Work from a number of laboratories has demonstrated that the use of equilibrium binding molecules can be an effective in vitro strategy to deliver alkylating agents (including antineoplastic drugs) to DNA. The goals of this proposal are to refine our understanding of the in vitro and in vivo interactions of alkylating agents, including alkylating- equilibrium binders, with DNA, and to obtain more control over the sequence selectivity of the alkylation process.
The specific aims of this proposal are to: (a) Determine the nature of the DNA lesions induced by 2- chloroethylnitrosoureas (CENU) attached to methidium and phenyl neutral red DNA intercalators. CENU-methidium reacts with linearized pBR322 (about 4.4 kB) to afford a DNA product that behaves on non-denaturing agarose gels as if it was > 100 kB. We hypothesize that the aberrant gel mobility results from the efficient formation of interduplex linkages. The mechanism of the reaction, as well as the toxicity, mutagenicity and mutational specificity of CENU-methidium and CENU-PNR will be assayed. In addition, the toxicity and mutational specificity of a series of minor groove binding alkylating agents will also be assayed to determine if their in vivo reactions with DNA mimic what we have observed in vitro. (b) Determine the role of Tris in the alkylation of DNA by CENU because we have found that Tris stabilizes CENU and significantly slows down the time course for DNA alkylation by CENU. No effect is seen with monofunctional analogues, e.g. MNU. The studies with Tris will provide information on the mechanisms of DNA alkylation by CENU and determine if CENU formulated with Tris can be used as a anticancer reagent. This would provide a clinical improvement in the delivery of these drugs to cancer patients. (c) Synthesize four C-glycoside analogues referred to as TRIPs that can be readily assembled into oligomers that are designed to bind with sequence specificity to duplex Watson-Crick DNA via a triple helix motif without the requirement for homopurine/homopyrimidine sequences. The synthetic oligomers containing the TRIPs will recognize major groove H-bonding information associated with the purine bases via Hoogsteen base pairing so that their sugar-phosphate backbone is (i) near the center of the major groove; and (ii) perpendicular to the H-bonds of the Watson-Crick duplex. The evaluation of the binding properties of the molecules will be performed in vitro using a variety of assays. The appendage of alkylating agents to the polymers is also described.
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