A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules Justyna K. Kulig a, Claudia Spandolf b, Ralph Hyde b, Antonio C. Ruzzini c, Lindsay D. Eltis c, Gunnar Grönberg d, Martin A. Hayes a, Gideon Grogan b,⇑ a

CVMD iMed DMPK, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden York Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD York, United Kingdom Department of Microbiology and Immunology, 2350 Health Sciences Mall, Life Sciences Centre, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada d RIA iMed MedChem, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden b c

a r t i c l e

i n f o

Article history: Received 23 April 2015 Revised 10 July 2015 Accepted 15 July 2015 Available online xxxx Keywords: P450 Biotransformation P450Rhf Imipramine N-demethylation Drug metabolism Diltiazem

a b s t r a c t The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme’s activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 lM) giving a 63% conversion to the N-desmethyl product. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Cytochromes P450 (P450s) are heme-thiolate proteins that are widely distributed in all forms of life, from viruses and prokaryotes to lower eukaryotes such as fungi and higher eukaryotes including humans.1 They catalyze numerous oxidative processes, including hydroxylation, epoxidation, dealkylation, sulfoxidation, deamination, desulfurization and dehalogenation in the metabolism of aliphatic, alicyclic and aromatic molecules.2 They are especially promising candidates for preparative biocatalysis, given that these systems catalyze such a vast and interesting set of reactions, and that their activity can now be modulated using a variety of structure-guided and random mutagenesis techniques.3–6 Obstacles to the application of P450s in biotechnology include poor stability, low turnover rates, and also the organisational complexity of the cofactors and auxiliary proteins required to constitute an active catalyst. P450s catalyze oxygenation reactions through the formation of ‘compound I’; an oxo-ferryl (FeIV) species formed at the ⇑ Corresponding author. Tel.: +44 1904 328256; fax: +44 1904 328266. E-mail address: [email protected] (G. Grogan).

heme iron through two single electron reduction steps and reaction with molecular oxygen.7 Electrons are delivered to the heme iron from the nicotinamide cofactor NAD(P)H via auxiliary redox transport proteins which, in most cases, are polypeptides separate from the heme-containing domain, and P450s can be classified according to the redox transfer protein systems on which they are dependent.8 Given that the expression and engineering of these multi-component systems presents complications with respect both to expression and electron transfer in the reconstituted system, much research has focused on the application of singlepolypeptide P450s, typified by ‘Class VIII’8 proteins such as the fatty acid hydroxylase P450BM3 (CYP102A1)9 from Bacillus megaterium and the ‘Class VII’8 enzymes represented by P450Rhf from Rhodococcus sp. NCIMB 978410,11 In each of these cases, the requisite electron transfer proteins (P450 reductases) form part of a single polypeptide with the heme domain that is primarily responsible for governing the substrate specificity of the enzyme(s). The existence of these naturally occurring ‘P450 fusions’ prompted investigations into the promiscuity of the P450 reductase domains in which the native heme domain of the P450 in question is swapped for an alternate one, with different

http://dx.doi.org/10.1016/j.bmc.2015.07.025 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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Figure 1. Oxidative activities attributed to cytochromes P450 from Rhodococcus jostii RHA1 in the literature. CYP125 catalyzes the oxidation of sterols such as cholesterol 1 at the C26 position.24 CYP257A1 catalyzes the N-demethylation of the alkaloid dextromethorphan 3.25

specificity, from another source. There are now many examples of P450 fusions therefore, in which the reductase system of P450Rhf, RhfRED, which is similar to that of a phthalate dioxygenase reductase, has been fused to heme domains from other organisms that are specific for the hydroxylation of terpenes,12–14 steroids,15 alkanes,16 aromatics17 and macrolides such as pikromycin18 and mycinamicins.19 In each of these cases the result has been a ‘redox-selfsufficient’ P450 fusion with which substrate turnover can be achieved through the addition of NAD(P)H to a mixture containing a single protein. The application of the fusion technology was facilitated by our development and application of a ligation-independent cloning vector (‘LICRED’),15 which permitted generic cloning protocols to be applied to the construction of larger libraries of diverse prokaryotic P450 fusions. Using this vector a P450 fusion library representative of the ‘CYPome’ of the actinomycete Nocardia farcinica was created, enabling new P450 catalysts for hydroxylation and dealkylation reactions.15 Bacterial species of the actinomycetes genus Rhodococcus have been shown to display a remarkable range of catabolic activity against natural and xenobiotic hydrophobic compounds.20 This catabolic versatility was attributed to the large number of genes encoding oxygenase-like proteins within its genome, first presented by Eltis in 2006.21 The catalytic diversity extant amongst these oxygenases was illustrated by studies of over twenty flavin-dependent Baeyer–Villiger monooxygenases22,23 from the organism. In addition to flavin-dependent oxygenases, open reading frames encoding at least twenty-five P450 heme domains could be identified within the genome. Included within the ‘CYPome’ of RHA1 are homologs of CYP125, which catalyzes the hydroxylation and possibly further oxidation of the C26 atom of sterols (1, Fig. 1)24 and CYP257A1, also thought to be involved in sterol metabolism, and which has been shown to possess N-demethylation activity towards the alkaloid substrate dextromethorphan (3, Fig. 1).25 The promising diversity and activity of R. jostii P450s prompted us to apply the LICRED technology to the creation of a P450RHA1RhfRED fusion library. The screening of these constructs against a large library of commercial drugs has allowed the identification of useful activities that might be of interest for the production of drug metabolites. 2. Results and discussion 2.1. Construction of a library of P450RHA1-RhfRED fusion proteins A survey of the genome of R. jostii RHA1 suggested the presence of at least twenty-five open reading frames encoding putative

cytochrome P450 heme domains. A list of the gene targets can be found in Table S1 and an alignment of the sequences is shown in Figure S1. A phylogenetic tree of the 25 targets, in which a representative number of cytochrome P450 heme domains with known structures and activities has been included, is also presented in Figure S2. R. jostii features, for example, one homolog of CYP51B (ro04671), which has been assigned the role of a sterol demethylase in strains of Aspergillus; four homologs of CYP125 (ro04679, ro04667, ro02651 and ro02355), including one that has been shown to initiate side-chain degradation in steroids through C26 hydroxylation,24 and one of CYP257A1 (ro11069), which has been shown to catalyze the N-demethylation of the alkaloid dextromethorphan.25 Genomic DNA from R. jostii RHA1 was used as a template for the amplification of twenty-three of the target genes by PCR. Two of the targets (ro05210 and ro03076) were not amplified successfully. Primers (Table S2) were designed such that the PCR products would be suitable for ligation independent cloning within the LICRED vector, equipping each with the RhfRED reductase fused at the C-terminus of each heme domain through a short linker.15 Expression conditions were tested in respect of Escherichia coli cell strain, growth medium and expression temperature for selected targets and analyzed using SDS–PAGE and Western Blots against an anti-His antibody sensitive for the N-terminal histidine tag on the gene fusion products. The heme precursor amino levulinic acid (ALA) and iron(III) chloride were added along with IPTG at induction, as detailed in the Experimental Section. Low-temperature gene expression (16 °C) in E. coli Rosetta (DE3), grown in M9 minimal medium proved to be the most efficient option overall in delivering soluble gene expression for the most targets. Significant signals for soluble expression of fusion proteins (named Ro(01-23)-RhfRED) of the predicted approximate molecular weight (between 80 and 90 kDa) were detected in Western Blot analysis for fusions of RhfRED with Ro01 (ro02510), Ro02 (ro02948), Ro04 (ro02604), Ro05 (ro04667), Ro06 (ro02651), Ro07 (ro11069), Ro08 (ro11320), Ro09 (ro04588), Ro11 (ro11277), Ro12 (ro00377), Ro16 (ro02355), Ro18 (ro08984), Ro20 (ro03876), Ro21 (ro03826) and Ro22 (ro 04671; Fig. 2). For Ro05-RhfRED, Ro08-RhfRED, Ro11-RhfRED, Ro12-RhfRED, Ro18-RhfRED and Ro22-RhfRED there was some evidence of cleavage at the heme domain-reductase linkage, illustrated by the blot response at approximately 45 and 55 kDa, the size of the heme domain, in these cases. Ro07-RhfRED appeared to be one of the more stable fusions, with less signal in the heme domain region of the blot. A sub-library of recombinant cell strains expressing seven of the soluble fusion proteins was then evaluated for oxidative activity as whole-cell biocatalysts in the biotransformation of drugs. 2.2. RhfRED fusion library Whole-cell preparations of resting recombinant strains expressing seven of the P450 fusion products in 50 mM Tris/HCl buffer at pH 7.5 were challenged with forty-eight commercially available drug compounds, including alkaloids (e.g., codeine, dextromethorphan), steroids (e.g., ethinylestradiol), anti-inflammatory(e.g., amodiaquine, diclofenac, indomethacin), antidiabetic(e.g., pioglitazone, rosiglitazone) and cardiovascular agents (e.g., clopidogrel, propranolol, verapamil) and natural antibiotics (e.g., erythromycin). A selection of these compounds is illustrated in Figure 3. In order to eliminate background reactions that may arise through either biotransformation by native E. coli enzymes or compound stability in buffers and solvents, control reactions were also set up, including a cell strain that had been transformed with the LICRED vector containing no heme domain gene, and a cell-free

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buffer sample. To obtain a first impression of the enzyme activity, cells were incubated with 12 lM compound overnight, after which mixtures were analysed by UPLC-MSE. Biotransformations of interest were observed for each of the seven recombinant cell strains, including hydroxylation and Ndemethylation reactions. Compounds that elicited the most interesting biotransformations from all strains included diltiazem 5, indomethacin 9, rosiglitazone 11, imipramine 13 and zafirlukast 15 (Fig. 3). Some of the most promising biotransformations with partial structure elucidation are listed in Table 1. Among these transformations it was observed that Ro05, Ro08, Ro11, Ro18 and Ro22 fusions were more likely to perform hydroxylation reactions. For example, Ro08-RhfRED catalyzed an 11.4% conversion of indomethacin 9 to a hydroxylated metabolite as analysed by UPLC-MSE and Ro18-RhfRED a 6.2% conversion of zafirlukast 15 to a hydroxylated product 16 when assessed against the controls. The Ro07-RhfRED fusion, by contrast, was more successful in catalyzing demethylation reactions, giving a 9.5% and 8.5% conversion to demethylated metabolites of diltiazem 5 and imipramine 13, respectively. This activity accorded with previous observations of demethylation activity of the alkaloid dextromethorphan by Ro07,25 although, in this screen, dextromethorphan itself was not transformed in significant quantity by the Ro07-RhfRED fusion protein in whole cells. This may be due to the substrate specificity of the heme domain being affected by its fusion to the reductase, as has been observed for P450MycG.19 The demethylation activity of Ro07-RhfRED towards a number of the drug targets prompted us

Figure 2. Western blot analysis of soluble cell extracts of strains of E. coli Rosetta (DE3) expressing P450RHA1-RhfRED fusions. Lanes: M = Low Molecular weight markers from BioRad. U = extract from non-induced cells; Lanes 1–12; 13–23 soluble extracts from strains expressing fusion proteins Ro01-RhfRED to Ro23RhfRED.

Figure 3. Drug molecules and biotransformation products from the screen that showed significant levels of oxidative transformation when incubated with P450RHA1-RhfRED fusions against negative controls. 5 = diltiazem; 9 = indomethacin; 11 = rosiglitazone; 13 = imipramine; 15 = zafirlukast.

Table 1 Biotransformation results from a screen of heterologously expressed cytochrome P450 fusion proteins RoXX-RhfRED from R. jostii RHA1 in whole cells of E. coli. Biotransformations using cells expressing the empty LICRED vector, overnight reactions in buffer and 40% acetonitrile were included as negative control Substrate

Conversion (%) Ro04

Diltiazem (5) Indomethacin (9) Rosiglitazone (11) Imipramine (13) Zafirlukast (15)

1.3 n.d. 0.4 6.0 n.d.

Ro05

0.3 5.7 n.d. 0.3 3.0

Ro07

9.5 n.d. 2.8 8.5 n.d.

Ro08

1.1 11.4 0.4 0.6 6.2

Ro11

0.9 7.2 0.5 0.5 3.9

Ro18

1.4 8.3 0.2 0.7 6.2

Product Ro22

0.9 5.6 0.4 0.7 4.6

Negative controls LIC1

C2

C3

0.6 n.d. 0.3 0.5 n.d.

n.d. n.d. n.d. 0.2 n.d.

0.2 n.d. n.d. 0.2 n.d.

N-Desmethyl diltiazem (6) Hydroxylated indomethacin (10) N-Desmethyl rosiglitazone (12) N-Desmethyl imipramine (14) Hydroxylated zafirlukast (16)

n.d.—not detected, LIC1—control biotransformations using empty vector LICRED, C2—control reaction in 40% acetonitrile, C3—control reaction in the buffer system.

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demethylation activity towards diltiazem 5 and imipramine 13, with an almost two-fold increase in conversion of 12 lM 13 to the N-demethylated product 14, compared to the results obtained with whole cells (13.6% vs 8.5%, respectively). Lower levels of demethylation of 12 lM diltiazem 5 by the purified Ro07-RhfRED were observed compared to the result with whole cells (5.4% and 9.5%, respectively), but were still detectable at levels well above that of controls. Imipramine and diltiazem were therefore selected as substrates for further detailed investigation of the catalytic properties of the Ro07-RhfRED fusion. 2.4. Biotransformation of imipramine 13

Figure 4. UV wavelength scan of the pure Ro07-RhfRED fusion in native state (. . .); after reduction with sodium dithionite (- - -) and after treatment with carbon monoxide (—), the latter displaying a characteristic absorption peak at 450 nm.

to focus on the activity of this fusion protein in more detail, through studies of the pure enzyme. 2.3. Purification and characterization of the Ro07-RhfRED fusion In order to investigate the physical and catalytic properties of Ro07-RhfRED, the production was scaled up and the enzyme purified using nickel affinity chromatography (Fig. S3). A sample of Ro07-RhfRED was reduced with sodium dithionite and then carbon monoxide (CO) was bubbled through the sample. Comparative analysis of the soluble protein before and after CO treatment revealed the signature Soret peak at 450 nm after CO complexation (Fig. 4). The spectroscopic characteristics suggest that Ro07-RhfRED is most likely folded in vitro and thus should be capable of binding substrates. We therefore performed a series of assays designed to test the ability of the pure Ro07-RhfRED to catalyze the demethylation reactions observed in whole cell preparations. The assays contained purified Ro07-RhfRED (0.8 mg mL1; 9.4 lM), NADPH (150 lM) and substrate (12 lM) derived from a stock in methanol [1% (v/v) final content of methanol in the assay]. The results obtained with the purified Ro07-RhfRED confirmed its

Conversions of imipramine 13 to N-desmethyl imipramine 14 were measured using different substrate concentrations and at different time points in the absence of co-solvent methanol, in order to exclude a possible negative effect of that additive on enzyme performance. N-demethylation proved to be strongly dependent on the substrate concentration (Fig. 5a). The highest conversions of over 30% were measured for imipramine concentrations of either 2.5 lM (36.2%) or 5 lM (35.4%) after 4 h incubation time. Higher substrate concentrations led to a decrease in conversion, with 17.5% and 13.8% observed for concentrations of 25 lM and 50 lM, respectively, after 4 h (Fig. 5a), after which no further conversion was observed. It proved possible to improve the conversion of 7 lM 13 to 14 up to 62.9% by increasing the concentration of purified Ro07-RhfRED to 1 mg mL1 (11.8 lM) (Fig. 5b). Conversions of up to 44% were observed at substrate concentrations of 50 lM, with the higher enzyme concentration, giving the best yield of 14 overall. 2.5. Biotransformation of imipramine analogues Encouraged by the performance of the purified Ro07-RhfRED towards imipramine we studied the biotransformation of ten further imipramine analogues (17–26, Fig. 6, left), each at a concentration of 10 lM, in order to investigate the influence of different chemical groups on the N-demethylase activity. The biotransformation of 13 to 14 was used as a reference, giving a conversion of 24.7% after 4 h incubation. Conversions were reduced markedly for hydroxylated derivatives 17 (2.8%) and 22

Figure 5. (a) Effect of concentration of imipramine 13 on the extent of bioconversion by purified Ro07-RhfRED (0.5 mg mL1; 5.9 lM). Imipramine concentrations: d = 1.0 lM, s = 2.5 lM, . = 5.0 lM, 4 = 7.5 lM, j = 10.0 lM, h = 25.0 lM and  = 50.0 lM. (b) Conversion (%) of imipramine 13 after 12 h by purified Ro07-RhfRED (1.0 mg mL1; 11.8 lM) at different substrate concentrations.

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Figure 6. Conversions of imipramine 13 and analogs 17–26 and their relationship to physico-chemical properties of the substrates transformed by Ro07-RhfRED ( = polar surface area (PSA), s = octanol–water partition coefficient (log P) and h = H-donors).

(4.1%), but only slightly for the derivative methylated in the alkyl chain 18 (18.8%). The stereoselectivity of Ro07-RhfRED was highlighted by a comparison of the conversions of enantiomeric substrates 23 (25.3%) and 24 (14.1%). The introduction of a cyclopropane ring into the imipramine side-chain, as in analog 26, decreased the conversion two-fold (13.5%). The presence of a chlorine atom on the aromatic ring, as in analog 19 (23.8%), did not affect levels of conversion significantly. The presence of a second side chain in the substrate (25) resulted in a substrate that was not converted. An analysis of the biotransformation results of the imipramine analogues revealed a clear correlation between the extent of conversion and their physico-chemical parameters, such as polar surface area (PSA), hydrogen donors (H-donors) and partition coefficient (log P) (Fig. 6, right). These data may be used in the future as a tool to inform the selection of new imipramine analogs prior to in vitro evaluation. Suitable explanations for the differential activity of Ro07-RhfRED towards these analogs await the determination of the crystal structure of the Ro07 heme domain in complex with the relevant substrates. It has been reported that imipramine is metabolized by primarily CYP2D6 in the human liver,26 which converts the drug to 2-hydroxyimipramine.27 10-Hydroxyimipramine is also formed, as well as the N-desmethyl metabolite 14, which is primarily the product of CYP3A4 metabolism.27 Bernhardt and co-workers have recently reported28 that recombinant prokaryotic P450 from Sorangium cellulosum can be used to prepare the 10-hydroxy derivative of imipramine, so Ro07-RhfRED may be candidate for a complementary role in the preparation of imipramine metabolites in the future.

to be strongly dependent on the initial substrate concentration (Fig. 7), with the most efficient reactions obtained at initial substrate concentrations of between 1.0 lM and 7.5 lM, giving conversions of 26.0% and 25.6%, respectively after 4 h. Using diltiazem at concentrations of 25 lM and 50 lM resulted in lower conversions to 6 of 12% and 8%, respectively after 4 h. However, as with the reactions with imipramine 13, the highest turnover was measured for diltiazem at an initial concentration of 50 lM after 4 h, after which no further product formation was observed (Fig. 7). During the progress of the reaction, the formation of deacetyldiltiazem 8 was detected, and was found not to be dependent on initial substrate concentration (Fig. S4). Subsequent experiments confirmed that this reaction was non-enzymatic and occurred spontaneously in the buffer solution at pH 7.5.

2.6. Biotransformation of diltiazem 5 Analysis of the biotransformation of diltiazem 5 by purified Ro07-RhfRED revealed the formation of one major metabolite, which was identified, according to observed mass shift by UPLCMSE as N-desmethyl diltiazem 6. This activity mirrors that of human liver CYP3A4 on diltiazem,29 which is also metabolized to the O-demethyl derivative by CYP2D6.30 Additional experiments demonstrated that Ro07-RhfRED was also capable of catalyzing a further N-demethylation of 6 to the di-desmethyl diltiazem 7, but only after the disappearance of the parent substrate 5 itself. The extent of the demethylation of diltiazem 5 was also shown

Figure 7. N-demethylation of diltiazem 5 by purified Ro07-RhfRED at different substrate concentrations. d—1.0 lM, s—2.5 lM, .—5.0 lM, 4—7.5 lM, j— 10.0 lM, h—25.0 lM and —50.0 lM.

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Interestingly, the deacetyldiltiazem formed as a consequence was not demethylated to desacetyl N-desmethyl diltiazem during the course of the reaction. The formation of a side-product caused by hydrolysis might have implications for metabolite purification from a preparative biotransformation, unless the reaction time was restricted to less than 4 h. Diltiazem has been shown to be metabolized in the liver by CYP3A429 and also CYP2D6,30 to the N-desmethyl and O-demethyl derivatives, respectively. 3. Conclusion Microorganisms such as R. jostii RHA1 have acquired a remarkable diversity of genes that encode oxidative enzymes for the breakdown of non-activated carbon compounds. This diversity can be examined for useful activities through the heterologous expression of individual genes. In the case of cytochromes P450, which require auxiliary electron transport proteins for full activity, the enzymes can be screened using protein fusion strategies, exploiting the P450 reductase components of other P450 enzymes. Such a strategy has led to the creation and screening of a library of diverse P450 fusions which, in addition to identifying enzymes competent for the demethylation of drug compounds such as imipramine and diltiazem, might be evaluated for further activities of use in the preparation of metabolites and other compounds of interest to medicinal chemistry. 4. Experimental section 4.1. Target selection, gene cloning and expression tests Details of target selection, gene cloning and expression protocols and Western Blot Analysis of expression tests can be found in the Supporting information. 4.2. Overexpression for whole purification of RhfRED-Ro07

cell

activity

assays

and

One colony was selected from plates with positive transformants to inoculate 5 mL of LB medium containing antibiotics. This starter culture was grown for 6 h at 37 °C and 180 rpm 500 lL starter culture was used to inoculate 10 mL of M9 medium preculture which was incubated overnight at 37 °C and 180 rpm 5 mL of the preculture was used as inoculum for a 500 mL culture in M9 medium. Cells were induced with 1 mM IPTG, 0.5 mM FeCl3 and 0.5 mM ALA an OD600 of 0.6–0.9 had been reached after shaking at 37 °C. The cultures were then incubated at 180 rpm at 16 °C overnight. Cells were then harvested by centrifugation at 5000 rpm for 15 min. The supernatant was discarded, the cell pellet was washed twice with Tris/HCl buffer (50 mM, pH 7.5, 300 mM NaCl) and subsequently resuspended in a proportionated volume of the buffer to give a cell density of 100 mg mL1. 4.3. Purification of RhfRED-Ro07 3 L of cell growth (6  500 mL) were resuspended in buffer as described above and sonicated on ice in 8  30 s intervals with 30 s delay between each interval. Soluble and insoluble fractions were then separated by high speed centrifugation at 16,000 rpm for 30 min at 4 °C. The obtained cell lysate was then filtered through a 0.22 lM membrane and loaded onto a 5 mL HisTrap FF Crude nickel column (GE Healthcare, Sweden) which was equilibrated with approximately 50 mL Tris/HCl buffer prior to use, which was then eluted with an optimized imidazole gradient to obtain the Ro07-RhfRED protein. The loaded column was initially

washed with 12 column volumes of Tris/HCl buffer pH 7.5, which also contained 300 mM NaCl. Some impurities were eluted by increasing the gradient of imidazole from 0 mM to 20 mM. By raising the imidazole concentration from 20 mM to 100 mM the Ro07RhfRED fusion protein could be eluted from the column. Fractions were analysed using SDS–PAGE and purified enzyme fractions were collected and concentrated by centrifugation using a Centricon with a 30,000 Da MW cut-off membrane (Millipore). The enzyme solution was finally washed three times with Tris– HCl buffer and reduced to a final concentration of 1 mg mL1 as determined by UV spectrophotometry. 4.4. Screening for drug metabolites using resting whole cells All of the whole-cell biotransformation tests were carried out with resting cells in 50 mM Tris/HCl buffer pH 7.5 containing 300 mM NaCl. 1 mM stock solutions in methanol of all substrates were prepared prior to biotransformation experiments. Biotransformations were carried out by addition of 3 lL of the relevant stock solution to a total volume of 247 lL of freshly harvested cells with a cell density corresponding to 100 mg mL1. Reactions were incubated with constant vigorous shaking at room temperature in deep 96-well plates (Thermo Scientific Nunc, USA) of 1 mL well volume. Samples of 50 lL were taken at intervals: t = 0 h, t = 3 h and t = 18 h and transferred to a conical 96-well plate (Thermo Scientific, Denmark). The biological material was precipitated by the addition of 100 lL acetonitrile and the samples centrifuged at 4000 rpm at 4° C for 20 min. 50 lL of the supernatant was then transferred into 150 lL 40% (v/v) acetonitrile in water and analyzed by UPLC-MSE using the conditions described below. 4.5. Screening for drug metabolites using purified Ro07-RhfRED 150 lM NADPH was added to a solution of pure Ro07-RhfRED (0.8 mg mL1, 9.4 lM) containing 12 lM of the drug compound, added from a 1 mM stock solution in methanol, to give a final reaction volume of 100 lL. Each reaction was incubated with shaking at room temperature (22–25 °C) in deep 96 well plates (Thermo Scientific Nunc, USA) of 1 mL well volume. Samples were taken at time point t = 0 and t = 18 h. 25 lL sample of the sample was then removed and the biological material precipitated by the addition of 50 lL 100% acetonitrile, after which the sample was centrifuged at 4000 rpm for 20 min at 4 °C. 25 lL of the supernatant was then transferred into 75 lL 40% (v/v) acetonitrile in water and analyzed by UPLC-MSE. 4.6. In vitro activity assays towards diltiazem 5 and imipramine 13 The N-demethylation activity of purified Ro07-RhfRED was tested at different concentrations of diltiazem 5 and imipramine 13 ranging from 1 lM to 50 lM using an enzyme concentration of 0.5 mg mL1 (5.9 lM) unless stated otherwise. All reactions were carried out in the biotransformation buffer containing Tris– HCl (50 mM) and sodium chloride (300 mM) at pH 7.5. Reactions were carried out as follows: To a 100 lL volume of buffer containing 0.5 mg mL1 of the purified enzyme was added 0.15 mM of NADPH, then 1–50 lM of diltiazem 5 or imipramine 13, from a 1 mM stock in methanol. The reactions were incubated at 30 °C with constant shaking at 500 rpm in 96-well plates (Nunc, Denmark), with a well volume of 300 lL, covered with a cap in a plate shaker (Grant-bio, UK). Reactions were carried out in duplicates and reaction progress was measured at intervals t = 0, 1, 2, 4, 6 and 24 h (unless stated otherwise). Reactions were stopped by addition of 100 lL of pure acetonitrile followed by removal of precipitated proteins by centrifugation of 96-well plates at 4 °C

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for 20 min at 4000 rpm (Hettich, Germany). Subsequently, the reaction mixtures were quenched using solution of 40% acetonitrile (v/v) in water. Samples were analysed using a UPLC-MS Synapt HDMS system (Waters, Milford, MA, USA). Metabolite identification and calculated conversions to respective demethylated metabolites were processed using MetaboLynx 4.1. Conversion was determined by calculating the fraction of a product peak area relative to the peak area of substrate.

This project was funded by the European Union FP7 People Inititiative as the Marie-Curie International Training Network P4FIFTY 289217, with awards of a studentship (to C.S.) and an Experienced Researcher position (to J.K.K.). R.H. was funded by the Industrial Affiliates of the Centre of Excellence for Biocatalysis, Biotransformation and Biomanufacture (CoEBio3).

4.7. In vitro activity assays imipramine analogues

Supplementary data

Reactions with imipramine analogues (17–26) were carried out as described above with final concentration of analogues at 10 lM in the biotransformation buffer. Reactions were carried out in triplicate. Samples for UPLC-MSE analysis were taken at t = 0 h and 4 h. Metabolite formation was assessed by calculation the fraction of a product peak area relative to the peak area of substrate.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.025.

4.8. Analysis using UPLC-MSE and data processing Samples (10 lL) were injected and analysed using a Waters ACQUITY UPLC liquid chromatography system coupled to a Waters Synapt HDMS instrument (Waters, Milford, MA, USA) and equipped with an electrospray ionisation (ESI) source. Chromatographic separation was carried using an ACQUITY UPLC BEH C18 column (130 Å, 1.7 lm  2.1 mm  100 mm; Waters, Milford, MA, USA) at flow rate of 0.5 mL min1 and column temperature of 45 °C. For separations using liquid chromatography mobile phases consisting of ultra-pure water supplemented with formic acid (0.1% v/v; mobile phase A) and pure acetonitrile (mobile phase B) were employed. The gradient applied for separation was as follows: 0.0–6.0 min (10–70% mobile phase B); 6.0– 6.7 min (70–90% mobile phase B), followed by a return to the initial mobile phase composition over 0.01 min. The MSE analysis was run on a Waters Synapt HDMS operating under positive electrospray ionization (ESI) conditions in V-mode. A generic method with two scan functions was used as follows: m/z 80–1000, cone voltage 20 V and 0.1 s scan time, the trap collision energy (CE) in function 1 was 20 V and in function 2 an energy ramp of 15–45 V was applied, the transfer cell CE was 12 V. Data were collected in a centroid mode. Leucine-enkephaline was used as a lock mass (m/z 556.2771) for internal calibration at a concentration of 250 pg mL1 and a flow rate of 0.04 mL min1. The MSE data were processed in MetaboLynx 4.1 (Waters, Milford, MA, USA) using both the mass defect filter (MDF) and the dealkylation tool.31 The list of proposed metabolites was reviewed manually. 4.9. Nuclear magnetic resonance 1 H nuclear magnetic resonance (NMR) spectra and 13C NMR spectra were recorded in DMSO-d6 at 25 °C on a Bruker Avance III 500 MHz spectrometer. Chemical shift values (d) were given in parts per million (ppm) relative to the residual solvent signal set to 2.50 ppm for 1H and 39.5 ppm for 13C.

Acknowledgements

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Please cite this article in press as: Kulig, J. K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.025