Evaluation of two xenobiotic reductases from Pseudomonas putida for their suitability for magnetic nanoparticle‐directed enzyme prodrug therapy as a novel approach to cancer treatment

Abstract Directed enzyme prodrug therapy (DEPT) is a cancer chemotherapy strategy in which bacterial enzymes are delivered to a cancer site before prodrug administration, resulting in prodrug activation at the cancer site and more localized treatment. A major limitation to DEPT is the poor effectiveness of the most studied enzyme for the CB1954 prodrug, NfnB from Escherichia coli, at concentrations suitable for human use. Much research into finding alternative enzymes to NfnB has resulted in the identification of the Xenobiotic reductases, XenA and XenB, which have been shown in the literature to reduce environmentally polluting nitro‐compounds. In this study, they were assessed for their potential use in cancer prodrug therapy strategies. Both proteins were cloned into the pET28a+ expression vector to give the genetically modified proteins XenA‐his and XenB‐his, of which only XenB‐his was active when tested with CB1954. XenB‐his was further modified to include a cysteine‐tag to facilitate direct immobilization on to a gold surface for future magnetic nanoparticle DEPT (MNDEPT) treatments and was named XenB‐cys. When tested using high‐performance liquid chromatography (HPLC), XenB‐his and XenB‐cys both demonstrated a preference for reducing CB1954 at the 4‐nitro position. Furthermore, XenB‐his and XenB‐cys successfully induced cell death in SK‐OV‐3 cells when combined with CB1954. This led to XenB‐cys being identified as a promising candidate for use in future MNDEPT treatments.


Approaches to overcoming the limitations of the NfnB/CB1954
combination have included the development of other prodrugs with greater dose potency such as the hypoxia-activated prodrugs PR-104A (Foehrenbacher et al., 2013;Guise et al., 2010;McKeage et al., 2011;Patterson et al., 2007;Singleton et al., 2009;Williams et al., 2015) and SN27686  or the identification of other bacterial enzymes that operate in combination with the CB1954 prodrug such as NfsA from E. coli (Copp et al., 2017;Vass et al., 2009), YfkO from Bacillus licheniformis (Emptage et al., 2009), or the novel nitroreductases from Bacillus cereus, BC_1619 and BC_3024, previously identified within our research group (Gwenin et al., 2015).

The two Xenobiotic reductases, XenA and XenB, from
Pseudomonas putida (P. putida) have been identified from the literature as being capable of reducing a nitro (NO 2 ) group, such as those found on explosive compounds like nitro-glycerin, in the presence of an NADPH cofactor to produce an NHOH derivative (Blehert, Fox, & Chambliss, 1999;Orville, Manning, Blehert, Studts, et al., 2004;Pak et al., 2000). Like NfnB, the two Xenobiotic reductases are flavoproteins but, unlike NfnB, which is a dimeric flavoprotein, they are monomeric flavoproteins Orville, Manning, Blehert, Studts, et al., 2004). While the ability of the XenA and XenB enzymes to reduce to nitro groups in explosive compounds in the presence of NADPH is well documented within the literature, their activity with nitroaromatic prodrugs such as CB1954 is yet to be established.
In this study, it was determined whether or not the two Xenobiotic reductases were able to readily reduce the CB1954 prodrug to its toxic metabolites to ascertain their potential for use in novel MNDEPT treatments. Firstly, for the ease of purification and isolation, both Xenobiotic reductases were modified to contain a His-tag, and only the enzyme which showed reactivity toward the CB1954 prodrug, XenB, was then further modified to contain an N-terminal Cys-tag for immobilization onto gold-coated magnetic nanoparticles (Gwenin, Gwenin, & Kalaji, 2011) for possible use in MNDEPT.

| Cloning of enzymes
Genomic DNA was isolated from P. putida using a Wizard® Genomic DNA Purification kit from Promega. The primers listed in Table 1 were synthesized by Eurofins Genomics and used to amplify the xenA and xenB genes from the genomic DNA using a Techne TC-3000 thermocycler. Amplification of the xenA and xenB genes from genomic DNA was achieved using PCR set up using thermic cycling parameters under the following conditions; 98°C for 30 s, 15 cycles of 98°C for 10 s (denaturing), 68-58°C for 30 s (annealing), and 72°C for 30 s (extension). This was then followed by a final hold at 4°C.
The PCR products were purified as previously described (Gwenin et al., 2015) using a GeneJet PCR purification kit and subjected to a restriction enzyme digest using the sites indicated in Table 1 before being analyzed using a 1% Agarose gel to confirm their size and purity.
After being purified, ligation between the PCR products and a pET28a + vector (Novagen, Merck, UK) was performed using t4 DNA ligase (New England Biolabs, UK) at 16°C overnight. To confirm successful ligation between the gene of interest and the plasmid vector, a PCR based on the T7 promoter and T7 terminator sequences, which flank the gene insert region contained within the vector, was performed using Taq DNA Polymerase Master Mix (Amplicon, Denmark). To amplify the recombinant plasmids, competent E. coli DH5α cells (200 µl) were transformed with a plasmid (~10 µl) and incubated on agar plates containing Kanamycin (50 µg/ml). Kanamycin is used to select for bacterial colonies containing the pET28a + plasmid with the Kan R gene.

| Transformation
The transformation of recombinant plasmids containing the cloned xenA and xenB genes into E. coli Rosetta pLysS (Novagen, Merck, UK) was done following the method described previously by Ball, Thompson, Anderson, Gwenin, and Gwenin (2019).

| Protein expression and purification
The expression and purification of all proteins used were done following the method previously described by Ball et al. (2019), with one minor modification. In this work, all Lysogeny Broth media used contained additional glucose (0.5%).

| Enzyme reactivity with CB1954
The ability of the purified enzymes to reduce the CB1954 prodrug was confirmed following the method previously described by Gwenin et al. (2011). Briefly, the proteins were incubated with NADPH (300 μM) and CB1954 (100 μM) in phosphate buffer (PB) (50 mM, pH 7.2) and scanned using UV-visible spectroscopy every 90 s for 15 min. For active enzyme/CB1954 combinations, product formation was measured at 420 nm (Gwenin et al., 2011(Gwenin et al., , 2015.

| CB1954 kinetics
All enzyme kinetics experiments were carried out following the method previously described by Ball et al. (2019), using a Thermo Scientific Varioscan 96-well plate microplate reader. The data were then transferred to SigmaPlot 12 (SPSS, Systat Software Inc.) where a non-linear regression tool was used to generate a Michaelis-Menten hyperbolic curve and a report containing the important kinetic information of the system under test.

| HPLC
All of the HPLC experiments in this study were carried out following the method previously described by Ball et al. (2019), using an HPLC machine (Dionex Ultimate 3000 HPLC system, ThermoScientific, USA) using a C18 column for analysis (Waters Spherisorb® 5 µm ODS2 4.6 mm × 250 mm C18 column, UK). The instrument was run using the following parameters: 50 µl injection volume, a fixed column oven temperate of 25°C, a run length of 45 min, and the UV wavelength for detection was 420 nm (Ball et al., 2019;Gwenin et al., 2015).
Next, medium (50 µl) containing the purified enzyme was added and after 4 h, the medium was removed, and cells were replenished with complete DMEM (100 µl). After 48 h, 20 µl of MTT (5 mg/ml) was added to each well and incubated at 37°C for 4 h. The formazan crystals that formed were dissolved in 100 µl of DMSO, after removing the media carefully, and the absorbance was read at 570 nm in a Thermo Scientific Varioscan 96-well plate microplate reader.

| Cloning and sequencing
Both the xenA and xenB genes were successfully amplified using PCR, purified, and inserted into the pET28a + expression vector, which adds an N-terminal histidine tag (his-tag) for ease of protein purification. The recombinant plasmids containing the xenA and xenB genes were sequenced verified to confirm their identity before they were used in other experiments.

| Protein expression and purification
The pET28a+ vector contains a his-tag that is added to all the recombinant proteins for ease of purification using metal ion affinity chromatography (IMAC). XenA-his and XenB-his are the native TA B L E 1 Primer sequences used for the cloning of XenB and XenA Primer name The restriction enzyme cut site is in italics and underlined. The addition of the cysteine-tag is in bold and underlined.
P. putida XenA and XenB cloned into the pET28a+ vector. The XenBcys, however, was genetically modified using PCR to contain an additional cysteine-tag (designed by our research group) (Gwenin et al., 2011) on the N-terminal of the protein which is 6 amino acids long.
The purpose of the cys-tag is to allow direct binding of the protein to metal surfaces such as gold. The additional weight of the amino acid tags causes small changes in migration on the SDS-PAGE as previously described (Gwenin et al., 2011). All genetically modified proteins were obtained at a yield of up to 5 mg/ml.

| Enzymatic reduction of the CB1954 prodrug
Initially, the reactivity of each enzyme with CB1954 in the presence of an NADPH cofactor was tested (Figure 1) following the method as previously described by V. Gwenin et al. (2011), Gwenin et al. (2015).
For active enzyme/prodrug combinations, NADPH consumption was observed at 340 nm and the formation of the CB1954 hydroxylamine products was seen at 420 nm.

| HPLC analysis
Following the method previously published (Ball et al., 2019;Gwenin et al., 2015), the ratio of the CB1954 NHOH derivatives formed after the reduction of the CB1954 prodrug by either XenB-his or XenBcys ( Figure 2) was analyzed and these ratios are presented in Table 3.
The hydroxylamine product ratios produced for XenB-his (3:97) and XenB-cys (6:94) were both extremely similar to one another, and in both cases, the enzyme demonstrated a preference for reducing CB1954 at the 4-position to give the 4-NHOH as the major product of the reaction. This is a result of clinical significance as the 4-NHOH derivative of CB1954 has been shown in the literature to be further reduced by intracellular thioesters such as Acetyl Coenzyme A to form a compound that can cross-link DNA (Anlezark et al., 1992;Knox et al., 1991;Race et al., 2007).

| Cell viability assays
Percentage cell viability of SK-OV-3 cells, relative to untreated controls, was determined in the presence of increasing concentrations of either XenB-his or XenB-cys enzyme, with a constant concentration of CB1954 present (Figure 3). Controls were cell culture medium only, the enzyme only and prodrug only and data points were plotted based on the averages taken from at least three repeats. The CB1954 concentration used was fixed at 10 µM so that the results of the cell viability assays could be related to clinical results, because the CB1954 concentration in vivo cannot exceed 10 µM due to the maximum tolerated dose observed in clinical trials thus far (Chung-Faye et al., 2001;McNeish et al., 1998;Searle et al., 1998Searle et al., , 2004Williams et al., 2015).

TA B L E 2
The Michaelis-Menten kinetic data obtained for XenB-his and XenB-cys (10 µg/ml) by varying the concentrations of the CB1954 prodrug in the presence of NADPH as the cofactor F I G U R E 2 HPLC chromatogram of the reaction between CB1954 and either XenB-his or XenB-cys, measuring absorbance at 420 nm. NADPH and phosphate buffer were detected at 2-4 min, unreacted CB1954 prodrug was detected at 10.5-14 min and DMSO was detected at 22.5-24.5 min. The 4-hydroxylamine was detected at 5-6 min and the corresponding amine was detected at 14.5-15.5 min. The 2-hydroxylamine was detected at 9-10 min with the corresponding amine being detected at 21-22 min unexpected result, it is a known phenomenon called the Hormetic effect, which has been well documented in the literature (Mattson, 2008). The Hormetic effect is evident in the biphasic response of the SK-OV-3 cells to the increasing amounts of enzyme used in the treatments, leading to the characteristic inverted U-shaped response on the graph (Mattson, 2008). Also seen from Figure 3 was that neither enzyme nor the CB1954 prodrug alone could cause significant cell death.

| DISCUSS ION
The major aim of this research was to clone and express genetically modified Xenobiotic reductases from P. putida and assess their ability to reduce the CB1954 prodrug and demonstrate an ability to cause cell death in the ovarian cancer cell line, SK-OV-3. It is of significance that these enzymes have never before been reacted with the Cb1954 prodrug, and it was hoped that the results obtained would show the recombinant enzymes to be suitable candidates for use in the novel-directed enzyme prodrug therapy approach, MNDEPT (Gwenin, Gwenin, & Kalaji, 2009;Gwenin et al., 2011). To achieve the aims, it was determined whether or not the enzymes could (a) effectively reduce CB1954 at low prodrug concentrations, (b) had a preference for reducing the prodrug at the 4-NO 2 position instead of the 2-NO 2 position, and (c) cause cell death in Sk-OV-3 ovarian cancer cells when reacted with the prodrug.
All of the aims were achieved and XenB-cys identified as a promising candidate for MNDEPT. Despite the poor activity of XenA-his leading to this enzyme being dropped from the study, XenB-his and subsequently XenB-cys have been shown to reduce the CB1954 prodrug. Both proteins showed a preference for reduction at the more desirable 4-NO 2 position and induced increased cell death in the ovarian cancer cell line SK-OV-3.
The fact that XenB-his and XenB-cys were shown to reduce CB1954 almost exclusively at the 4-NO 2 position was a significant result because intracellularly the 4-NHOH derivative of CB1954 is further reduced by thioesters to a DNA crosslinking species which is highly cytotoxic (Anlezark et al., 1992). It has been shown previously by Ball et al. (2019) that the hydroxylamine product ratio produced after the reduction of the CB1954 prodrug changes over time in favor of the 4-NHOH product as the 2-NHOH product is more rapidly converted via a non-enzymatic reduction into the corresponding amine (2-NH 2 ) than the 4-NHOH product. In the case of XenB-his and XenB-cys, a negligible amount of 2-NH 2 was obtained, which indicated that little-to-no 2-hydroxylamine had been formed and that both proteins reduced CB1954 almost exclusively at the 4-NHOH position. It should be noted that while reduction at the 4-NO 2 position is more favorable in terms of generating a more potent DNA-crosslinking product, literature has suggested that there may also be negatives to using enzymes that favor 4-NO 2 reduction over 2-NO 2 reduction because of the significantly higher bystander NfnB NTR. Again this result is of clinical significance as any enzyme used in cancer prodrug therapy treatments in combination with CB1954 must be effective at lower prodrug combinations due to the dose-limiting toxicity of CB1954 restricting the concentration of the prodrug which can be used in treatments.
As well as characterizing the reaction between the Xenobiotic reductases, XenB-his or XenB-cys, with the CB1954 prodrug, it was important to ascertain if the enzyme/prodrug combinations could cause cell death in a cancerous cell line, in this case, the ovarian cancer cell line SK-OV-3. It was decided that the cancer cell kill experiments would be carried out without the addition of extracellular cofactor so that any cell-killing observed could be attributed to the successful uptake of the enzymes into the cancer cells, thus accessing the intracellular cofactor. The results obtained for the 2D cell culture experiments confirmed that XenB-his and XenB-cys were successfully taken up into SK-OV-3 cells and induced cell death after reducing the CB1954 prodrug to its toxic products, thus identifying them as promising enzymes for use in cancer prodrug therapy treatments.
In conclusion, two Xenobiotic reductases were identified, cloned, expressed, and tested with the CB1954 prodrug. Unfortunately, the XenA-his reductase was shown not to reduce the CB1954 prodrug at detectable levels, even though it was capable of reducing nitro-glycerin. Together with other literature, it was thus hypothesized that this enzyme was unable to reduce nitroaromatic compounds. XenBhis and subsequently XenB-cys were shown to be able to readily reduce CB1954, primarily at the 4-NO 2 position, and these enzyme/ prodrug combinations were shown to cause significant cell death in SK-OV-3 cells without the addition of extracellular cofactor thus confirming they are promising candidates for use in DEPT strategies. The next stage of this research will be to assess the XenB-cys enzyme when immobilized onto gold-coated magnetic nanoparticles (AuMNPs) to see if this leads to any loss of activity with the CB1954 prodrug or a change in the performance in cell viability assays.

ACK N OWLED G M ENTS
The authors would like to thank the School of Chemistry at Bangor University for their support throughout this project as well as funding from the Life Sciences Research Network Wales and the Knowledge Economy Skills Scholarship 2 fund.'

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data generated or analyzed during this study are included in this published article.