Mol Cancer Ther. Epub Oct 6. Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency.
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Abstract Background The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli.
To address this limitation we evaluated other nitroreductase candidates from E. Findings Initial screens of candidate genes in the E.
In monolayer cytotoxicity assays, human colon carcinoma HCT cells transfected with P. In three dimensional mixed cell cultures, not only were the P. Conclusion We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures.
This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. A key aspect of GDEPT is the bystander effect, the ability of activated prodrugs to transport either passively or actively out of the cell of origin and into neighbouring non-transfected cells, which provides an elegant solution to the unavoidable issue of low cell transfection rates [ 1 ].
Studies employing prodrugs with high bystander effects have demonstrated that significant tumour reduction can occur when less than 0. Bacterial type I nitroreductase enzymes, which catalyse the simultaneous two-electron bioreductive activation of nitroaromatic prodrug substrates, hold great potential for GDEPT.
However, the intrinsically oxygen insensitive nature of the two-electron reduction mechanism enables nitroaromatic prodrugs that were originally designed to target tumour hypoxia i. The main class of hypoxia-activated prodrugs to have been considered in this context is the dinitrobenzamide mustards DNBMs e.
The black dotted line indicates the basal SOS activity in the empty plasmid control. Full size image Also promising for GDEPT are the nitro-chloromethylbenzindolines nitro-CBIs , originally designed to be hypoxia-activated prodrugs [ 9 ] of amino analogues of the cyclopropylindoline anti-tumour antibiotics, exemplified by CC and duocarmycin SA [ 10 , 11 ].
Findings To identify novel nitroreductase enzymes we previously constructed an over-expression library of eleven candidate oxidoreductases from E. Consistent with the SOS assays, the two E.
Figure 2 Bacteria-delivered enzyme prodrug cytotoxicity assay. The sensitivity of each cell line towards nitro-CBI-DEI was then evaluated using an in vitro proliferation assay as described [ 3 ]. The 5. In contrast, in co-cultures of target cells and a minority 5. The target cells in this 3D co-culture were nearly as sensitive as the activators, their C10 value of 0.
The target cell population was calculated by subtracting the number of puromycin resistant activator cells colonies counted on selective plates from the total cell population colonies counted on non-selective plates. The dashed line represents one log of cell kill.
We surmise that the cytotoxic potential of nitro-CBI-DEI may be dramatically underestimated in low cell density proliferation assays due to washout and loss in the media, as suggested for other high-bystander prodrugs [ 3 ]. Should it prove necessary to further enhance enzyme activity via directed evolution or targeted mutagenesis studies, our demonstration that nitro-CBI-DEI induces the E.
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