Results and Discussion

Inhibition of CYP1B1 is proposed as one of the strategies in the treatment of cardiovascular diseases and cancer chemoprevention (Mikstacka et al., 2007). In this regard, we tested the capacity of both enantiomers of 19-HETE to inhibit the catalytic activity of human recombinant CYP1B1, on the basis of our previous finding that 19(R)- and 19(S)-HETE protected against Ang II-induced cellular hypertrophy via decreasing the level of cardiotoxic midchain HETEs, the major metabolites of CYP1B1 (Shoieb and El-Kadi, 2018). For that purpose, the inhibition kinetics of both enantiomers of 19-HETE on CYP1B1 activity were carried out using CYP1B1 substrate 7-ER, a compound that is metabolized via oxidative reaction by this enzyme (Shimada et al., 1997). The experimental conditions, such as the concentrations of 7-ER, concentration of the recombinant enzyme, in addition to the incubation time, were all optimized before the addition of either 19(R)-HETE or 19(S)-HETE.

Inhibition of EROD is a well reported assay carried out predominantly to examine the inhibitory potencies of CYP1B1 inhibitors (Androutsopoulos et al., 2011). Inhibitory activity of 19(R)-HETE (Fig. 2A) and 19(S)-HETE (Fig. 2B) on CYP1B1 was studied. Foremost experiments involved the assessment of 7-ER-O-deethylase kinetics by determination of the rate of resorufin formation over time. On the basis of the nonlinear regression analysis and fitting to the Michaelis-Menten model, the maximal EROD activity (V_max) in the control containing 0.1% dimethyl sulfoxide was 16.7 pmol resorufin/pmol P450 per minute (R² = 0.9888) (Table 1). The Michaelis-Menten constant (K_m) value for the current EROD reaction is 86.6 nM (Table 1). During the coincubation of either 19-HETE enantiomers at 10 nM concentration with different concentrations of 7-ER, we observed a significant inhibition of CYP1B1 enzymatic activity. This finding prompted us to test increasing concentrations (0–40 nM) of either 19(R)-HETE or 19(S)-HETE with different concentrations (0–100 nM) of 7-ER to characterize the mode of inhibition by 19-HETE enantiomers.

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Fig. 2.

Inhibitory effect of 19(R)-HETE and 19(S)-HETE on EROD activity mediated by human recombinant CYP1B1. In 96-well solid black polystyrene plates, the reaction mixture containing 100 mM potassium phosphate (pH 7.4) buffer supplemented with 5 mM magnesium chloride hexahydrate and 1 pmol of human CYP1B1 was incubated with 10–100 nM 7-ER. In addition, either 19(R)-HETE (A) or 19(S)-HETE (B) at 0, 5, 10, 20, or 40 nM concentration was added to the reaction. The reaction was initiated by the addition of 100 μl of 2 mM NADPH, the fluorescent signal related to the formation of resorufin was measured every minute (excitation and emission wavelengths of 550 and 585 nm, respectively) for 30 minutes at 37°C using a BioTek Synergy H1 Hybrid. The quantity of formed resorufin was measured by the construction of standard curve of 0–200 nM resorufin dissolved in the same incubation buffer. Each point represents the mean of six independent experiments ± S.E.

Nonlinear regression analysis and comparisons showed that the mode of inhibition for 19(R)-HETE and 19(S)-HETE is noncompetitive inhibition of CYP1B1 enzyme. Dixon plots showed that 19(R)-HETE (Fig. 3A) and 19(S)-HETE (Fig. 3B) have K_i values of 89.1 and 37.3 nM, respectively (Table 1). The K_i values of both enantiomers showed that the S-enantiomer is more potent than the R-enantiomer by approximately 2.4-fold. This comes in agreement with our previous work demonstrating that the S-enantiomer of 19-HETE has a preferential effect over the R-enantiomer in inhibiting the formation of midchain HETEs (Shoieb and El-Kadi, 2018). In accordance with being noncompetitive inhibitors, the in vitro stability study showed that both enantiomers of 19-HETE are metabolically stable in solution during the 30-minute time course of the kinetic experiment as there was no significant difference in the level of both enantiomers in the duration of the experiment (Supplemental Figs. 1 and 2; Table 2). Discovery of selective and potent inhibitors of CYP1B1 enables the design of CYP1B1-targeted cardiovascular treatment and prevention in addition to cancer chemotherapy (Pingili et al., 2017; Ware, 2017).

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Fig. 3.

Dixon plots representing the inhibitory effect of 19(R)-HETE and 19(S)-HETE on EROD activity mediated by human recombinant CYP1B1. The y-axis represents the reciprocal of EROD activity expressed in picomoles of resorufin/picomoles CYP1B1/minute and the x-axis represents 19(R)-HETE (A) or 19(S)-HETE (B) concentrations in nanomolar concentrations in the presence of increasing concentration of the substrate 7-ER (10–100 nM). Each point represents the mean of six independent experiments ± S.E.

To date, more than 50 synthetic and natural compounds have been designed or recognized as inhibitors of CYP1B1 (Li et al., 2017). Various CYP1B1 inhibitors with varying mode of inhibition have been identified. For instance, trans-resveratrol inhibited CYP1B1 enzymatic activity via mixed-type inhibitory mechanism, and the K_i was 0.75 μM (Chang et al., 2000). Also, myricetin and quercetin with K_i 230 and 120 nM, respectively, exhibited mixed-type inhibition, whereas apigenin with K_i 60 nM acted as a competitive inhibitor of CYP1B1 (Chaudhary and Willett, 2006). Moreover, tetramethoxystilbene (TMS) noncompetitively inhibited EROD activity of CYP1B1 with an IC₅₀ value of 2 nM, when 2 μM 7-ER was used as a substrate (Chun et al., 2009). In the present study, both enantiomers of 19-HETE inhibited CYP1B1 enzymatic activity in the nanomolar range and more potently than some of the previously mentioned CYP1B1 inhibitors, which recommends their introduction as therapeutic and preventive modalities in CYP1B1-targeting therapies.

In the current study, there is a significant difference in the inhibitory activity between R- and S-enantiomers of 19-HETE. To the best of our knowledge, the current study is the first to characterize the enantioselective pattern of inhibition of CYP1B1 by enantiomers of 19-HETE. Enantioselective inhibition of cytochrome P450 enzymes has been previously documented. S002-333, a novel and potent antithrombotic agent, inhibited CYP2B6-mediated bupropion 6-hydroxylation in a stereoselective manner. Although the S-enantiomer showed strong inhibition with IC₅₀ value of 5.28 μM, the R-enantiomer showed much less inhibition on CYP2B6 catalytic activity with IC₅₀ value of more than 50 μM (Bhateria et al., 2016).

It is noteworthy that CYP1B1 regulates several metabolic pathways. For example, AA is oxidized by CYP1B1 to produce the cardiotoxic midchain HETE metabolites. In rat neonatal cardiomyocytes, 15-HETE has been reported to be capable of elevating isoproterenol-induced β-adrenergic response sensitivity. Moreover, 5-, 12-, and 15-HETE were capable of inducing cellular hypertrophy in RL-14 cells, human ventricular cardiomyocytes, via mitogen-activated protein kinase- and nuclear factor κB-dependent pathways (Zhang et al., 2014; Maayah and El-Kadi, 2016). Furthermore, CYP1B1 is responsible for some vital physiologic processes in blood vessels important to the development and progression of hypertension. In Ang II-induced hypertension, CYP1B1 mediates cell migration, induces proliferation, and increases protein synthesis in vascular smooth muscle cells through regulation of the metabolism of AA (Yaghini et al., 2010). The fact that CYP1B1 expression is vastly enhanced in several tumors, such as prostate, breast, and colon cancers, suggests that CYP1B1 inhibitors could be considered an accepted therapeutic strategy in treatment and prevention of cancer (Gibson et al., 2003; Tokizane et al., 2005).

In summary, CYP1B1 is considered one of the important cytochrome P450s. It is involved in the metabolism of many endobiotics, xenobiotics, and in the activation pathways of various procarcinogens. The findings of the current study show that both enantiomers of 19-HETE noncompetitively inhibited CYP1B1 enzymatic activity with the S-enantiomer having a higher potency. The current study suggests that 19(R)-HETE and 19(S)-HETE could be considered a novel therapeutic modality in the treatment of hypertension, cardiac hypertrophy, and cancer. Moreover, given that a noncompetitive inhibitor might bind to the enzyme regulatory region, 19(R)-HETE and 19(S)-HETE could be the focus for unraveling the obscure mechanisms of CYP1B1 enzymatic reaction.