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.

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.

Rapid Detection of Botulinum Neurotoxins-A Review.

A toxin is a poisonous substance produced within living cells or organisms. One of the most potent groups of toxins currently known are the Botulinum Neurotoxins (BoNTs). These are so deadly that as little as 62 ng could kill an average human; to put this into context that is approximately 200,000 × less than the weight of a grain of sand. The extreme toxicity of BoNTs leads to the need for methods of determining their concentration at very low levels of sensitivity. Currently the mouse bioassay is the most widely used detection method monitoring the activity of the toxin; however, this assay is not only lengthy, it also has both cost and ethical issues due to the use of live animals. This review focuses on detection methods both existing and emerging that remove the need for the use of animals and will look at three areas; speed of detection, sensitivity of detection and finally cost. The assays will have wide reaching interest, ranging from the pharmaceutical/clinical industry for production quality management or as a point of care sensor in suspected cases of botulism, the food industry as a quality control measure, to the military, detecting BoNT that has been potentially used as a bio warfare agent.

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug.

Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-activating enzymes, or their encoding genes, are directed to the tumour before administration of a prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target effects. The bacterial nitroreductases are a class of enzymes used in this therapeutic approach and although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, NfnB from Escherichia coli (NfnB_Ec), is a major limit to this technology. There is a continual search for enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and shown to reduce the CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 4'-hydroxylamine products. Both enzymes shared features characteristic of the nitro-FMN-reductase superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one)), and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was identified as a promising candidate for future enzyme prodrug therapy.

Botulinum neurotoxin serotypes detected by electrochemical impedance spectroscopy.

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A-E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.

A label free colorimetric assay for the detection of active botulinum neurotoxin type A by SNAP-25 conjugated colloidal gold.

Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring the activity of the toxin is the mouse bioassay, which is lengthy and has ethical issues due to the use of live animals. This report demonstrates a novel assay that utilises the endopeptidase activity of the toxin to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of 373 fg/mL and has been further developed into a high throughput method using a microplate reader detecting down to 600 fg/mL of active toxin. The results show clear differences between the toxin product and the placebo, which contains the pharmaceutical excipients human serum albumin and lactose, showing that the assay detects the active form of the toxin.