Fig 1.
Curcumin analogs employed in this study.
Curcumin (Sigma-Aldrich) was initially identified as a hit in an in vitro screen for inhibitors of the plasma membrane H+-ATPase. Curcumin (CM, 1) demethoxycurcumin (DMCM, 2), bisdemethoxycurcumin (BDCM, 3) and 1,5-dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-4,6-heptadien-3-one (6) were then purified as previous described [39, 62]. 6-shogaol (4) and dibenzylideneacetone (9) were obtained from Sigma-Aldrich. Tetrahydrocurcumin (5), 1,7-bis(3',4'-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadien-3,5-dione (7) and 1-(3',4'-dimethoxyphenyl)-4,4-dimethyl-7-(4'-methoxyphenyl)-1,6-heptadien-3,5-dione (8) were synthesized as previously described in the literature[63].
Fig 2.
Curcumin analogs inhibit plasma membrane H+-ATPases activity in a dose dependent manner.
(A) ATPase activity measured on yeast plasma membranes from yeast strains expressing either A. thaliana PM H+-ATPase isoform AHA2 (left panel) or the endogenous yeast plasma membrane Pma1p (right panel) in the presence of 50 μM of the curcumin analogs, compounds 1–9, or vanadate, a P-type ATPase inhibitor. Values are mean ± S.E. (n = 3). Student’s t tests:*, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to the control. (B) Dose-dependent inhibition of H+-ATPase activity of AHA2 (left) and Pma1p (right). Experiments were carried out in triplicates at different concentrations of CM (1), DMCM (2) and BDCM (3). Data were analyzed by using nonlinear regression tool and fitted to log(inhibitor) vs. normalized response (variable slope) for determination of IC50 values.
Table 1.
IC50 values for compounds 1–3 in ATP hydrolysis assays.
Fig 3.
Curcumin analogs also inhibit proton pumping.
(A-C) Proton mediated accumulation of ACMA in spinach plasma membrane vesicles after incubating with various concentrations of CM (1), DMCM (2) and BDCM (3). ATP stimulated H+-pumping is initiated upon addition of MgSO4. One representative of three independent experiments is shown for each compound (left panels). Proton transport activity was determined as initial rates of fluorescence quenching of ACMA probe (right panels). Values are mean ± S.E. (n = 3). Student’s t tests:*, p < 0.05; **, p < 0.01; ***, p < 0.001 relative to the control.
Fig 4.
Kinetic analysis of PM H+-ATPases inhibition by DMCM.
ATP hydrolysis rate was plotted as function of [ATP] in the presence of the indicated amounts of DMCM; Yeast expressing A. thaliana PM H+-ATPase isoform AHA2 (A) and the endogenous yeast isoform Pma1p (B). Error bars represent standard errors of the mean for three independent trials.
Table 2.
Effect of DMCM on the ATP hydrolysis of the PM H+-ATPases.
Table 3.
Effects of compounds 1–3 on PM H+-ATPases in vitro and in in vivo drop test assay.
Fig 5.
Dose-dependent inhibition of SERCA Ca2+-ATPase activity.
Inhibition of ATP hydrolytic activity by addition of increasing amounts of compounds. Experiments were carried out in triplicate at different concentrations of CM (1), DMCM (2) and BDCM (3). Data were analyzed by using nonlinear regression tool and fitted to log(inhibitor) vs. normalized response (variable slope) for determination of IC50 values.
Fig 6.
Drop tests showing the sensitive response of S. cerevisiae mutant cells (encoding AHA2, aha2∆92 or Pma1p) to compounds 1–3.
Serial dilutions of the yeast cultures are spotted on galactose (YPG) or glucose (YPD) media containing the indicated concentrations of CM (1), DMCM (2) or BDCM (3). Growth results were recorded after incubation for 3 days at 30°C. Results shown are representative data of three independent experiments for each compound.