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Statin Lactonization by Uridine 5′-Diphosphoglucuronosyltransferases (UGTs) Tom J. J. Schirris,†,‡ Tina Ritschel,§ Albert Bilos,† Jan A. M. Smeitink,∥,‡ and Frans G. M. Russel*,†,‡ †

Department of Pharmacology and Toxicology, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands Center for Systems Biology and Bioenergetics of the Nijmegen Center for Mitochondrial Disorders, 6500 HB Nijmegen, The Netherlands § Computational Discovery and Design Group, Center for Molecular and Biomolecular Informatics (CMBI), Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands ∥ Department of Pediatrics, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands ‡

S Supporting Information *

ABSTRACT: Statins are cholesterol-lowering drugs that have proven to be effective in lowering the risk of major cardiovascular events. Although well tolerated, statin-induced myopathies are the most common side effects. Compared to their pharmacologically active acid form, statin lactones are more potent inducers of toxicity. They can be formed by glucuronidation mediated by uridine 5′-diphospho-glucuronosyltransferases (UGTs), but a systematic characterization of subtype specificity and kinetics of lactonization is lacking. Here, we demonstrate for six clinically relevant statins that only UGT1A1, 1A3, and 2B7 contribute significantly to their lactonization. UGT1A3 appeared to have the highest lactonization capacity with marked differences in statin conversion rates: pitavastatin ≫ atorvastatin > cerivastatin > lovastatin > rosuvastatin (simvastatin not converted). Using in silico modeling we could identify a probable statin interaction region in the UGT binding pocket. Polymorphisms in these regions of UGT1A1, 1A3, and 2B7 may be a contributing factor in statin-induced myopathies, which could be used in personalization of statin therapy with improved safety. KEYWORDS: statins, HMG-CoA reductase inhibitors, uridine 5′-diphospho-glucuronosyltransferase (UGT), lactonization, glucuronidation, acyl-coenzyme A, UGT homology models, statin-induced myopathy

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lactone forms are more potent inducers of in vitro cytotoxicity.8 Moreover, increased systemic statin lactone concentrations are observed in patients with statin-induced myopathies.9 Most statins, except for simvastatin and lovastatin, are administered in their pharmacologically active acid form. Lactonization has been suggested as an intermediate step of statin metabolism resulting in a better substrate for CYP3A4 and therefore enhancing overall clearance.10 Several mechanisms for in vivo statin lactonization have been proposed.11−13 These include intestinal nonenzymatic lactonization due to the low pH, and enzymatic mechanisms catalyzing the formation of coenzyme A or acyl glucuronide intermediates, which are unstable and rapidly decay to the lactone form. Glucuronide conjugation of statins, mediated by uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) in the liver, appears to be a universal mechanism in multiple species.13 The human UGT1A1*28 and

INTRODUCTION Statins lower blood cholesterol levels by inhibition of the enzyme HMG CoA reductase and have proven to be very effective in reducing the risk of major cardiovascular events. Although the number of people using statins is not exactly known, they belong to the most commonly prescribed drugs.1 Moreover, recent adaptation of the guidelines on the treatment of blood cholesterol levels will probably lead to a further increase in their use, already accounting for more than 38 million users in the US and many times more worldwide.2,3 Although statins are generally well tolerated, myopathies are the most common adverse effects, ranging from myalgias to potentially life-threatening rhabdomyolysis.4 In contrast to the incidences found in initial clinical trials, the real burden is probably much larger with rates up to 26%, if less severe types of myopathies are included.5−7 The high morbidity rates associated with statin use will probably also lead to a decreased treatment adherence, putting more individuals at risk of cardiovascular disease. While the exact mechanism underlying statin-induced myopathies remains unknown, it has been observed that the © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4048

June 18, 2015 September 12, 2015 September 27, 2015 September 27, 2015 DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

Article

Molecular Pharmaceutics

units), respectively. Argon was used as collision gas at a pressure of 1.5 mTorr. The most abundant product ion was used for quantification, which was performed using peak areas. A second product ion was used for qualification purposes. See Table 1 for the optimal SRM transitions, polarity, and collision energies (CE).

UGT1A3*2 polymorphisms were associated with a relative decrease or increase in systemic atorvastatin lactone concentrations, respectively.9,14 Genetic differences in UGT-mediated formation of the lactone forms could therefore influence the individual safety and efficacy of statin therapy. Since a systematic comparison of the conversion of different statins by separate UGT subtypes is lacking, we aimed to study the kinetics of lactone formation of six clinically relevant statins by 13 human UGTs belonging to the major drug metabolizing subfamilies 1A and 2B.

Table 1. LC−MS/MS Conditions for Determination of Statin Acids and Lactones

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CEa (m/z) (eV)

EXPERIMENTAL SECTION Compounds and Supersomes. Atorvastatin acid and lactone, lovastatin acid and lactone, rosuvastatin lactone, and simvastatin lactone were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Cerivastatin acid and lactone, pitavastatin acid and lactone, rosuvastatin acid, and simvastatin acid were from Sequoia Research Products (Pangbourne, United Kingdom). Supersomes were derived from insect cells (BTI-TN-5B1-4) infected with baculovirus (Autographa californica)-expressing human UGT (wild type, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, UGT2B17) and were purchased from Corning (Amsterdam, The Netherlands). In Vitro Statin Metabolism. To determine statin lactone formation rates, supersomes of 13 human recombinant UGTs were incubated with various statins with minor modifications according to a previously described method.15 Briefly, the reaction mixture contained 1 mg/mL supersomes, 10 mM uridine diphosphate glucuronic acid (UDPGA, Sigma-Aldrich, Zwijdrecht, The Netherlands), 10 mM magnesium chloride, and 50 mM Tris (pH7.5). The reaction was started by the addition of a 100 times concentrated atorvastatin, cerivastatin, lovastatin, pitavastatin, rosuvatatin, or simvastatin acid stock in DMSO to the reaction mixture, at 37 °C for the indicated incubation times (see figure legends). The reaction was quenched adding two times the reaction volume of ice-cold acetonitrile, after which the samples were kept at ice. Samples were centrifuged for 3 min at 10000g, and the statin lactone concentration in the supernatant was determined using liquid chromatography−tandem mass spectrometry, as described below. Determination of Statin Concentrations Using Liquid Chromatography−Tandem Mass Spectrometry (LC− MS/MS). For liquid chromatography an Accela UPLC system (Thermo Scientific, Waltham, MA, USA) was used equipped with a HSS T3 analytical column (Acquity UPLC) coupled with a VanGuard HSS T3 precolumn (Acquity UPLC) at 30 °C, with a mobile phase solvent A (0.1% formic acid in water) and solvent B (0.1% (v/v) formic acid in acetonitrile) using the following gradient: 0 min 30% B, 5.5 min 85% B, 9 min 30% B, and a constant flow rate of 250 μL/min. Next, the effluent from the HPLC was passed into the electrospray ion source for tandem mass spectrometry using a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, Waltham, MA, USA). MS parameters using negative ion mode were optimized for achieving good sensitivity for simvastatin acid; positive ion mode was used for all other statins. Heated electrospray ionization (HESI) was operated at a spray voltage of 3.5 kV, a capillary temperature of 207 °C, and a vaporizer temperature of 382 °C. Nitrogen was used as sheath and auxiliary gas with a gas pressure of 20 and 15 AU (arbitrary

compd name atorvastatin acid atorvastatin lactone cerivastatin acid cerivastatin lactone lovastatin acid lovastatin lactone pitavastatin acid pitavastatin lactone rosuvastatin acid rosuvastatin lactone simvastatin acid simvastatin lactone a

precursor ion (m/z)

product 1

product 2

polarity (+/−)

559.1

440.2 (21)

292.1 (31)

+

541.2

448.1 (17)

276.0 (41)

+

460.1 442.1

356.1 (35) 354.1 (33)

280.0 (66) 292.1 (54)

+ +

445.1 405.1

343.1 (21) 143.0 (42)

383.1 (20) 128.0 (65)

+ +

422.1 404.1

274.0 (47) 272.0 (44)

290.0 (27) 260.0 (49)

+ +

482.1

258.0 (34)

270.0 (35)

+

464.1

270.0 (32)

282.0 (33)

+

435.2 419.2

319.1 (18) 199.0 (14)

115.0 (29) 142.9 (40)

− +

CE: collision energy.

Multi Sequence Alignment UGTs. The UGT fasta files were obtained from UniProt (www.uniprot.org16), i.e., UGT1A1 (entry: P22309), UGT1A3 (P35503), UGT1A4 (P22310), UGT1A6 (P19224), UGT1A7 (Q9HAW7), UGT1A8 (Q9HAW9), UGT1A9 (O60656), UGT1A10 (Q9HAW8), UGT2B4 (P06133), UGT2B7 (P16662), UGT2B10 (P36537), UGT2B15 (P54855), UGT2B17 (O75795), and a multi sequence alignment was performed using Clustal Omega (version 1.2.1).17,18 The multi sequence alignment file is included in Figure S1. Homology Modeling of UGTs. Homology models were built with the automated protocol of Yasara (version 13.9.8)19 using the GT1 family plant flavonoid glucosyltransferases (VvGT1, Vitis vinifera, pdb-code 2c1z) as template.20 Molecular Docking. The homology models were used for docking of the various statins transition states using MOE (version 2013.0802).21 Before docking, structure preparation was performed using the MOE protonate 3D protocol. Docking was performed using the induced-fit protocol. The transition states of the different statins were prepared in MOE by assigning PEOE charges and energy minimization. Statistical Analysis. Curve-fitting and statistical analysis was performed using GraphPad prism 5.02 software (GraphPad Software Inc., San Diego, CA). Lactonization rates due to sample handling and conditions were evaluated for all statins. Only for simvastatin was a correction needed, as for this statin considerable background levels were observed. Briefly, the fraction of the background simvastatin lactonization rate due to assay buffer addition was calculated as [(lactonization rate in assay buffer) − (lactonization rate in DMSO)]/(lactonization 4049

DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

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Molecular Pharmaceutics Table 2. Kinetic Parameters of Dose-Dependent Statin Lactone Formation by UGT1A1, 1A3, and 2B7a Vmax ± SE (pmol·s−1·(mg protein−1))

Km ± SE (μM) uncorrected UGT1A1 UGT1A3 UGT2B7

4±4 4±2 44 ± 35

UGT1A3

12 ± 2

UGT1A1 UGT1A3

77 ± 72 27 ± 21

UGT1A3 UGT2B7

11 ± 6 220 ± 130

UGT1A1 UGT1A3

18 ± 12 191 ± 90

Vo-corrected

uncorrected

Atorvastatin 2 ± 10 4±5 20 ± 8 Cerivastatin 12 ± 4 Lovastatin 220 ± 450 22 ± 11 Pitavastatin 10 ± 9 220 ± 30b Rosuvastatin 16 ± 7c 220 ± 40

Vo-corrected

4.8 ± 1.1 41 ± 5 10 ± 3

2±2 38 ± 10* 3.7 ± 0.5***,c

37 ± 2

36 ± 3***

26 ± 12 43 ± 13

14 ± 21 30 ± 6***

910 ± 130 13400 ± 5800

880 ± 200* 12900 ± 1300***

20 ± 12 102 ± 90

17 ± 2**,b 105 ± 15**

Km and Vmax were determined using the Michaelis−Menten curve fit based on the uncorrected lactonization rates (see Figure 2) as well as on the values corrected for the nonenzymatic statin lactone formation (Vo). One-sample t-test was used to examine whether Vo-corrected Vmax rates were significantly different from zero (*p < 0.05, **p < 0.01, ***p < 0.001). bKm or Vmax significantly different compared to UGT1A3 (p < 0.001). cKm or Vmax significantly different compared to UGT1A3 (p < 0.01). a

rate in assay buffer), which resulted in a value of 0.53. The background levels were calculated for each experiment by multiplication of the control (UGTCT) simvastatin lactonization rates by 0.53. Next, these background levels were subtracted from all samples. Steady-state rates were logtransformed to correct for the positively skewed distribution of the data. Concentration-dependent curves for all statins were corrected for nonenzymatic lactonization (Vo) as observed with the UGTCT samples (see Table 2 for kinetic parameters based on corrected and uncorrected values). Unless indicated otherwise, all results are presented as mean ± SEM, and differences between groups were tested using one-way ANOVA analysis with appropriate post hoc tests.

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RESULTS Time-Dependent Statin Lactonization. We measured time-dependent atorvastatin lactone formation as a representative example for the relationship between time and statin conversion rate (Figure 1). Lactonization rates were determined using UGT1A1 and UGT1A3 to account for the differences between slow and fast lactonization rates, as observed previously.14 Up to 1 h, for both isotypes a linear relationship was found between time and the amount of lactone formed. Based on these results an incubation time of 15 min was chosen for all statins in the kinetic experiments. Steady-State Statin Lactonization by UGT1A and 2B Subfamily Members. UGT-mediated lactonization rates of six statins were determined for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Moderate statin lactonization could be observed with UGT1A1, mediating the conversion of three statins, with comparable rates (0.8 ± 1.0 up to 2.6 ± 1.5 pmol·s−1·(mg protein−1)). Moderate, but statistically significant, atorvastatin lactonization could also be observed with UGT2B7 (0.3 ± 1.1 pmol·s −1 ·(mg protein−1)) (Figure 2A). The highest lactonization rates were observed with UGT2B7 for pitavastatin (163 ± 4 pmol·s−1·(mg protein−1)). Remarkably, simvastatin was not lactonized by any of the UGTs tested (Figure 2F). UGT1A3 appeared to be the

Figure 1. Time-dependent atorvastatin lactone formation by recombinant human UGT1A1 and 1A3. Time-dependent atorvastatin acid (10 μM) lactone formation was determined for recombinant human UGT1A1 (○), UGT1A3 (□), or control membrane fractions (UGTCT, ●) up to 60 min. Lines through the data points were fitted by linear regression analysis. Mean ± SEM; n = 3 independent experiments.

most important subtype in in vitro lactonization, resulting in statistically significant conversion of five statins. Only with simvastatin acid was no lactone formation found (Figure 2F). Large differences between the statins were observed for their lactonization rates by UGT1A3, ranging from 166 ± 3 to 3 ± 1 pmol·s−1·(mg protein−1), according to the following ranking: pitavastatin ≫ atorvastatin > cerivastatin > rosuvastatin > lovastatin (Figure 2). Kinetics of Statin Lactonization by UGT1A1, 1A3, and 2B7. Next, we selected the UGT subtypes that exhibited 4050

DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

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Molecular Pharmaceutics

Figure 2. Steady-state statin lactone formation by human UGT1A and 2B subfamily isotypes. Statin lactone formation was determined after 1 h of incubation with indicated recombinant human subfamily 1A and 2B UGTs or control membrane fractions (CT) in combination with (A) atorvastatin acid, (B) cerivastatin acid, (C) lovastatin acid, (D) pitavastatin acid, (E) rosuvastatin acid, or (F) simvastatin acid at equimolar concentration (10 μM). Statistical analysis: one-way ANOVA with Dunnett’s post hoc analysis was applied to compare values to control membrane preparations. *p < 0.05, ***p < 0.001. Mean ± SEM; n = 3−5 independent experiments.

difference in absolute isotype expression in the supersomes as the Vmax values for atorvastatin for 1A3 and 2B7 were comparable. As the Km values of 1A3 and 2B7 were 10 ± 9 vs 220 ± 30 pmol·s−1·(mg protein−1), respectively, the intrinsic clearances (Clint = Vmax/Km) of both isotypes were in the same range (80 ± 40 vs 60 ± 40 μL·s−1 ·(mg protein−1), respectively). Like for atorvastatin, no significant lactonization could be observed for lovastatin by UGT1A1. Sequence and Structure Analysis of UGT Isotypes To Explain the Interaction with Statins. To gain insight into the molecular interactions of statins with the different UGT isotypes, a sequence and structure analysis was performed using different in silico approaches. It is important to consider that the UGTs have two domains (Figure 4A), a highly conserved UDP-glucuronic acid binding (C-terminal) domain and a highly variable N-terminal domain, which determines the substrate specificity of the different UGT isotypes. Up to now only the C-terminal domain of UGT2B7 is available as X-ray structure.22 In order to compare the different substrate binding sites of UGT isotypes, a multi sequence alignment was performed. Next, homology models were built of all UGTs investigated, using the GT1 family plant flavonoid glucosyltransferases (VvGT120). These models were used to review the

significant lactonization rates to further determine the kinetics of the conversion (Figure 3). Concentration-dependent conversion rates could be described according to simple Michaelis−Menten kinetics, and the apparent affinity (Km) and maximum lactonization rate (Vmax) for the different statin− UGT subtype combinations were determined after correction for the nonenzymatic lactonization (Vo, Figure 3 and Table 2). For atorvastatin the highest maximum rate was obtained with UGT1A3 (38 ± 10 pmol·s−1·(mg protein−1)), which was considerably higher than the maximum rate obtained with UGT2B7 (3.7 ± 0.5 pmol·s−1·(mg protein−1)). Contradictory to the results obtained in the steady-state experiments, no significant atorvastatin lactonization could be observed by UGT1A1. No difference between the apparent affinities of atorvastatin for UGT1A3 and 2B7 was found. Comparable affinities and maximal lactonization rates could be observed for conversion of cerivastatin and lovastatin by UGT1A3. The maximum rate of rosuvastatin lactonization by UGT1A3 was 2fold higher, but with a lower affinity (Table 2). The highest UGT1A3 Vmax was found for pitavastatin (880 ± 200 pmol·s−1· (mg protein−1)). The Vmax for pitavastatin lactonization by UGT2B7 was substantially higher (12900 ± 1300 pmol·s−1· (mg protein−1)), which does not seem to be the result of a 4051

DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

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Figure 3. Concentration-dependent statin lactone formation by recombinant human UGT1A1, 1A3, and 2B7. Statin lactone formation was determined after 15 min incubations with a concentration range of (A) atorvastatin acid, (B) cerivastatin acid, (C) lovastatin acid, (D) pitavastatin acid, or (E) rosuvastatin acid in combination with recombinant human UGT1A1 (○), UGT1A3 (□), UGT2B7 (◇), or control membrane fractions (UGTCT, ●). Statistical analysis: Data points were fitted to simple Michaelis−Menten kinetics. See Table 2 for Km and Vmax values obtained from curve fitting. Mean ± SEM; n = 3 independent experiments.

poses of the statin intermediates investigated above (e.g., coupled to UDP-glucuronic acid). Both methods showed the conserved amino acids in the UDP-glucuronic acid binding site (Figure S1). In the substrate binding site, amino acids of helix 5 and 6 appear to be less conserved (Figure S1). These two helices and the short loop in between form the entrance of the substrate binding site. In the VvGT1 X-ray structure the atoms in this region have high b-factors, which is an indicator of enhanced flexibility23 leading to poor helix 6 formation. Due to the lack of a crystal structure of the N-terminal domain of human UGT and limited availability of such structures of the Cterminal domain, homology modeling of helix 5/6 is rather difficult and an interpretation of the models on a molecular level to detect amino acids that influence substrate recognition is hardly possible. However, this helix 5/6 and the loop in between are most likely involved in the substrate specificity of the binding site (Figure 4B). Additionally, docking the different statins into the substrate pocket of the different UGT homology models showed that all statins investigated could fit into this binding site (Figure 4B, UGT1A3 was selected as representative UGT). For the docking, the statins were covalently bound to the UDPglucuronic acid site to model the binding mode of the transition state. Based on the docking, the hydrophobic moiety of the

statins could make several interactions with helix 5, once more suggesting its importance.

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DISCUSSION Up to now statin lactonization has mainly been attributed to UGT1A1 and UGT1A3, as reported for atorvastatin, cerivastatin, simvastatin, and pitavastatin.13−15 Although we observed a more modest role for UGT1A1, we could confirm statin lactonization by UGT1A3 for most statins. Only for simvastatin did we not observe any UGT-mediated lactone formation, whereas previously lactonization by UGT1A1 and 1A3 was reported.13 However, in line with our results, low rates just above background were also observed for simvastatin lactonization in other studies.14 These ambiguous findings may be the result of differences in assay conditions for simvastatin lactonization. Clinically, simvastatin lactone formation seems less relevant, because it is administered as the lactone form, already resulting in relatively high plasma lactone levels.24,25 The observed patterns of steady-state lactonization with atorvastatin, cerivastatin, and pitavastatin are similar to previous findings, except for the absence of cerivastatin lactone formation by UGT1A1.13−15 Here, we found that the extensive lactonization of pitavastatin by UGT2B7 was not observed with any of the other statins. An explanation for this remarkable 4052

DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

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Molecular Pharmaceutics

Moreover, contribution to the total atorvastatin lactonization is expected to be limited, because of the much higher (∼200 times) rates observed with UGT1A3. The major role of UGT1A3 is further emphasized by the identical ranking of statin lactonization rates compared to previously reported lactone formation in human liver microsomes.14 Besides confirming the role of UGT1A1 and 1A3, our results also show that many UGTs (UGT1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B10, 2B15, and 2B17) are not involved in the lactonization of any of the six statins used. We are the first to report the UGT-mediated lactonization of lovastatin and rosuvastatin. In agreement with the other statins, UGT1A1, UGT1A3, and UGT2B7 are the main metabolizing enzymes. The lactonization rates of lovastatin and rosuvastatin are, however, an order of magnitude lower than found for the other statins. This could explain the lower plasma fraction of the lactone forms (∼10%) for both statins as compared to other statins, such as atorvastatin (∼40%).14,24,32−35 These lower plasma lactone levels are also in line with the low incidence of statin-induced myopathies observed with lovastatin in several large postmarketed studies.36,37 However, for rosuvastatin the incidence of muscle toxicities is similar to that for other statins, suggesting that other mechanisms also play a role in the etiology of these side effects.36−38 Using in silico multiple sequence alignments and homology models, we identified helices 5 and 6 of the UGT substrate binding pocket as a possible site for the interaction of the statins with the UGTs. The relevance of this site for glucuronidation kinetics of UGTs is emphasized by the observation that two single nucleotide polymorphisms (SNPs) in this region are associated with low in vivo statin lactonization.14,39 The first SNP, resulting in the replacement of a phenylalanine by an isoleucine residue at position 110, is characteristic for the UGT1A3*7 genotype, which was associated with low atorvastatin acid lactonization rates (Figure 4B, yellow residue).14 The second polymorphism, resulting in the replacement of a methionine by an isoleucine residue at position 114, is characteristic for the UGT1A3*11 genotype, which was associated with low intrinsic clearance rates of estrogen, a model substrate for drug glucuronidation conversions (Figure 4B, cyan residue).39 Although these polymorphisms confirm the relevance of this region, the constructed homology models can only propose that helix 5/6 and the loop in between play an important role, because of the limited availability of human UGT crystal structures, as discussed above. To gain more insight into the statin binding mode to UGTs, mutation studies combined with cocrystallization data could provide a final proof of the importance of this region. To conclude, we report a major role of UGT1A1, 1A3, and 2B7 in statin lactonization and identified a region of the UGT substrate binding site that probably is involved in the interaction with the statins. Further research on the helix 5 and 6 region of the UGT binding site indicated the presence of clinically relevant polymorphisms. Eventually, insight into interdrug and interindividual variation in lactonization could help to personalize statin treatment with improved efficacy and reduced side effects.

Figure 4. Schematic presentation of the UGT binding pocket based on homology modeling. (A) Overview of VVGT1 X-ray structure with Cterminal domain in green and N-terminal domain in pink. (B) Homology model of UGT1A3 (gray) with docking pose of atorvastatin intermediate (orange). Part of helix 5/6 and the loop between the two helices, which are part of the substrate binding site, are colored blue. SNP in position 110 (yellow) and 114 (cyan).

difference could be the unique cyclopropyl moiety of pitavastatin, which not only increased the inhibitory potency against HMC-CoA-reductase but also resulted in diversion from CYP3A4 metabolism.26−28 Another study indicated that this group possibly also influenced the bioavailability and hepatocellular uptake by the organic anion transporting polypeptide (OATP)1B1.29 Furthermore, replacement of the cyclopropyl moiety by an isopropyl side chain significantly decreased its HMG-CoA reductase inhibitory potency.30,31 Future studies are needed to indicate whether such a replacement of the cyclopropyl moiety also decreases the pitavastatin lactonization by UGT2B7. Moreover, none of the other UGT2B family members showed this activity toward pitavastatin. The region of helix 5−6 is more strongly conserved among the UGT2B than the UGT1A family members, however, some differences in the amino acid sequence can be observed. Amino acids of UGT2B7 that are different compared to other UGT2B family members might play a role in pitavastatin binding. Positions Q93, I94, and S117 could be involved, since they are not present in the other UGT2B family members (bold amino acids, Figure S1). Although the lactonization of atorvastatin by UGT2B7 is significant, it is considered minor compared to pitavastatin. 4053

DOI: 10.1021/acs.molpharmaceut.5b00474 Mol. Pharmaceutics 2015, 12, 4048−4055

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00474.

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Figure S1 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Radboud University Medical Center, Department of Pharmacology and Toxicology, Geert Grooteplein 21, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: frans.russel@ radboudumc.nl. Phone: +31 (0) 24 361 36 91. Fax: +31 (0) 24 361 42 14. Notes

The authors declare the following competing financial interest(s): J.A.M.S. holds a (partial) position at Khondrion, a Radboud University Medical Center spin-out company founded by J.A.M.S.

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ACKNOWLEDGMENTS This research was supported by a grant from The Netherlands Organization for Scientific Research NWO Centers for Systems Biology Research initiative (CSBR09/013 V). The authors would like to thank NBIC and DFG for funding. T.R. is recipient of a personal grant Ri 2087/1-1 from the DFG.

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ABBREVIATIONS USED ANOVA, analysis of variance; AU, arbitrary units; BD, Becton Dickinson; CE, collision energy; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; HESI, heated electrospray ionization; HMG-CoA reductase, 3-hydroxy-3-methyl-glutarylCoA reductase; Km, Michaelis constant; LC−MS/MS, liquid chromatography−tandem mass spectrometry; MOE, Molecular Operating Environment; MS, mass spectrometry; PDB, Protein Data Bank; SEM, standard error of the mean; SNP, single nucleotide polymorphism; SRM, selected reaction monitoring; UDPGA, uridine diphosphate glucuronic acid; UGT, uridine diphosphate (UDP)-glucuronosyltransferases; UniProt, Universal Protein Resource; Vmax, maximal velocity

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