Interactions of Dihydroxybenzenes with the Ca2+-ATPase: Separate Binding Sites for Dihydroxybenzenes and Sesquiterpene Lactones

Ym, Khan; Wictome, M; Jm, East; Ag, Lee

doi:10.1021/bi00044a015

Cited by 34 publications

(49 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Kinetic studies indicate that although inhibition by a variety of hydrophobic molecules is generally related to interference with global conformational changes of the enzyme (38), TG and DBHQ may actually bind to separate sites (39). We then tested whether the F256V mutation would interfere with the inhibitory effect of DBHQ.…”

Section: Tg Interferes With Conformationalmentioning

confidence: 99%

Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Inesi

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca 2؉ ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1⅐2Ca 2؉ transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase.Sesquiterpene lactone thapsigargin (TG), 1 derived from the plant Thapsia garganica (1, 2), is a potent and highly specific inhibitor of the endo-(3) and sarcoplasmic reticulum (4 -6) Ca 2ϩ ATPases (SERCA). Because of the role of intracellular Ca 2ϩ stores in control and regulation of numerous cellular functions (7,8), TG is used extensively as a pharmacological tool in physiological experimentation (9, 10). Furthermore, TG is being considered for targeted cancer treatment (11) because of its inhibitory effect on cell proliferation (12).Inhibition of Ca 2ϩ ATPase is observed at subnanomolar concentrations of TG and is attributed to drug binding to the enzyme with a 1:1 molar ratio (4). TG induces stabilization of the ATPase protein in a state similar to that of the Ca 2ϩ -free enzyme (i.e. E2), facilitating formation of ordered arrays (13) for structural studies by electron microscopy and x-ray diffraction (14, 15).Spectroscopic (16), photolabeling (17), chimerization (18), and mutational studies (19,20) suggested that the TG binding site resides near the M3 segment at the cytosolic membrane surface. TG was then clearly identified by diffraction studies (15) in a cavity delimited by the M3, M5, and M7 transmembrane helices near the cytosolic surface of the membrane (Fig. 1). We report here a mutational study, including construction and characterization of 20 mutations within the region surrounding the TG binding pocket. The aim of this study was to evaluate specific structural features that may be involved in determining the ATPase affinity for TG and its inhibitory mechanism. The specificity of the TG inhibitory mechanism was fur...

show abstract

Section: Tg Interferes With Conformationalmentioning

confidence: 99%

Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Inesi

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…By far the best known and most potent inhibitor is thapsigargin (TG), a plant sesquiterpene lactone (18), with the dissociation constant in the subnanomolar range (15). Another commonly used inhibitor is 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ), an antioxidant, which has a much simpler chemical structure and a lower affinity (19). We have determined the crystal structures of the ATPase with either one [E2(TG) or E2(BHQ)] or both [E2(TGϩBHQ)] of them bound (4,13).…”

mentioning

confidence: 99%

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

Takahashi

Kondou²,

Toyoshima³

2007

Proc. Natl. Acad. Sci. U.S.A.

111

View full text Add to dashboard Cite

Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum is an ATPdriven Ca 2؉ pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca 2؉ , the three cytoplasmic domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1-M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1 helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca 2؉ -ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations.Ca 2ϩ -ATPase ͉ ion pump ͉ membrane protein C a 2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) is structurally and functionally the best studied member of the P-type ion-translocating ATPases (1, 2). Located in the sarcoplasmic reticulum membrane, SERCA1 pumps Ca 2ϩ released into muscle cells during contraction and thereby causes relaxation. The reaction cycle consists of at least eight intermediates, and crystal structures have been determined for six of them (3-9). These crystal structures show that SERCA1 consists of three cytoplasmic domains (A, actuator; N, nucleotide binding; and P, phosphorylation) and 10 (M1-M10) transmembrane helices (3). They also demonstrate that ATPase undergoes large and global conformational changes during the reaction cycle. In conventional E1/E2 theory (10-12), active transport of Ca 2ϩ is achieved by alternating the affinity and accessibility of the transmembrane Ca 2ϩ -binding sites. That is, in E1, the binding sites have high affinity and face the cytoplasm; in E2, the binding sites have low affinity to Ca 2ϩ and face the lumen of the sarcoplasmic reticulum. Although H ϩ and water, instead of Ca 2ϩ , stabilize the binding sites in E2 (13), this form of the ATPase is unstable, particularly when solubilized (14). Therefore, so far all successful crystallization experiments in the absence of Ca 2ϩ used inhibitors that stabilize the transmembrane domain.Several inhibitors specific to SERCA1 are available (15-17). By far the best known and most potent inhibitor is thapsigargin (TG), a plant sesquiterpene lactone (18), with the dissociation constant in the subnanomolar range (15). Another commonly used inhibito...

show abstract

“…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.…”

Section: A Synthesis Of 25-disubstituted Hydroquinonesmentioning

confidence: 99%

“…The observed trend suggested an optimum number of six carbon atoms total (five ring plus one methyl carbon atom) in the substituent, a size which is most likely dictated by the size of the binding pocket. It should be noted that 4 is about as potent as the most active non-cyclic hydroquinone-based SERCA inhibitor known to date, 2,5-diamylhydroquinone (IC 50 : 0.34 M) [10], [29]. …”

Section: A Synthesis Of 25-disubstituted Hydroquinonesmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Paula¹,

Elam²,

Woeste³

et al. 2013

IJBBB

View full text Add to dashboard Cite

Abstract-Derivatives of the compound 2,5-di-tert-butylhydroquinone (BHQ) are inhibitors of the sarco/endoplasmic reticulum calcium ATPase (SERCA). Previous work identified BHQ analogs with two alkyl substituents composed of four to six carbon atoms as the most potent representatives from this inhibitor class. In this study, we explored the effects of introducing two cycloalkyl substituents on the hydroquinone scaffold. Since these substituents have limited conformational flexibility, the entropy penalty upon binding of these compounds to SERCA was expected to be smaller than for non-cyclic BHQ analogs, potentially conveying higher inhibitory potency. We synthesized a congeneric series of four 2,5-disubstituted BHQ analogs and determined their inhibitory potencies against SERCA in bioassays. Potencies were found in the low micromolar and submicromolar concentration ranges, making the new compounds some of the most potent hydroquinone-based inhibitors to date. Computational docking facilitated a detailed analysis of enzyme/inhibitor interactions at the molecular level and showed that the entropic effect was noticeable but too small to increase potency in a substantial manner.Index Terms-Calcium homeostasis, enzyme inhibition, P-type ATPase, medicinal chemistry.

show abstract

Interactions of Dihydroxybenzenes with the Ca2+-ATPase: Separate Binding Sites for Dihydroxybenzenes and Sesquiterpene Lactones

Cited by 34 publications

References 29 publications

Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

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

Contact Info

Product

Resources

About