Several polyphenolic compounds, including twelve flavonols and a variety of lower molecular weight compounds were found to diminish the effects of CuZnSOD on NMR relaxation of fluoride ion in a pH and concentration dependent manner. While we originally thought the effect arose from binding of the compounds to the enzyme active site, several lines of evidence indicated that active polyphenols reduced the paramagnetic copper(II) form of the enzyme to the diamagnetic copper(I) form, thereby giving false positive indications of enzyme inhibition in the NMR assay. First, docking experiments failed to provide a satisfactory explanation of the SAR. Second, effects on the enzyme’s EPR spectrum indicated that catechols could bind the active site copper leading to enzyme reduction. Third, while these reactions did not proceed to completion in aerobic solution, they did so under inert atmosphere. Fourth, experiments employing superoxide producing compounds demonstrated that loss of NMR activity did not prevent the enzyme from redox cycling. Thus, while the polyphenols appeared to inhibit the enzyme in the NMR assay, the compounds did not inhibit the enzyme’s reactions with superoxide.
While inhibitors of superoxide dismutase enzymes (SODs) could be valuable tools for the study and management of oxidative stress, few compounds with this activity have been discovered. Based on the report that the steroid metabolite 2-methoxyestradiol (2ME) and related compounds inhibited CuZnSOD [
We began our study by looking at a number of flavonoids, a class of polyphenolic natural products. Flavonoids display both antioxidant and prooxidant activities [
Conventional assays for SOD activity use redox-active chromaphores to monitor the fate of superoxide generated in situ by enzymatic or chemical means [
Viglino et al. [
While the original objective of this work was to use NMR methods to search for inhibitors of CuZnSOD, redox reactions involving the enzyme and compounds under investigation led to several false positives. We believe that paramagnetic NMR relaxation can be used as a convenient measure of active site accessibility, but investigators must be careful that reactions that produce the diamagnetic reduced form of the enzyme do not interfere with the assay.
CuZnSOD from bovine erythrocytes was purchased from MP Biologicals. Flavonoids were purchased from Indofine Chemical Corporation. Buffers, NaF, trifluoroacetate (tfa), and other reagents were purchased from com- mercial sources and used without purification. 19F NMR spectra were acquired at 283 MHz (300 MHz 1H frequency) using a JEOL Eclipse NMR spectrometer. NMR solutions were prepared with 20 mM buffer, 20 mM NaF, 10% (v/v) D2O, and sufficient enzyme to give an easily measurable increase in transverse relaxation rate (R2 = 1/T2). Typically, the effects of potential inhibitors were measured under conditions where inhibitor-free, enzyme-containing control gave T2 values between 7 and 50 ms.
Screening experiments employed a one-dimensional experiment using 2 mM trifluoroacetate (tfa) as an internal reference. While the one dimensional experiments employed the CPMG pulse sequence, spectra were acquired at a single relaxation delay. The 19F resonance integrals for fluoride and tfa were compared to determine the effects of potential inhibitors on the NMR relaxation activity of the enzyme.
CuCl2 or CuZnSOD (50 µM), were allowed to incubate for known times with or without 10 mM polyphenol in 100 mM buffer, (either Tris, pH 8 or glycine/NaOH, pH 11) at room temperature. Samples without buffer were prepared using the same concentrations in deionized water, and the pH was adjusted by addition of NaOH to the appropriate value. Sample volumes were 0.2 mL. EPR spectra were measured at 120 K on a Bruker EMX spectrometer in quartz EPR tubes (0.3 mm i.d.). EPR samples were flash-frozen in liquid nitrogen and loaded into a pre-cooled cavity. Reported spectra are the average of five scans with a modulation amplitude of 10 G, modulation frequency of 100 kHz, microwave power of −1.0 mW, microwave frequency of 9.432 GHz, time constant of 40.96 ms, conversion time of 81.92 ms, and a sweep width of 1000 G centered at 3100 G. Spectra were corrected for residual cavity signal.
A 2 mL sample containing approximately 4400 units of SOD (20 mM glycine buffer, pH 10) was divided into two aliquots and 16.2 μL of either DMSO or 123 mM methyl 3,4-dihydroxybenzoate (4, MDHB) solution in DMSO were added. The control and 4 containing solutions were allowed to equilibrate overnight at room temperature. Aliquots of the experimental and control solutions were removed for analysis of total copper and the high molecular weight components were removed from the remaining samples using YM-10 membrane filtration devices (10 KDa molecular weight cutoff, Millipore). Aliquots of the four samples were treated with aqueous HNO3 and copper concentrations were determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) using a Perkin Elmer ICP-OES Optima 4100 DV spectrometer.
DMSO solutions with a range of polyphenol concentrations were prepared by serial dilution at 50 times their intended final concentration. NMR samples were prepared by addition of 12 µL aliquots of these DMSO solutions to 588 µL enzyme/buffer/fluoride solution. Thus, NMR solutions containing varied concentrations of polyphenol were prepared with a constant 2% (v/v) DMSO. Fluoride ion 19F transverse relaxation rates were determined by the standard CPMG method [
where R2,obs, R2,+ and R2,− are the transverse relaxation rates of the sample and controls containing either the same amount of enzyme or none, respectively. Inhibition profiles were determined by measuring fractional activities of samples as a function of polyphenol concentration.
The Molecular Operation Environment (MOE) program (Chemical Computing Group, Montreal) was used to dock nine flavonol structures to the active site of molecule 1 from the 1PU0 structure of CuZnSOD. Flavonolstructures were built and energy minimized in MOE as anions, deprotonated at the 4’ position. The enzyme structure was energy minimized after proton positions and partial charges had been assigned. Atoms within 12 Å of the copper atom were considered part of the active site. The flavonols were docked to the selected active site using the Triangle Matcher placement method. The docked poses were energy minimized using Force Field minimization and the energies of the poses were scored using the London DG method.
Samples of enzyme in buffered solution were sparged with argon and aliquots of either MTHB or quercetin were added anaerobically. Using the NMR assay, samples were tested periodically for several hours, and again after overnight incubation.
The effects of oxidized CuZnSOD on fluoride ion 19F NMR relaxation can be monitored using one-dimensional experiments if an internal reference is present in the assay solution. To illustrate, the effects of CuZnSOD on 19F NMR spectra of solutions containing F− and tfa are presented in
A variety of polyphenolic organic compounds were tested for their abilities to inhibit 19F NMR relaxation by CuZnSOD. In an initial screen of seven natural products (apigenin, biochanin a, coumestrol, coumestrol dimethyl ether, quercetin, genistein, and daidzein), only quercetin: 1) affected a diminution of the 19F NMR relaxation rate. We note that the loss of activity did not occur in the absence of 1, nor does it appear to result from non- specific interaction governed by hydrophobicity; Overnight incubation of enzyme with more hydrophobic compounds such as apigenin; 2) had no effect on the relaxation activity of the enzyme. Subsequently, we measured the effects of a series of related compounds, including a variety of flavonoid natural products and lower molecular weight species. The effects of the compounds on the NMR relaxation rate were strongly dependent on pH, molecular structure, compound concentration, and dissolved oxygen. Structures of these compounds and further details of their interactions with the enzyme in aerobic solution are presented in the Supplemental section of this manuscript.
The effects of pH on the NMR activity were consistent with activity requiring deprotonation of a phenol group, 1 was less potent at pH 7 than at pH 8, and inhibitory activity dropped off even more significantly under acidic conditions. The pH dependence should not be surprising since flavonols typically have pKa values around 7, 9, and 11 [
enzyme. Results of these experiments indicated that: 1) deprotonation of a carboxylic acid was not sufficient to induce inhibitory activity; 2) phenolic deprotonation is required; and 3) deprotonation of the enzyme at high pH renders it more susceptible to reaction. These results are described in greater detail in Supplemental Section 5.7.
The effects of twelve flavonoids and nine additional low molecular weight compounds on CuZnSOD NMR activity in aerobic aqueous solution were measured at pH 8.0. Inhibition of the NMR activity under these conditions depended strongly on compound structure; flavonols having hydroxyl groups at the 3, 3’ and 4’ positions were more effective than lower molecular weight polyphenols or flavonoids lacking any of these groups. Representative results are shown in
As shown in
Since the 3’ and 4’ hydroxyl groups constitute a catechol group on the flavonoid B ring, and since catechols (including 1 [
the active site metal ion. Examples of species that inhibit CuZnSOD by interacting with the active site copper ion include low molecular weight anions (like cyanide and azide [
While the low molecular weight CuZnSOD inhibitor diethyldithiocarbamate (DDC) acts by sequestering Cu2+ from the enzyme active site [
To evaluate the feasibility of an inhibition mechanism where the compounds bound the metal in the active site, we used the Molecular Operating Environment (MOE, Chemical Computing Group, Montreal, Canada) suite of programs to dock the flavonoids to the 1PU0 structure of CuZnSOD. While these experiments indicated that the active site could accommodate the flavonols, most poses generated by docking calculations did not present the flavonol in contact with the enzyme copper atom. While it was possible to generate the proposed active site by tethering the flavonol B ring to the copper via the 3’ and 4’ hydroxyl groups, energy minimization did not return significantly stabilized structures. Thus, the docking results did not help rationalize the observed structure activity relationship.
To investigate the effects of the compounds on the active site copper, we measured the EPR spectrum of the oxidized enzyme in the presence and absence of two low MW polyphenols (4, MDHB and 5, MTHB) at pH 8 and 11. Representative EPR spectra collected at pH 8 are presented in
While the similarity of the spectrum to that of the Cu2+ complex of 4 suggests coordination, the centrifugal concentration experiments described above indicate that copper is not lost from SOD under these conditions. Thus, 4 does appear to coordinate copper in the active site of the protein.
In contrast to the effects of 4, addition of 10 mM 5 (MTHB) at pH 8 (
The effects of oxygen on inhibition of the NMR activity were studied using either 1 mM 5 (MTHB) or 50 µM 1 (quercetin) (concentrations where ~50% inhibition of the NMR activity is observed in aerobic solution). In contrast to the behavior in aerobic environment, overnight incubation of CuZnSOD with either 1 or 5 under inert atmosphere resulted in complete loss of NMR activity. The activity versus time profiles for these experiments is presented in
Our results suggest that redox reactions (Equation (2) and Equation (3)) account for NMR activity inhibition.
Copper(II) salts will oxidize catechols [
Since the Cu+ form of the enzyme is an intermediate in the enzymatic reaction, the redox hypothesis predicts that reaction with the polyphenols would not affect the ability of the enzyme to catalyze superoxide dismutation. To test the redox competence of the inhibited enzyme, we measured the effect of 1 on the steady state concentration of oxidized CuZnSOD under redox cycling conditions. Rigo et al. [
In summary, we have demonstrated that enhancement of the fluoride ion 19F NMR relaxation rate by CuZnSOD can be assayed in one dimensional experiments using tfa as an internal reference. Using this technique, we have screened a variety of compounds for CuZnSOD inhibitory action and have discovered that flavonols inhibit the
NMR relaxation activity of the enzyme. Our results indicate that inhibition of the NMR relaxation activity occurs via electron transfer. Interpretation of the inhibition data was complicated by aggregation of the inhibitors. Experiments with superoxide generating compounds indicated that reactions with the flavonoids did not prevent subsequent redox cycling of the enzyme.
We are grateful for support from the North Carolina Biotechnology Center (Basic Research Grant 556,603) and from the NIH (Academic Research Enhancement Award 1 R15 GM094034-01).
Jack S. Summers,Benjamin Hickman,Megan E. Arrington,Bradley S. Stadelman,Julia L. Brumaghim,Michelle R. Yost,Jeffrey D. Schmitt,Mariah Hornby,Stacy Sprague, (2016) Reaction of Oxidized CuZnSOD with Polyphenols. Natural Science,08,359-379. doi: 10.4236/ns.2016.88041
Interpretation of inhibition constants (Kinh); The effects of flavonols and lower molecular weight compounds on NMR relaxation enhancement by CuZnSOD were measured as described in the Experimental section of the manuscript. Normalized activities of inhibitor containing solutions were calculated as;
Our work (described in the Results and Discussion section), however, subsequently showed that NMR inhibition likely results from both binding to the enzyme and from electron transfer to the paramagnetic copper, with the latter probably giving the more important contribution to the most active compounds. If, as we propose, the relaxation activity of the enzyme is determined by steady state ratio of oxidized to reduced enzyme in aerobic solution is determined by the rates of the reactions in Equation (S2) and Equation (S3), then differences between apparent inhibition constants should reflect differences in the rates of reaction (S2) (Equation (S4)):
Chemical structures of the flavonoids used in this study are presented in
As discussed in the Results and Discussion section of this manuscript, plots of 1/A-1 were distinctly non-linear with polyphenol concentration. We attribute this to dimerization and higher order aggregation of the compounds in aqueous solution. Equation (S1), above describes how the inhibitory activities of flavonoids could be affected by dimerization, this is a simplified scenario and the fluorescence data indicate that higher order aggregation occurs for some compounds. Characterizing such behavior requires consideration of trimerization, characterized by an equilibrium constant (Ktrimer, defined in Equation (S5)). In the general case, the total mass of material must be distributed between monomer, dimer, trimer, etc., as in Equation (S6). While a fuller description of aggregation would require an equilibrium constant for each multimer considered, our data could be modeled adequately assuming that only monomer, dimer and trimer were present in significant concentrations. For example, more than 98.8% of the variance in the fluorescence spectra of solutions containing from 1 to 512 M 3 could be accounted for by the three species.
A complete description of the behavior predicted by the involvement of trimers would require solution of a third order polynomial. While roots of the cubic equation can be calculated, the exact solution does not lend itself to least squares refinement since solutions having an imaginary component can arise under different scenarios. To simplify the calculation, we made two simplifying assumptions. First, we limited our model by assuming 1/Ktrimer would always be greater than or equal to 1/Kdimer. Since the individual units all carry a negative electronic charge, it is reasonable that electrostatic repulsion will destabilize larger aggregates relative to smaller ones. Our second assumption was that the concentration of monomer could be approximated by that calculated in the absence of trimerization (the dimer only case). The validity of this assumption can be assessed by comparing calculated distribution information for different assumed values of Ktrimer.
In the dimerization only case, Equation (S7) gives a quadratic and the concentration of monomer can be estimated using Equation (S8). The remaining material can be partitioned between dimer and trimer using Equation
(S5) and Equation (S6). Modeling the data in this way allowed us to approximate concentrations of monomer, dimer, and trimer for any total inhibitor concentration given an assumed combination of equilibrium constants. Molar fluorescence coefficients for the monomer, dimer, and trimer were then estimated by least squares optimization to minimize the difference between predicted and measured spectra. To illustrate, we will consider two cases, one where trimerization was not important at low concentrations (myricetin, 3) and the other where trimerization was important (gossypin, 6). The observed and predicted fluorescence data for 6 are presented in
In contrast to the behavior of 6, 3 appeared to only form significant amounts of trimer at the highest concentrations where fluorescence spectra were recorded. The effects of concentration on the emission spectrum of 3 are presented in
Uncertainties in 1/Ktrimer were too great to justify reporting their values. Even when we have evidence for significant trimerization at lower concentrations, our fitting parameter (SSR) was relatively insensitive to changes in this parameter. As shown in Panel C of
Uncertainties in the results of NMR experiments arise from a number of factors. For these experiments, samples were prepared by a series of steps. The mass of solid inhibitor was measured to ~1%. The solid sample was dissolved in a volume of DMSO that is known to ~2%. DMSO solutions of descending concentration were prepared by serial dilution. NMR solutions were prepared by dilution of one of the DMSO solutions in enzyme containing buffer. If each dilution added another 2% uncertainty, then the highest concentration NMR sample should have an inhibitor concentration known to approximately 7%, and each subsequent sample is known to 2% lower accuracy. Thus, accumulation of errors leads the concentrations of more dilute solutions to be less certain than those of more concentrated samples. Measured relaxation rates (R2 values) were reproducible to within ~3% on a given sample and activities were determined from measurements of three samples (the experiment and two controls). Since the 1/A-1 value is the ratio of two differences (Equation (1), Experimental Section), the greatest accuracy is achieved at unity with uncertainty increasing at both larger and smaller values. In the worst case, we estimate that 1/A-1 values should be good to within 10%. Experimental reproducibility can be gauged by comparing the results of independent measurements of inhibition by the same compound.
Using limits on 1/Kdimer established by the fluorescence experiments (20 and 40 M), it is possible to estimate limits on acceptable values of Kinh. The dependence of enzyme activity on total added inhibitor comes from substituting the monomer concentration from the fluorescence experiments into Equation (S1). Trend lines in
Comparing the 1/Kinh values in
Compound | IC50 (M) | Kdimer−1 (M)a | 1/Kinh (M)b | Ep/2 vs NHE (V)c |
---|---|---|---|---|
1, Quercetin | 12 | 30 | 12 | 0.271 |
2, Apigenind | >1000 | 59 | >200 | |
3, Myricetin | 6.7 | 24 | 6.8 | 0.211 |
6, Gossypin | 39 | 12 | 13 | |
7, Fisetind | 69 | 0.361 | ||
8, TetHF | 140 | 0.6 | 10 | |
9, Taxifolin | 200 | 0.391 | ||
10, Kaempferold,e | 660 | 0.361 | ||
11, TriHFd | 820 | 7 | 9 | |
12, Morind | >1000 | 330 | >200 | |
13, Luteolind,e | >1000 | 0.421 | ||
14, Syringetin | >1000 |
Notes: a) 1/Kdimer values should be accurate to within a factor of three. b) 1/Kinh values should be accurate to within a factor of two c) Half wave potentials for flavonoid oxidation, adjusted for comparison to the normal hydrogen electrode, NHE. Values are from reference S7. d) Kdimer could not be determined accurately from fluorescence data. e) IC50 estimated by extrapolation of reciprocal activity versus [Inh]0.5 plot.
of 1 (1/Kinh = 24 M) to that of 13 (1/Kinh > 200 M). These two compounds only differ at the 3 position where 1 has the hydroxyl group and 13 has hydrogen. We considered the possibility that the importance of the 3-hydroxyl group to inhibition arises from its ability to tautomerize to give a ketone at this position. To test this hypothesis, we measured the effect of 9 on the enzyme. The structure of 9 differs from that of 1 in that the bond between the 2 and 3 position carbon atoms is a single bond in 9 whereas it is a double bond in 1. Thus, the 3 hydroxyl group on 1 may tautomerize to give a ketone while 9 cannot. Comparing IC50 values for the compounds in
It is apparent that compounds containing the catechol moiety on the B ring are more effective inhibitors than are compounds that do not. For example, 10 (having a single B ring hydroxyl group) is much less effective against CuZnSOD than 1 (having B ring hydroxyl groups at the 3’ and 4’ positions). Further, the importance of the catechol can be seen by comparing the inhibitory activities of two isomeric flavonols, 12 and 1. The structures of the compounds differ in the positions of the hydroxyl groups on the B ring. While 1 has hydroxyl groups on the 3’ and 4’ positions, the 12 hydroxyls are at the 2’ and 4’ positions. As was the case with 10, 12 is also a poor inhibitor.
In contrast to the dramatic effects observed when substitution patterns on the B ring are changed, the number and positions of hydroxyl groups on the A ring had a lesser impact on inhibition. We measured the inhibitory activities of five active compounds that only differ in A ring substitution patterns (1, 6, 7, 8, 11). While IC50 values (reported in
To determine what structural features of the flavonols contributed to their activities toward the enzyme, we investigated a variety of other low molecular weight compounds. Structures of compounds used in this study are presented in
At pH 9 or below, 4 reacts with CuZnSOD in such a way as to prevent NMR relaxation activity of the enzyme. The reaction appears reversible, with an equilibrium constant of ~3 mM. At pH greater than 9, a second, irreversible reaction occurs. The rate of this second reaction is pH dependant in a way consistent with deprotonation occurring at the enzyme and not at the inhibitor.
We measured the effects of 4 concentration on the activity of CuZnSOD at pH ranging from 7.0 to 11.5. Our results indicate that a deprotonated form of the compound is responsible for SOD inhibition. At pH of 7 or less, 4 is predominantly charge neutral (pKa = 8.13) and a concentration of 4 mM does not appreciably diminish SOD activity. At pH 8 and 9, the activity of CuZnSOD decreased with increasing concentration of 4. A plot of the reciprocal of the normalized activity (A, defined in the experimental section) versus inhibitor concentration was linear (
Effects of pH on inhibition by 4: At pH greater than 10, a second reaction appears to occur between 4 and CuZnSOD. The kinetics of the second reaction could be conveniently monitored by NMR. Representative kinetic results are presented in
At a constant 4 concentration (1.0 mM), the extrapolated value of the activity at the time of mixing was not
influenced by pH over the range from 9 to 11.5 (data not shown) indicating that the binding constant is not grossly affected by pH. Continuation of the study to pH greater than 11.5 was not possible due to the pH dependent loss of enzyme activity documented elsewhere [
When compared to 4, 5 is a much more effective inhibitor of SOD NMR relaxation activity at low concentration. The pronounced curvature of the plot suggests that 5 is strongly aggregated at mM concentration. If this is the case, then NMR inhibition at the lowest concentrations of 5 should best reflect the effects of the monomeric compound. The data from
We examined the effects of a series of chelating agents that are known to inhibit zinc enzymes by binding the active site metal. While acetohydroxamic acid (25), maltol (26), thiomaltol (27), and 2-mercaptopyridine N- oxide (28) have been reported to inhibit zinc containing metalloproteinases with IC50 ranging from 25 mM to 35 M [
We measured the effects of 29, 30, and 31 (
EPR data for CuCl2 and CuZnSOD upon addition of MTHB and MDHB. Numbers in brackets are estimated values.
asamples were prepared in Tris buffer (pH 8) or in glycine/NaOH buffer (pH 11) unless otherwise specified; bg value for MTHB or MDHB semiquinone radical; cestimated g and A values for Cu2+-buffer complex; destimated g and A values for MTHB, MDHB or EC hyperfine coupling with Cu2+.