Targeting the substrate preference of a type I nitroreductase to develop antitrypanosomal quinone-based prodrugs.

Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, having K(cat)/K(m) values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO2 or 4-NO2 positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO2 group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs

TRISPEC: A Simultaneous Optical and Near-Infrared Imager, Spectrograph, and Polarimeter

Original article can be found at : http://www.journals.uchicago.edu/ Copyright The Astronomical Society of the PacificPeer reviewe