Epigallocatechin Gallate Inhibition of SULT1A1.

Catechins are water soluble flavanols that constitute ∼25% of the dry weight of green tea, and epigallocatechin gallate (EGCG) accounts for approximately half of the tea catechins (Sabhapondit et al., 2012). Previous work on the interactions of SULTs and dietary chemicals revealed that EGCG is a potent inhibitor of SULT1A1 (K_i = 42 nM). The mechanism of inhibition appeared to be uncompetitive versus PAPS; inhibition versus acceptor was not investigated. Initial-rate studies of EGCG inhibition versus both PAPS and acceptor (para-nitrophenol, pNP) are presented in Fig. 1, A and B. Inhibition versus PAPS is well fit using an uncompetitive model, which assumes that EGCG binds only to the PAPS-bound forms of the enzyme. In contrast, inhibition versus acceptor is well fit using a pure noncompetitive model, indicating that EGCG and acceptor bind at separate sites and do not influence one another’s affinity for the enzyme. The initial-rate inhibition parameters are listed in Table 1. The mechanism of SULT2A1 is rapid-equilibrium random (Wang et al., 2014), and it has been argued, on the basis of conservation of structure, the equivalence of initial-rate and thermodynamic parameters, and the partial substrate inhibition common within the family, that other SULTs, including SULT1A1 (Gamage et al., 2003), have similar mechanisms. Isotope-exchange experiments have been interpreted in favor of an ordered mechanism for SULT1A1; however, these results are also consistent with a random-binding mechanism that includes a dead-end complex (Cook and Cleland, 1981), which is the case with SULT1A1 (Gamage et al., 2005). For these reasons, we assume that the binding mechanism of SULT1A1 is random.

Although the inhibition studies are revealing, they leave several key mechanistic issues unresolved. For example, a parallel-line inhibition pattern (Fig. 1A) indicates only that inhibitor binds significantly more tightly to the nucleotide-bound than nonbound forms of the enzyme. Further, the data do not address whether the enzyme is partially inhibited (i.e., turnover is reduced to a nonzero value at saturating inhibitor) or totally inhibited by EGCG.

To identify the enzyme forms to which EGCG binds and obtain its binding affinities, equilibrium-binding studies were performed using the enzyme forms typically associated with the substrate section of the catalytic cycle (E, E∙pNP, E∙PAPS, and E∙pNP∙PAP). It should be noted that PAP is an excellent surrogate for PAPS in ternary-complex binding studies (Cook et al., 2013a). Binding was monitored via ligand-induced changes in the intrinsic fluorescence of SULT1A1(Cook et al., 2013a). In all cases, substrate-ligand concentrations were ≥15 × K_d (Cook et al., 2013a, c). The titrations are shown in Fig. 2, and the affinity constants are compiled in Table 2.

The studies reveal that EGCG binds to all four enzyme forms—the mechanism is depicted in Fig. 3. Its affinities for nucleotide-bound forms are identical within error, as are its affinities for the nucleotide-free forms; however, EGCG binds 21-fold more tightly to the enzyme when nucleotide is bound. The 21-fold difference in affinity provides an important clue as to the molecular basis of the inhibition. SULTs harbor a conserved 30-residue active-site cap that is positioned over both the nucleotide- and acceptor-binding pockets. Nucleotide binding stabilizes the cap in a “closed” position that encapsulates the nucleotide and acceptor and forms a pore that sterically restricts access to the acceptor-binding site (Cook et al., 2013c; Leyh et al., 2013; Wang et al., 2014). The cap can isomerize into an open state when nucleotide is bound, and it is from the open position that nucleotide escapes. The equilibrium constant for this isomerization, K_iso, has been measured for SULT1A1 and it equals 21 in favor of the closed position (Cook et al., 2013c, a). The fact that the value for the PAPS-induced enhancement in EGCG affinity and K_iso are the same strongly suggests that EGCG binding is linked to cap closure. It is particularly interesting that previous studies demonstrate that EGCG exhibits high affinity for SULT1A1, but not 1A2 or 1A3 (Coughtrie and Johnston, 2001), which are closely related isozymes whose caps differ slightly from that of SULT1A1. These facts, when taken together, are consistent with a model in which the isozyme specificity of the EGCG is determined by its interactions, either direct or indirect, with the cap.

MEF Inhibition of SULT1A1.

A broad-based study of NSAID inhibition of SULT1A1 revealed that MEF is particularly potent [IC₅₀ = 20 nM, (Vietri et al., 2002)]; however, the mechanism of its inhibition is not known. MEF inhibition of the initial rate of SULT1A1 turnover is plotted versus PAPS and pNP in Fig. 4, A and B. The velocities were determined in triplicate, averaged, and were well fit using a noncompetitive model, which assumes that the binding of substrate and inhibitor are entirely independent. The resulting affinity constants are compiled in Table 2. The simplest interpretation of these findings is that MEF binds to all four substrate forms of the enzyme at a site that is separate from those of the substrate-binding sites, and that MEF binding is not influenced by bound substrates. A notable feature of such mechanisms is that such inhibition cannot be “overcome” by an accumulation of substrate caused by restricted metabolic flow at the point of inhibition. It is interesting to note that the fact that MEF inhibits turnover without altering substrate affinities suggests that it perturbs only protein elements that control chemistry. Deeper mechanistic work will test this linkage.

To confirm the implications of the initial-rate findings, MEF binding to same four enzyme forms used in the EGCG study was investigated in equilibrium-binding studies. Here again, binding was monitored via changes in SULT1A1 intrinsic fluorescence. The results of the titration (Fig. 5) are consistent with the predictions of the initial-rate study—MEF binds to all four forms (Fig. 3) and has nearly the same affinity (∼23 nM) for each complex (Table 2).

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Fig. 5.

The binding of MEF to SULT1A1. The protocol was virtually identical to that described in Fig 2. Binding was monitored via ligand-induced changes in the intrinsic fluorescence of SULT1A1 (λ_ex = 295 nm, λ_em = 345 nm). Each point is the average of two independent determinations. The line through the data represents a least-squares fit using a model that assumes a single binding site per subunit. Conditions were as follows: SULT1A1 (10 nM, dimer), PAP (0 or 10 μM, 33 × K_d), pNP (0 or 45 μM, 30 × K_d), MgCl₂ (5.0 mM), NaPO₄ (50 mM), pH 7.2, T = 25 ± 2°C.

EGCG and MEF Are Partial Inhibitors.

Partial inhibitors reduce turnover of an enzyme to a fixed, nonzero value at saturating inhibitor concentrations. The preceding initial-rate experiments do not have the resolution needed to distinguish between partial and total inhibition in cases where turnover is reduced to less than ∼10% of noninhibited turnover. To address this issue, the initial-rate of pNPS synthesis was determined at EGCG and MEF concentrations that ranged as high as 110 × K_i (Fig. 6). Velocities were determined in triplicate and averaged. The data were fit using a model that assumes a single inhibitor-binding site per subunit, and the best-fits are shown as solid lines passing through the datasets. Turnover clearly decreases to a nonzero value at saturating inhibitor; thus, EGCG and MEF are partial inhibitors. At saturating concentrations of MEF and EGCG, SULT1A1 turnover is reduced to 6 ± 1 and 12 ± 2%, respectively, of their uninhibited values.

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Fig. 6.

EGCG and MEF are partial inhibitors. Reaction progress was monitored at 405 nm. The conditions were as follows: SULT1A1 (1.0 nM, dimer), PAPS (10 μM, 625 × K_m), PnP (30 μM, 22 × K_m), MgCl₂ (5.0 mM), NaPO₄ (50 mM), pH 7.2, T = 25 ± 2°C. Less than 5% of the substrate converted at the endpoint of the reaction was consumed during the rate measurements. Each point represents the average of three independent determinations. The lines through the points indicate the behavior predicted by a least-squares fit using a model that assumes a single binding site per subunit. K_i values in the model were fixed using constants in Table 1, and data were fit only for the maximum inhibition value. The best-fit, maximum inhibition values for EGCG and MEF were 88 ± 2 and 94 ± 1%, respectively.

EGCG and MEF Bind at Separate, Noninteracting Sites.

The mechanisms of EGCG and MEF inhibition are similar in that they each bind to the four enzyme forms studied; however, the fact that their inhibition mechanisms differ (that is, only EGCG exhibits enhanced affinity when PAPS is bound) suggested that they might bind at separate sites. If so, and if they operate independently (i.e., they do not influence one another’s affinity, or influence on turnover) their effects on SULT1A1 turnover will be additive. If, on the other hand, they bind at the same site, or at separate sites that are interactive, the effects will be nonadditive. To assess the additivity of their effects, the inhibitors were used in combination, and the results were compared with the predictions of same- and separate-site binding models. The study simultaneously varied inhibitor concentrations in equal K_d increments using the constants in Table 1. Assuming separate, noninteracting sites, this design causes the distribution of the inhibitor-bound forms of the enzyme to shift from predominantly single- to double-inhibitor occupancy as the concentration increases from low to high K_d equivalents. As the shift occurs, deviations from simple additivity can be observed. The patterns predicted by separate noninteracting, and same-site binding models are shown, in Fig. 7, in solid and dashed lines, respectively. The models were parameterized using the constants obtained from the single-inhibitor studies (Table 1). The same-site model poorly describes the data; the separate noninteracting site model provides an excellent fit. Thus, each inhibitor binds at a separate site, and their actions are largely independent.

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Fig. 7.

EGCG and MEF bind at separate and noninteracting sites. The pattern of SULT1A1 inhibition by EGCG and MEF in combination was used to assess their binding independence. EGCG and MEF were added simultaneously in equal K_i-equivalents over a concentration range that (as suggested by single-inhibitor studies) would cause the enzyme to transition from singly- to doubly-inhibitor bound. The curving solid lines are the predictions of a same-site (dashed line) and independent-site (solid line) binding models that were parameterized using the constants in Table 1. The experimental data (black dots) is in strong agreement with the additive model. Reaction conditions: SULT1A1 (20 nM), PAPS (10 μM, 625 × K_m), pNP (36 μM, 22 × K_m), MgCl₂ (5.0 mM), and NaPO₄ (50 mM), pH 7.2, T = 25 ± 2°C. Reactions were monitored at 405 nm. Each point is the average of three independent trials.

Inhibition by two independent, partial inhibitors differs substantially from that of a single inhibitor. At an inhibitor concentration equal to its affinity constant, assuming the enzyme concentration is negligible, half of the enzyme will be inhibitor-bound. This is also the case for the second inhibitor, since it binds independently. The fraction of the enzyme bound to both inhibitors is given by the product of the fraction-bound for the individual inhibitors—in this case, 0.25. Thus, one quarter of the total enzyme will be in each of the four possible forms: E, E∙I_A, E∙I_B and E∙I_A∙I_B. Turnover is given by the sum of the fraction of enzyme in each state weighted by its turnover. Given EGCG and MEF each at its K_d, turnover will be 29% of the uninhibited enzyme, which is nearly one-half (0.52) of that predicted for EGCG alone. An important consequence of double partial-inhibition is that turnover of double-inhibitor–bound enzyme is given by the product of the fraction-turnover associated with each inhibitor. Individually, EGCG and MEF reduce turnover to 0.12 and 0.06 times the noninhibited value, respectively; together, they reduce turnover to near zero, 0.0072.

The affinities of EGCG (34 nM) and MEF (27 nM) for SULT1A1 are well below their normal plasma concentrations. Unmodified EGCG achieves a peak concentration of ∼300 nM (8.8 × K_i) following consumption of 400 mg of pure compound, and consumption of MEF (500 mg) results in a peak concentration of 28 μM (1000 × K_i). Although inhibition studies have not yet been performed in humans, the plasma concentrations of EGCG and MEF are sufficient to inhibit 1A1 in vivo. It is notable that studies that used human liver extracts determined that the IC₅₀ of MEF for SULT1A1 is approximately 20 nM (Vietri et al., 2002; De Santi et al., 2000).

Therapeutic Relevance.

As our society evolves toward greater drug dependency, predicting drug-drug or drug-xenobiotic interactions becomes increasingly complex. The drug regimen of an average nursing home resident in the United States includes routine administration of 8.3 drugs and an additional 3.2 drugs that are given pro re nata (Jones et al., 2009). The better we understand the interactions of these compounds with their cellular counterparts the more able we will be to predict whether compounds will interact, and the consequences of those interactions.

The neutralization of toxins, which occurs in a variety of ways, is among the primary functions of SULT1A1. Consider, for example, its role in preventing acetaminophen-induced hepatotoxicity—the most prevalent over-the-counter drug-induced hepatotoxicity in the United States (Larson et al., 2005). Acetaminophen is sulfonated (∼40%) by SULT1A1, and glucuronidated (∼60%) by UGT1A1 (Rogers et al., 1987). Whereas the conjugated form is nontoxic, the unconjugated compound is oxidized in liver, primarily by CYP3A4 (Larson et al., 2005), to N-acetyl-p-benzoquinone, which is cytotoxic. As the catalytic capacity of the conjugating systems becomes overwhelmed, either by overdose or inhibition owing to a coadministered compound, fatal toxicity can ensue (Larson et al., 2005). Thus one should carefully consider whether individuals taking acetaminophen are also consuming catechin-rich foods and liquids, and/or NSAIDs.

Sulfonation is a primary pathway for the activation of polyaromatic procarcinogens. The sulfonated derivatives of these compounds are unstable and thus disproportionate (heterolytically) into sulfate and highly reactive, planar electrophiles that covalently attach to DNA. sult-gene knockin and knockout studies (Sachse et al., 2014), and work with SULT-specific inhibitors, demonstrated that DNA adduct formation decreases dramatically when only the 1A1 isoform is inhibited. In this connection, it is notable that prostate cancer is 5- to 10-fold more probable in individuals who express high, versus low, levels of SULT1A1 activity in serum (Nowell et al., 2004). On the basis of these and similar findings, it is often suggested that, depending on diet, the routine consumption of SULT1A1 inhibitors contributes to a reduced incidence of cancer (Thorat and Cuzick, 2013; Pasche et al., 2014).

Recent work has shown that 76 of the 1211 FDA-approved small-molecule drugs are sulfonated by SULT1A1, and an additional 136 have been shown or are predicted to be SULT1A1 inhibitors (Cook et al., 2013c). In many instances, sulfonation inactivates these drugs by preventing them from binding to their target receptors, and it can dramatically shorten their terminal half-lives. The extent of sulfonation is idiosyncratic to both the compound and its cellular locale. In certain cases, UDP-glucuronosyltransferases compensate for lowered SULT activity by glucuronidating the moiety that would otherwise have been sulfonated (Kane et al., 1995). In many cases, SULT inhibition is expected to enhance the efficacy of a drug. In cases like propofol, where rapid inactivation by 1A1 is desirable for quickly bringing patients out of anesthesia (Vree et al., 1987), SULT1A1 inhibition is detrimental. Alternatively, using inhibition of SULT1A1 to substantially lengthen the half-life and efficacy of apomorphine could lead to a more stable anti-Parkinson therapeutics and a substantial reduction in the tremors associated with the disease (Calabresi et al., 2010).