Characterization, and Evaluation as Calcium–ATPase Inhibitors

“…1, a series of four 2,5-disubstituted hydroquinones with cyclic substituents was synthesized by Friedel-Crafts alkylation of hydroquinone with commercially available cyclic tertiary alcohols. Since previous studies had shown that the most active hydroquinone-based SERCA inhibitors with non-cyclic residues carried between four and six carbon atoms in their side chains [10], [13], we varied the ring size of the newly synthesized compounds from four to seven carbon atoms to be within this size range. Disubstituted hydroquinones 1-4 were prepared in low yields by small scale (100-200 mg) reaction of hydroquinone with slightly more than two equivalents of the appropriate cycloalkanol under acidic conditions.…”

“…The protein structure was prepared for docking by removing all non-protein entries, adding hydrogen atoms, and optimizing the position of the latter by molecular mechanics as described previously [13,26]. The scoring function used for docking was ChemScore [27,28] and the genetic algorithm of GOLD was executed at the default settings, performing 20 independent and identical repeats for each compound.…”

“…Due to their relatively small size, BHQ derivatives can be synthesized in a few steps from inexpensive starting materials, which constitutes a considerable advantage over TG-based inhibitors. Structure-activity relationships are available for sizeable collections of hydroquinones obtained from virtual screens of large compound collections or from organic synthesis [13]- [15]. Among the hydroquinones tested in enzyme inhibition assays, the highest potency inhibitors with submicromolar potencies are 2,5-disubstituted compounds that carry two alkyl residues with four to six carbon atoms each.…”

“…Examples include, but are not limited to, the fungal metabolite cyclopiazonic acid, bisphenols, curcumins, and hydroquinones [8], [9]. The most prominent representative from the latter class is the compound 2,5-di-tert-butylhydroquinone (BHQ) [10]- [13]. Due to their relatively small size, BHQ derivatives can be synthesized in a few steps from inexpensive starting materials, which constitutes a considerable advantage over TG-based inhibitors.…”