Predicting Oxidation Sites with Order of Occurrence Among Multiple Sites for CYP4A-Mediated Reactions

Drug Metab. Pharmacokinet. 26 (4): 351363 (2011). Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Predicting Oxidation Sites with Order of Occurrence among Multiple Sites for CYP4A-mediated Reactions

Yoshiyuki YAMAURA1,2,KouichiYOSHINARI1 and Yasushi YAMAZOE1,* 1Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2DMPK Research Group, Discovery Technology Laboratories, Ono Pharmaceutical Co., Ltd., Osaka, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: To predict CYP4A-mediated reactions, we developed a two-dimensional template scoring system based on published data. The system predicts the order of occurrence among multiple oxidation sites, as well as the regioselectivity. The template has a linearly arranged honeycomb shape and an adjacent area. Molecules are overlaid on the template with the locations of the atoms restricted to the corners of hexagonal blocks. The overlaid conformers are then checked to determine whether they reside within the template area, and their position occupancy and position function scores are calculated. The position occupancy score is determined based on occupation of the respective positions on the template. The functional and steric properties are reﬂected in the position function score. The sum of these scores is compared among possible conformers, and the conformer with the highest total score is predicted to be preferentially metabolized. In the present study, prediction of sites of CYP4A-mediated oxidation and classiﬁcation into substrates and non-substrates were performed for collected compounds, and agreement between predicted and experimental data exceeded 95% for substrates and non-substrates. The template scoring system can be easily linked to databases of two-dimensional chemical structures, and thus this system may be useful for drug development and studies of drug metabolism.

Keywords: cytochrome P450; CYP4A11; fatty acid ½-hydroxylation; drug metabolism prediction; regioselectivity; template and scoring system; in silico method

sites of oxidation of a substrate is not well handled in most Introduction cases, and classification of molecules as substrates and non- Cytochrome P450 4A ¤CYP4A¥ enzymes selectively substrates is also difficult. These studies show that current catalyze Ý-hydroxylation of fatty acids such as lauric acid.1,2¥ approaches are not sufficiently developed for exact The selectivity of CYP4A for Ý-hydroxylation is particularly prediction of the regioselectivity of substrate biotransforma- striking since the electron density of the C-H bonds is much tion. This is partly because of the conformational flexibility lower at the Ý-terminal methyl group than the adjacent Ý%1 of CYP enzymes, including adaptive changes of the enzyme methylene group.3¥ CYP4A enzymes are also involved in the conformation and multiple binding sites. A clear advantage metabolism of physiologically important lipid components of studying human P450 enzymes compared with other such as eicosanoids, prostaglandins and arachidonic acid4®6¥ enzymes is the ample experimental data for specific and also of drugs such as febuxostat.7¥ Therefore, prediction recombinant forms. In addition, data consistency is now of CYP4A-mediated metabolism is important at the established between human-derived preparations and re- discovery stage of new drug development. combinant forms. These data may allow mapping of the Methods for prediction of CYP metabolism have used active sites of CYP isoforms from substrate structures only. ligand-property-based approaches such as quantitative Substrates are often oxidized at multiple sites at varying structure-activity relationships ¤QSAR¥ or X-ray crystallog- rates by a single CYP isoform. Thus, a substrate may raphy-based docking. However, the prediction of multiple interact with a CYP isoform in multiple conformations.

Received; January 19, 2011, Accepted; February 28, 2011 J-STAGE Advance Published Date: March 14, 2011, doi:10.2133/dmpk.DMPK-11-RG-004 *To whom correspondence should be addressed: Yasushi YAMAZOE, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel. +81-22-213-6827, Fax. +81-22-217-6826, E-mail: [email protected]

351 352 Yoshiyuki YAMAURA, et al.

Current docking models show the three-dimensional Table 1. List of compounds with published data for CYP4A interactions of a CYP isoform with a substrate in a certain activity conformation, but these data are not readily comparable for Compounds References multiple conformations of a substrate. In the present study, a Substrates Lauric acid ¤1,2,8¥ two-dimensional template and scoring system was developed ¤ ¥ to examine the regioselectivity of CYP4A-mediated reac- Caproic acid 1,2 ¤ ¥ tions based on published data. Undecanoic acid 1,2 Tridecanoic acid ¤1,2¥ Materials and Methods Myristic acid ¤1,2,8¥ Experimental data for CYP4A recombinant enzymes were Pentadecanoic acid ¤1,2¥ obtained from the literature. If the sites of oxidation of a Palmitoic acid ¤1,2,8¥ substrate were unknown, the data were excluded. Most data 3-Hydroxy palmitoic acid ¤9¥ were from assays using the human CYP4A11 recombinant Isolaurate ¤1,10¥ enzyme. Some data derived from rat CYP4A1 recombinant ¤ ¥ fi Isomyristate 1 and puri ed enzymes were also used to compensate for Arachidonic acid ¤4,11¥ information missing for the human enzyme. Data from Prostaglandin A1 ¤4¥ CYP4A1/NADPH-P450 reductase-fusion protein or liver Oleic acid ¤8¥ microsomes were used if only these were available for a ¤ ¥ particular substrate. According to the experimental data, the Linoleic acid 8 ¤ ¥ substrate concentration did not affect the priority of All-trans-retinoic acid 12 reactions for the substrate. Thus, substrate concentration 13-cis-Retinoic acid ¤13¥ was not considered in this study. Data for a total of 108 Phytanic acid ¤14¥ compounds ¤52 substrates, 45 non-substrates and 11 Z9¤10¥-EpSTA ¤15¥ ® ¥ inhibitors¥ were used.1,2,4,5,7 63 These compounds are listed Luciferin-ME ¤16¥ in Table 1. Luciferin-CEE ¤16¥ ¤ ISIS/Draw ver. 2.5 MDL Information Systems, San Luciferin-4A ¤17¥ Leandro, CA¥ was used to generate secondary structures of ¤ ¥ fi Luciferin-MultiCYP 17 the compounds and to examine the suitability of speci c Febuxostat ¤7¥ conformers as a substrate. Substrates with rigid structures U44069 ¤18¥ were used for generation of the initial template through U51605 ¤18¥ overlays. Typical substrates of CYP4A enzymes such as ¤ ¥ lauric acid have a flexible structure and are not easily used Leukotriene B4 15 ¤ ¥ for mapping of the CYP4A binding site. Arranging the 5,8,11-Eicosatrienoic acid 5 carbonyl group and possible sites of oxidation in substrates in Eicosapentaenoic acid ¤5¥ specific regions gave a template that could be used to Docosahexaenoic acid ¤5¥ approximate the allowable ligand shape and size. Scores Farnesol ¤19¥ were counted manually for all possible conformers. For 7-¤p-Tolyloxy¥heptanoic acid ¤10¥ verification of a compound as a CYP4A substrate, atoms of 7-¤m-Tolyloxy¥heptanoic acid ¤10¥ fi fi ¤ the substrate were rst tted to a corner of the template for m-¤n-Heptyloxy¥-benzoic acid ¤10¥ ¥ example, to the site of oxidation and then oriented so that p-¤n-Heptyloxy¥-benzoic acid ¤10¥ the atoms matched the corners of the hexagons in the 4$-Methoxy-4-biphenylcarboxylic acid ¤10¥ template while staying within the border. Inverted 11,11-Dimethyllauric acid ¤10¥ structures were also evaluated in this way. 11-Dodecenoic acid ¤20¥ A conformer was judged to be a candidate substrate if all ¤ ¥ parts of the structure remained within the border of the 11-Dodecynoic acid 20 ¤ ¥ template. Position occupancy and position function scores Methyl laurate 2 were calculated for different conformers to predict the order Lauryl alcohol ¤2¥ of reactions. Hydrogen atoms on substrates were not 2,2-Dimethyllauric acid ¤2¥ included. In compounds with rings other than six-membered trans-2-Dodecenoic acid ¤2¥ rings, each atom on the ring was associated with the nearest Dodecane ¤2¥ corner on the template. 10-Methoxydecanoic acid ¤2,21¥ ¤ ¥ Results 12-Chlorododecanoic acid 22 12-Bromododecanoic acid ¤22¥ General CYP4A-ligand interactions: Most CYP4A : substrates have a carboxyl group at the opposite end of the Continued on next page

Continued: Continued:

Compounds References Compounds References 12-Iodododecanoic acid ¤22¥ Endosulfan ¤57¥ 10-Methylthiodecanoic acid ¤21¥ 4-Ipomeanol ¤56¥ 8,9-Epoxyeicosatrienoic acid ¤23¥ m-¤n-Amyloxy¥hydrocinnamic acid ¤10¥ 11,12-Epoxyeicosatrienoic acid ¤23¥ p-¤n-Amyloxy¥hydrocinnamic acid ¤10¥ 14,15-Epoxyeicosatrienoic acid ¤23¥ 9-Phenylnonanoic acid ¤10,20¥ Lauramide diethanolamine ¤24,25¥ 8-Phenyloctanoic acid ¤10¥

Non-substrates Valsartan ¤26¥ Inhibitors 17-Octadecynoic acid ¤58¥ alpha-Tocopherol ¤27¥ HET0016 ¤59,60¥ gamma-Tocopherol ¤27¥ 1-Aminobenzotriazole ¤61¥ Beraprost ¤28¥ TS-011 ¤62¥ Estrone ¤29¥ Sesamin ¤63¥ DB289 ¤30¥ Naproxen ¤10¥ Selegiline ¤31¥ Ibuprofen ¤10¥ Diphenhydramine ¤32¥ Cimetidine ¤10¥ Clopidogrel ¤33¥ 10-Imidazolyldecanoic acid ¤10,20¥ SCH351125 ¤34¥ 11,12-Methanolauric acid ¤2¥ Vitamin K1 ¤35¥ N-Methylsulfonyl-15,15-dibromopentadec-14-enamide ¤54,55¥ ¤ ¥ Emetine 36 The template scoring system was developed using data for the 34 underlined Zotepine ¤37¥ compounds 9cUAB30 ¤38¥ ¤ ¥ Aristolochic Acid ¤39¥ molecule from the oxidation site Table 1 . CYP4A Astemizole ¤40¥ enzymes metabolize carboxylic acid esters such as methyl N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide ¤41¥ laurate and Luciferin-MultiCYP and alkylamides including lauramide diethanolamine;2,17,24,25¥ these enzymes are in- Cilostazol ¤42¥ ¤ ¥ ﬁ ¤ ¥ hibited by N-hydroxyformamidines HET0016 and TS-011 Ne racetam 43 and acylsulfonamide ¤N-methylsulfonyl-15,15-dibromopen- ¤ ¥ Melatonin 44 tadec-14-enamide¥.55,59,62¥ Thus, a charge-pair interaction Meperidine ¤45¥ between the carboxylate anion and a positively charged OSU6162 ¤46¥ group on the enzyme is unlikely to be required for ligand Quinacrine ¤47¥ recognition of CYP4A. Instead, dipole interactions and NE-100 ¤48¥ hydrogen bonding between sites on CYP4A ¤referred to as Arecoline ¤49¥ trigger sites¥ and a carbonyl group or homologous polar Indiplon ¤50¥ moiety of the substrate were assumed to be the initial Resveratrol ¤51¥ requirements for a molecule to be a substrate. CYP4A 2¥ CP-122,721 ¤52¥ enzymes also metabolize lauryl alcohol, and thus hydrogen ¤e.g. Imatinib ¤53¥ bond donors as well as hydrogen bond acceptors methyl ester¥ may be able to interact with the CYP4A trigger site. Stearic acid ¤8¥ ¤ ¥ Allowable ligand size: The template was generated 3-Hydroxystearic acid 9 through overlays of substrates with rigid structures. Move- ¤ ¥ Prostaglandin E1 4 ment of atoms was restricted and the atoms were assumed to 9-cis-Retinoic acid ¤13¥ sit only on the corners of the honeycomb structure. This Luciferin-BE ¤16¥ approach yielded a template with a linearly arranged Luciferin-H ¤17¥ honeycomb structure and an adjacent area ¤Fig. 1¥. The Luciferin-ME-EGE ¤16¥ rings in the template were deﬁned as Rings A to G. To Lauramide ¤2¥ prevent confusion with naming of template rings and 6-¤2-Propargyloxyphenyl¥-hexanoic acid ¤54,55¥ substrate/ligand rings, a capital letter is used for the ¤ ¥ N-Methanesulfonyl-6-¤2-proparglyoxyphenyl¥- template ring. Location numbers from 1 to 29 were ¤55¥ hexanamide assigned to the corner of each ring to identify modes of Continued on next column: interaction.

Fig. 1. Template for simulated interactions of CYP4A with substrates Cavities in CYP4A for ligand binding were determined using substrates with relatively rigid structures. Arrows indicate experimentally reported oxidation sites. Asterisks show potential sites of oxidation (interaction with heme-oxygen). The substrate boundary is shown as a green broken double line. The bold lines of the template indicate the region for substrate recognition. The position given the highest occupancy score of 5 is shown in red, and positions given a score of 3 are shown in blue. An unoccupied position (score of 0) is shown in gray. Other positions are given a score of 1 and are shown in black. The details are discussed in the text (Scoring system for the CYP4A template).

Prostaglandin A1 may arrange its carboxylic acid chain on Ring F and an adjacent area ¤Ring G¥ of the template ¤Fig. 2B¥. The limitation at position 26 of Ring D was consisted with luciferin-ME ¤Fig. 2C¥. CYP4A isoforms were assumed to recognize substrates at a trigger site present on Ring F, and to oxidize them at the bottom of Ring A or B ¤positions 1 to 3¥. For example, all- trans retinoic acid ¤Fig. 3A¥ and 13-cis retinoic acid ¤Fig. 3B¥ were placed within the template, whereas no conformations of 9-cis retinoic acid ¤Fig. 3D¥ could be fitted within the template, since the cyclohexenyl group extended beyond the boundary of the template. These results are consistent with experimental data12,13¥ and suggest the fi Fig. 2. Proposed modes of interaction with prostaglandin E1 validity of this template for an initial classi cation of (A), prostaglandin A1 (B) and luciferin-ME (C) Various con- compounds into substrates and non-substrates. formers were mapped on the template Oxidation site: CYP4A11 mediates selective 4- Placements fulfilling the occupancy requirements for triggering and hydroxylation of 13-cis retinoic acid ¤Fig. 3B¥. Thus, site of oxidation are shown. Arrows indicate experimentally reported position 1 on the template was assumed to be a site of sites of oxidation. A reaction not mediated by CYP4A11 is indicated by X. The substrate boundary is shown as a broken double line. oxidation. For all-trans retinoic acid, 18-hydroxylation is the primary reaction, followed by 4-hydroxylation and 5,6- epoxidation ¤Fig. 3A¥. Based on the simulated interactions CYP4A11 catalyzes Ý-hydroxylation of prostaglandin A1, of both compounds, positions 1, 2 and 3 were judged to be but not of its 11-hydroxyl derivative, prostaglandin E1.4¥ sites of oxidation, with position 3 preferred over positions 1 The hydroxyl group of prostaglandin E1 was located at and 2. position 26 on the template ¤Fig. 2A¥. These data suggest Overlays of caproic acid showed that position 2 becomes that CYP4A substrates cannot enter position 26 of Ring D. the only site of oxidation for Ý-hydroxylation under

result is consistent with experimental data showing that CYP4A11 oxidizes caproic acid selectively to the Ý-hydroxy metabolite.1¥ CYP4A11 also oxidizes Ý-regions of fatty acids with 11 to 20 carbon atoms. For example, CYP4A11 oxidizes lauric acid primarily at the terminal ¤Ý¥ site rather than the energetically favorable Ý%1 site. This phenomenon can be explained if the terminal region of lauric acid occupies positions 2, 7 and 8 of the template, and this supports the idea of positions 1 to 3 as sites of oxidation ¤Figs. 5A to 5D¥. The structures of m-¤n-heptyloxy¥benzoic acid and m-¤n- amyloxy¥hydrocinnamic acid closely resemble each other, except for the location of the phenylene unit. However, Fig. 3. Proposed modes of interaction with all-trans retinoic CYP4A1 mediates Ý-hydroxylation of the former, but not of acid (A), 13-cis retinoic acid (B and C) and 9-cis retinoic acid (D) the latter.10¥ m-¤n-Heptyloxy¥benzoic acid can be placed on Arrows indicate experimentally reported sites of oxidation. A ¤ ¥ reaction not mediated experimentally by CYP4A11 is indicated by the template at positions 2, 7 and 8 Fig. 6A . The terminal X. The substrate boundary is shown as a broken double line. residue of m-¤n-amyloxy¥hydrocinnamic acid can reach the site of oxidation through positions 9 and 29 to fulﬁll the need for Ring F triggering by the carboxylic acid moiety conditions without occupancy of positions 1 and 3 ¤Figs. 4A ¤Fig. 6B¥, but not through positions 7 and 8 ¤Fig. 6C¥. and 4B¥. Ý-Hydroxylation at position 2 may also occur with These results further support a regional requirement of occupancy of position 1 ¤Figs. 4C to 4F¥. Other placements positions 2, 7 and 8 for oxidation. ¤Figs. 4G to 4J¥ suggested Ý-hydroxylation at position 1. As The overlays of these substrates on the template suggest a shown in Figure 1, placement at positions 7 and 8 on the mutual occupancy of a region located from positions 7 to 13 template were suggested for CYP4A11 substrates. These of Rings B to D, which are shown in bold on the template results suggest that CYP4A11-mediated oxidation of caproic ¤Fig. 1¥. This region of CYP4A1 may hold alkyl chains acid occurs as shown in Figures 4A or 4B. The predicted through hydrophobic interactions.

Fig. 4. Proposed modes of interactions with caproic acid Possible modes of interactions are shown with the suggested site of the oxidation (indicated by an arrow). Estimated scores (see text) are also indicated. Circles with a dotted line (A to F, I and J) indicate scoring corrections (−2points)relatedtotriggersiteafﬁnity to compensate for a discrepancy between experimental results and predicted scores. The conformation in (K) may not produce a metabolite, but may be an inhibitory interaction.

Fig. 5. Proposed modes of interaction with lauric acid Possible modes of interaction are shown with the suggested site of oxidation(indicatedbyalargearrow).Estimatedscores(seetext)arealso indicated. Circles with a dotted line indicate scoring corrections (−2points)relatedtotriggersiteafﬁnity (E to M) or limited levels of thickness of position 4 (L and M). The conformation in (N) may not produce a metabolite, but may be an inhibitory interaction.

extents, 4-hydroxylation and 5,6-epoxidation.12¥ In contrast, this enzyme rather selectively catalyzes 4-hydroxylation for 13-cis retinoic acid.13¥ These differences in regioselectivity suggest the possibility of a shift of the trigger site to positions 21®22 for 13-cis retinoic acid ¤Fig. 3B¥. Placement of the carbonyl moiety of 13-cis retinoic acid at positions 23®22 allowed overlay of the trimethylcyclohexene moiety on Ring A ¤Fig. 3C¥. This interaction is likely to generate the 18-hydroxy derivative as well as the 4-hydroxy derivative, but CYP4A11 selectively mediates 4-hydroxylation of 13-cis retinoic acid. These results suggest reduced trigger capacity for interaction at positions 23®22. Similarly, overlays of Fig. 6. Proposed modes of interaction with m-(n-heptyloxy)- p-¤n-heptyloxy¥benzoic acid and prostaglandin A1 for Ý- benzoic acid (A), m-(n-amyloxy)hydrocinnamic acid (B and C) ® ® p n hydroxylation suggest that positions 22 21 and 22 23 accept and -( -heptyloxy)benzoic acid (D) ¤ ¥ Various conformers were mapped on the template. Placements trigger interactions Figs. 6D and 2B . CYP4A11 mediates fulﬁlling the occupancy requirements for triggering and site of Ý-oxidation and slight Ý%1 oxidation of caproic acid. The oxidation are shown. Arrows indicate experimentally reported predicted placement of caproic acid on the template is shown oxidation sites. A reaction not mediated by CYP4A11 is indicated in Figures 4A and 4B. These interactions suggest triggering by X. by the carbonyl group of caproic acid at positions 15 and 23 or 15 and 19. Thus, collectively the results suggest trigger Trigger site: The carboxyl group of CYP4A substrates roles for all positions on Ring F ¤positions 15, 19, 20, 21, 22 was arranged on Ring F on the template. All-trans retinoic and 23¥. acid can ﬁt on the template only in the conformation shown Scoring system for the CYP4A template: The in Figure 3A. Thus, positions 19®20 were judged to be a template with Rings A to G was generated using typical trigger site for interaction with the carbonyl carbon-oxygen. substrates for CYP4A11 and CYP4A1. Simulated inter- CYP4A11 mainly mediates 18-hydroxylation and, to lesser actions suggested a contribution of multiple conformers of

CYP4A substrates to metabolite formation, but did not Table 2. Summary of position and function points in the address the priority of these conformers. To predict the CYP4A scoring system preference of interactions among these conformers, a scoring Classification Position Points system was devised. In this system, the highest score among 10 the possible interactions is taken to indicate the primary 2 %2 Compound occupies positions 1 and/or 3 metabolite. The total score is obtained as the sum of the Oxidation sites 3 %1 position occupancy score and the position function score. Position occupancy reflects the distribution of atoms at the 2 0 Compound does not occupy positions 1 and 3 corners of the template. Position function is used to 15®23 %2 compensate for the limitations of a two-dimensional model 23®22 %2 by introducing physicochemical information for residues at ® % fi Trigger sites 21 20 2 Carbonyl carbon-oxygen speci c positions. Both scores have distinct values that 15®19 %2 depend on the contribution of the simulated interaction. 22®23 %2 Both caproic acid ¤C10¥ and lauric acid ¤C12¥ are Accept only a flat structure coplanar to the preferentially oxidized regioselectively at the Ý-carbon and 2 to lesser extents at the Ý%1 position.1¥ To reproduce these alkyl backbone % experimental results, positions 7, 8, 9 and 10 were scored as 4 2 Flexible residue ¤e.g. terminal alkyl chain¥ at 3 points and position 2 as 5 points to reflect the hydrophobic 5 %2 this position interaction with the terminal carbon. Other positions Steric properties thought to interact with ligands were scored as 1 point. 15 Cannot accept bulky residues The position occupancy points are shown using coloring of 26 Cannot accept any compound the position number in Figure 1 ¤5 in red, 3 in blue, 1 in ¥ Ring A black and 0 in gray . The position function points are Aromatic ring is not oxidized described below and summarized in Table 2. Ring B 18-Hydroxylation is the preferential reaction for all-trans 1 %2 retinoic acid, followed by 4-hydroxylation and 5,6- 2 %2 Halogen atom in this position fl % % % epoxidation. To re ect this regioselectivity, 2 and 1 Chemical 3 2 points were assigned to positions 2 and 3, respectively, when properties a molecule occupies position 1 and/or 3. 29 %2 Iodine atom in this position fi Af nities of the trigger interaction may also vary among 2 ¦2 Sulfur atom in this position the positions. Thus, to improve the fit between experimental and simulated results, positions 23®22, 15®19, 15®23, 21® 20 and 22®23 were assigned %2 points ¤see Affinity of trigger site and regioselectivity of saturated fatty acids¥. 8B¥. Ring E was thus assumed to have a certain level of Steric properties at specific positions on the depth. CYP4A enzymes also catalyze Ý-hydroxylation of template: To compensate for the relatively limited 11,11-dimethyllaurate10¥ ¤Fig. 8C¥. This is consistent with chemical information in a two-dimensional model, position the idea that positions 6 and 7 of Ring A have space to function points were introduced to reflect steric and accept bulky residues. In contrast, CYP4A only slightly chemical properties. Positions 4 and 5 of Ring A are metabolizes 2,2-dimethyllaurate2¥ ¤Fig. 8D¥, suggesting that expected to accept a cyclohexene ring based on the position 15 is likely to accept planar groups only. simulated interaction of all-trans retinoic acid ¤Fig. 3A¥. Affinity of trigger site and regioselectivity of CYP4A11 metabolizes luciferin-ME, while this enzyme only saturated fatty acids: Fourteen simulated interactions slightly oxidizes luciferin-CEE16¥ ¤Fig. 7¥. Positions 4 and 5 that gave the highest score among all possible conformers of of Ring A were thus assumed to have limited depth and lauric acid are shown with their scores on the template in flexibility for accepting flexible residues such as a chloroethyl Figure 5. These interactions for Ý-hydroxylation ¤Figs. 5A group. Similarly, the terminal alkyl chain of pentadecanoic to 5I¥ and ¤Ý%1¥-hydroxylation ¤Figs. 5J to 5M¥ gave the acid and palmitoic acid ¤C15 and C16 saturated straight- highest scores of 24. Experimental data suggest preferential chain fatty acids¥ were not expected to fit into positions 4 Ý-hydroxylation of this fatty acid.1¥ To explain the differ- and 5 of Ring A. Thus, overlay of a terminal alkyl chain at ence, trigger interactions of the carbonyl group at positions positions 4 and 5 of the template was scored as %2 points. 15®19 and 15®23 ¤Figs. 5E, 5J, 5K, 5L and 5M¥ were This scoring system reflects the experimental preferences for assumed to have lower affinities compared with those at oxidation sites of fatty acids ¤see Affinity of trigger site and positions 19®20, 20®21 and 22®21 ¤Figs. 5A to 5D¥.As regioselectivity of fatty acids¥. shown for 13-cis retinoic acid, trigger interactions at CYP4A11 oxidizes the prostaglandin analogs U44069 and positions 23®22 ¤Figs. 5F and 5G¥ also had lower affinities. U51605 to their Ý-hydroxy metabolites18¥ ¤Figs. 8A and Thus, %2 points were assigned to trigger interactions at

Hydroxylation

Fig. 7. Proposed modes of interaction with luciferin derivatives Arrows indicate experimentally reported sites of oxidation. A reaction not mediated by CYP4A11 is indicated by X. The substrate boundary is shown as a broken double line.

modes of interaction for ¤Ý%1¥-hydroxylation ¤each 22 points, Figs. 5B, 5J and 5K¥. Caproic acid has two modes of interaction for Ý-hydroxylation ¤each 20 points¥ and two modes of interaction for ¤Ý%1¥-hydroxylation ¤each 16 points¥¤Fig. 4¥. Kawashima et al. 1¥ reported regioselectivities of saturated straight-chain fatty acids of various lengths ¤C10 to C16¥ using recombinant human CYP4A11 enzyme. Caproic acid, undecanoic acid and lauric acid ¤C10 to C12¥ are metabolized selectively by CYP4A11 at the Ý-carbon Fig. 8. Proposed modes of interactions with U44069 (A), U51605 ¤Ý/¤Ý%1¥ ratios of 15 to 17¥, whereas tridecanoic acid, (B), 11,11-dimethyllaurate (C) and 2,2-dimethyllaurate (D) myristic acid, pentadecanoic acid and palmitoic acid ¤C13 to Various conformers were mapped on the template. Placements C16¥ have reduced selectivity ¤Ý/¤Ý%1¥ ratios of 2 to 3¥. fulﬁlling the occupancy requirements for triggering and site of % oxidation are shown. Arrows indicate experimentally reported sites Simulated interactions with fatty acids suggested 2 points of oxidation. A reaction not mediated by CYP4A11 is indicated by X. for the position function for trigger interactions at positions 21®20 and 22®23. To verify this choice, simulated results obtained using the scoring system were compared with positions 15®19, 15®23 and 23®22. As described above, the experimental data ¤Table 3¥. terminal alkyl chain of lauric acid is not thought to enter Caproic acid, undecanoic acid and lauric acid exhibited positions 4 and 5 of the template. Thus, interactions shown higher scores for Ý-hydroxylation ¤scores of 20, 23 and 24, in Figures 5L and 5M were given an additional %2 points respectively¥ than for ¤Ý%1¥-hydroxylation ¤scores of 16, 19 ¤see Steric properties at speciﬁc positions on the template¥. Other and 22, respectively¥. These results are consistent with trigger interactions shown in Figures 5H and 5I ¤positions experimental data for CYP4A11. Tridecanoic acid, myristic 23®22¥ were given %2 points. Thus, lauric acid may have acid, pentadecanoic acid and palmitoic acid, which showed three distinct modes of interactions for Ý-hydroxylation reduced selectivities, had equivalent highest scores for ¤each scoring 24 points, Figs. 5A, 5C and 5D¥ and three Ý- and ¤Ý®1¥-hydroxylation.

Table 3. Comparison of experimental data with scores ob- CYP4A1 oxidizes 12-chloro-, 12-bromo- and 12-iodo- tained from the template dodecanoic acids to 12-hydroxydodecanoic acid ¤Ý-oxida- ¥ ª¤Ý% ¥ « 22¥ Simulated results Experimental tion and 12-oxododecanoic acid 1 -oxidation . The ÝÝ%1 results alcohol is hypothesized to arise from oxidation of the halide Compound Number of Number of Ý/¤Ý%1¥ to an oxohalonium species that is hydrolyzed by water, Score Score interactions interactions ratio whereas the aldehyde arises by a conventional carbon 22¥ Caproic acid ¤C10¥ 20 2 16 hydroxylation-elimination mechanism. 12-Chloro- and 12- bromododecanoic acids are oxidized preferentially at the 16 0 16 2 Ý%1 position over the Ý position ¤oxidation of halogen¥.To Undecanoic acid 23 1 % ¤C11¥ account for these phenomena, 2 points were assigned to a 21 1 halogen atom located at the oxidation site ¤positions 1 to 3 of 20 1 the template, Figs. 9A to 9F¥. For 12-iodododecanoic 19 0 19 2 acid, the Ý- and ¤Ý%1¥-oxidations occur to similar extents, Lauric acid ¤C12¥ 24 3 11®19 suggesting that iodine is too large to enter position 29 on 23 1 the template. Thus, %2 points were also given for this 22 3 22 3 interaction ¤Fig. 9I¥. 21 2 21 0 CYP4A1 oxidizes 10-methylthiodecanoic acid primarily at Ý% Tridecanoic acid the 1 site, whereas 10-methoxydecanoic acid undergoes 25 4 25 1 3 ¥ ¤C13¥ Ý-oxidation.21 These results are explained by the greater 24 3 24 0 ¤ !¥ ¤ !¥ 23 2 23 3 length of the C-S bond 1.81 relative to the C-C 1.54 or C-O ¤1.41 !¥ bond and by the greater atomic radius and 22 0 22 1 polarizability of the S atom vs. OorCH, which places the Myristic acid ¤C14¥ ® 2 26 7 26 3 2 2.5 reactive lone pair of electrons on the S atom close enough to 25 4 25 0 the oxoiron ferryl group that they become the energetically 24 3 24 4 favored target. Thus, ¦2 points were given for a sulfur atom 23 2 23 3 at position 2. Pentadecanoic acid 27 5 27 4 2 Steric conﬁguration of Ring A or Ring B relative ¤C15¥ 26 3 26 0 to the heme plane: CYP4A1 Ý-hydroxylates both 7- 25 1 25 9 ¤p-tolyloxy¥heptanoic acid and 7-¤m-tolyloxy¥heptanoic acid 24 1 24 1 at their benzylic methyl group, but not at their aromatic 10¥ Palmitoic acid 28 4 28 3 3 ring. CYP4A1 does not oxidize 8-phenyloctanoic acid or ¤C16¥ 27 3 27 0 9-phenylnonanoic acid, but these molecules strongly inhibit 10¥ 26 9 26 4 lauric acid oxidation. The latter observation suggests that these compounds enter the active site of CYP4A1 and 25 4 25 6 interfere with lauric acidös access to the active site. In these interactions, the phenyl ring may be lying in a plane that is

Fig. 9. Proposed modes of interaction with 12-chlorododecanoic acid (A–C), 12-bromododecanoic acid (D–F), and 12-iodododecanoic acid (G–I) Various conformers were mapped on the template. Placements fulﬁlling the occupancy requirements for triggering and site of oxidation are shown. Modes of interactions are shown with scores. Arrows indicate the expected sites of oxidation. Circles with a dotted line indicate corrections (−2 points) related to the size limitation of position 29 (I).

Fig. 11. Proposed modes of interaction with febuxostat (A) and 4′-methoxy-4-biphenylcarboxylic acid (B) Arrows indicate experimentally reported sites of oxidation. A reaction not mediated by CYP4A11 is indicated by an X.

Table 4. Prediction accuracy for 23 substrates and 40 non- substrates

Predicted result Correctly predicted Experimental result ¤ ¥ Substrate Non-substrate % Substrate 22 1 95.7 Non-substrate 1 39 97.5

boundary at Ring G. Luciferin-H was too short to reach Fig. 10. Proposed modes of interaction with 7-(p-tolyloxy)hep- m the oxidation site of the template. tanoic acid (A), 7-( -tolyloxy)heptanoic acid (B), 9-phenylnona- fi noic acid (C) and 8-phenylnonanoic acid (D) Veri cation of the template scoring system: The Arrows indicate experimentally reported sites of oxidation. A template scoring system was developed using data for the 34 reaction not mediated by CYP4A11 is indicated by an X. compounds shown underlined in Table 1 ¤29 substrates and 5 non-substrates¥. All the predicted results for these compounds were consistent with experimental results. Data perpendicular to the heme plane. Thus, the C-H bonds on for 23 substrates and 40 non-substrates were not used for the edge of the phenyl ring, rather than the p-orbitals of the template construction, and these were applied to verify the aromatic system, are oriented toward the heme ferryl accuracy of the template. As shown in Table 4, the group. In this arrangement, the ferryl group is not able to template approach predicted the site of oxidation in 22 of the attack the Ô-electron system, which is the typical pathway 23 substrates found experimentally to undergo CYP4A- leading to aromatic hydroxylation by CYP enzymes.64,65¥ mediated oxidation, while 39 of the 40 non-substrates could Aromatic C-H bonds are very much stronger than aliphatic not be fitted to the template and were predicted to be C-H bonds, and therefore the aromatic ring is not a non-substrates. Thus, agreement between predicted and substrate for CYP4A1. These ideas are consistent with the experimental data exceeded 95% for substrates and non- steric configuration of oxidation sites for CYP4A1 proposed substrates. by Bambal and Hanzlik.10¥ Possible inhibitory interactions: Possible interac- As shown in Figures 10C and 10D, overlays of 8- tions of inhibitors of CYP4A-mediated oxidation are phenyloctanoic acid and 9-phenylnonanoic acid on the shown in Figure 12. Naproxen ¤Fig. 12A¥ and ibuprofen template allowed the phenyl rings to sit on Ring A or ¤Fig. 12B¥, which inhibit lauric acid hydroxylation,10¥ have a Ring B. Therefore, Rings A and B were assumed to be carboxylic acid residue that is recognized at the trigger located in a plane perpendicular to the heme plane. site. Thus, these structures bind to CYP4A1, but are Application of the template to compounds with too short to reach the oxidation site of the template. condensed rings or biphenyl groups: CYP4A iso- HET0016 ¤Fig. 12C¥ and TS-011 ¤Fig. 12D¥, which are forms also oxidize condensed-ring or biphenyl compounds potent CYP4A inhibitors,59,62¥ were also too short to reach such as luciferin derivatives, febuxostat and 4$-methoxy- the oxidation site. These two compounds have N-hydroxy- 4-biphenylcarboxylic acid.7,10,16,17¥ Simulated interactions of formamidine groups, and this moiety may interact at the these compounds are shown in Figures 7 and 11. These trigger site with an affinity greater than those of carboxyl substrates could not be overlaid perfectly on the template, and carbonyl groups. 11,12-Methanolauric acid, a cyclo- but did not exceed the allowable size or location. Parts of propane fatty acid, inhibits lauric acid hydroxylation.2¥ the structures sat over the central portion of Ring D, but This compound could fit onto the template, but with the not position 26, where the substrate is not accepted. cyclopropyl moiety at position 6 ¤Fig. 12F¥, rather than Luciferin-BE is unlikely to interact with CYP4A because the position 2 ¤Fig. 12E¥. Only a flat structure coplanar with benzyl group crossed the boundary of the template. The the alkyl backbone is allowed to enter close to the heme in ethylene glycol of luciferin-ME-EGE also crossed the the area of Ring A.

Fig. 12. Proposed modes of interactions with naproxen (A), ibuprofen (B), HET0016 (C), TS-011 (D) and 11,12-methanolauric acid (E and F)

oxidation sites or in the regioselectivity of substrates. Discussion Moreover, the template scoring system predicted exper- In this study, we developed a two-dimensional template imentally reported sites for CYP4A11-mediated oxidation to identify a ligand as a CYP4A substrate. Distinct roles of for almost all substrates. Thus, it appears that the active sites CYP4A regions were assumed for triggering, interaction and of CYP4A11 and CYP4A1 resemble each other in terms of oxidation based on different positions on the template. Our the properties considered in development of the template. results suggest a primary role for a trigger site to promote a Two molecules showed inconsistency between the CYP4A-mediated reaction. Thus, CYP4A substrates may predicted and experimental results. Lauramide should change conformation to interact with the CYP4A active site recognize the trigger site of CYP4A because it can both after triggering. Alternatively, CYP4A isoforms may anchor donate and accept hydrogen bonds. However, it is not a substrates through an initial interaction around Ring F, with substrate for CYP4A and does not inhibit hydroxylation of a subsequent change in enzyme conformation to open up lauric acid.2¥ Second, dodecane does not have polar moieties other parts of the active site. In the template, substrates are to interact with the trigger site, but CYP4A slowly oxidizes anchored through interactions at positions 7 to 13. this compound to lauryl alcohol.2¥ The reasons for these Oxidation is accomplished if the interaction allows place- discrepancies are unclear, but they suggest that more work is ment of an atom at the oxidized site ¤positions 1 to 3¥. needed to improve the template and scoring system. Within The template also allows prediction of the priority of this limitation, the template developed in the study is a regioselective reactions based on a scoring system. The total versatile tool for studying CYP-mediated metabolism. This score is the sum of the position occupancy score and the approach is likely to be useful for drug design and position function score on the template. The highest total development, since the template can be easily applied to score was used to define the primary reaction. Several databases of secondary structures. substrates are oxidized at multiple sites by CYP4A, and the predicted orders of reactions were largely consistent with Acknowledgments: We would like to thank Yoshikazu experimental data for CYP4A-mediated reactions, including Takaoka, Soonih Kim and Koji Yano for their support of this for flexible substrates such as saturated straight-chain fatty study, and Hiroshi Ochiai and Motohiko Morihara for acids. Experimentally reported sites of CYP4A-mediated helpful discussion and comments. oxidation were predicted for 51 of 52 substrates. Previous studies of fatty acid derivatives have suggested that activity References requires a certain distance between the carboxylic acid and 1¥ Kawashima, H., Kusunose, E., Kikuta, Y., Kinoshita, H., Tanaka, terminal moiety. In the template, the properties of these fi fi S., Yamamoto, S., Kishimoto, T. and Kusunose, M.: Puri cation regions were de ned as stereochemical and positional and cDNA cloning of human liver CYP4A fatty acid omega- requirements. Our results suggest that an intermediate area hydroxylase. J. Biochem., 116:74®80 ¤1994¥. ¤Rings B, C and D on the template¥ is also essential for the 2¥ Alterman, M. A., Chaurasia, C. S., Lu, P., Hardwick, J. P. and CYP4A-substrate interaction. Hanzlik, R. P.: Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH- In this study, we used data that were mostly obtained with P450 reductase fusion protein. Arch. Biochem. Biophys., 320: 289® human CYP4A11 recombinant enzyme, but some data from ¤ ¥ ¥ 296 1995 . rat CYP4A1 were also included. Dierks et al.61 found 3¥ de Visser, S. P., Kumar, D., Cohen, S., Shacham, R. and Shaik, S.: differences in the susceptibility of these two CYP4A enzymes A predictive pattern of computed barriers for C-h hydroxylation by ® to inhibition by substituted imidazoles. These results suggest compound I of cytochrome p450. J. Am. Chem. Soc., 126: 8362 8363 ¤2004¥. that the human CYP4A11 oxidation site is somewhat 4¥ Palmer, C. N., Richardson, T. H., Griffin, K. J., Hsu, M. H., different from that of rat CYP4A1. However, we did not Muerhoff, A. S., Clark, J. E. and Johnson, E. F.: Characterization find a marked difference in the order of reaction at multiple of a cDNA encoding a human kidney, cytochrome P-450 4A fatty

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