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.

FIGURE 1

UV/Vis spectra showing the enzymatic reduction of CB1954 to its hydroxylamine derivatives (λ_max = 420 nm) by XenB‐his in presence of NADPH (λ_max = 340 nm)

FIGURE 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

FIGURE 3

Percentage cell survival relative to untreated control cells of SK#x2010;OV‐3 cells after a 4 h incubation with prodrug only, the enzyme only and increasing concentrations of either XenB‐his or XenB‐cys (25–200 nM) in presence of CB1954 (10 µM). All data points are taken from the averages of at least three repeats, and the error bars represent the standard deviation

FIGURE A1

SDS‐PAGE of the purifications of (a) XenA‐his, (b) XenB‐his and (c) XenB‐cys. Flowthrough (FT) 1 and 2 were the first eluted fractions collected after adding the protein to the IMAC column. Imidazole concentration was then steadily increased from 50 mM to 800 mM to elute the pure his‐tagged proteins