This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to characterize the DNA binding capacity and mutational activity of four novel bioreductive anticancer drugs. These novel compounds belongs to a new family of unnatural cationic alkaloids known as benzazolo[3,2-a]quinolinium salts (BQS's). In this study, we will test four novel nitro containing BQS derivatives, namely nitrobenzazolo[3,2-a]quinolinium salts (NBQ's): NBQ-25, NBQ-38, NBQ-59, and NBQ-97. The basis for the selective toxicity of these drugs depends on the reduction of the nitro group and their potential ability to bind covalently to DNA. In addition, the cationic nature of these NBQs has been implicated in intramitochondrial retention, which allows selective killing of cancer cells. These two characteristics make NBQ's strong candidates as anticancer drugs. In this project we will develop an analytical LCMS method that will allow us to assess the interaction between DNA and these novel NBQ's. The collaborators of this project and the mentor, Dr. Gonzalez have been working on the synthesis and biological testing of these new anticancer drugs. In their research they have shown that NBQ-91 can bind covalently to calf thymus DNA at anaerobic conditions and in the presence of the reductive enzymes hypoxanthine/xanthine oxidase (HX/XO). However structural identification of their covalent DNA adducts formed has not been presented until now. Recently Dr. Zayas has developed a high performance liquid chromatography mass spectrometry (HPLC-MS) method to characterize the nucleoside interaction capacity of one of the novel quinolinium (NBQ91). Our preliminary studies clearly indicated by LCMS analysis, the formation of an NBQ91-guanosine adduct. The formation of a prominent molecular ion at 413.02 19I/Z, as supported in the literature is consistent with a chemical structures in which the NBQ91-guanosine adduct has been formed and fragmented after the loss of the D-ribose group. In addition to the DNA adduct forming capacity we will study the mutation induction capacity of these novel compounds. We propose to apply the HPRT somatic mutation assay to evaluate the capacity of the NBQ's to induce mutation in solid tumor cell culture lines. The applicant's research area and long term research goals focuses on the study of the interaction of environmental chemicals and anticancer drugs with biomolecules including DNA. Dr. Zayas brings to this project her previous experience in the area of HPLC-MS techniques and mutation induction analysis. She has applied this technique both on the study of novel anticancer drugs and to monitor DNA adducts from environmental carcinogens in human tissues and cells.
Specific Aims are:
Aim 1 : Develop and validate an HPLC analytical method that can be applied to monitor the formation of deoxynucleosides adducts treated with the novel NBQ's: NBQ25, NBQ38, NBQ95, and NBQ2.
Aim 2 : Develop and validate an Electro Spray Ionization ?Mass Spectrometry (ESI-MS) method that will be applied for structural analysis and identification of the deoxynucleosides-NBQs adduct formed.
Aim 3 : Characterize the interaction of the NBQ's with calf thymus DNA by applying the optimized analytical conditions (aim 2) to enzymatically digested Calf Thymus DNA.
Aim 4 : Citotoxicity on A431 cells and mutational induction capacity at the HPRT gene on solid tumor cell lines treated with BQ's. New Objective 5: Determine the Mitochondrial Damage and Apoptosis induction on A431 cells treated with BQs. This objective was not proposed originally however the observed results have lead us to evaluate the apoptotic induction and mitochondrial damage on treated cells. New Objective 6: Synthetic Methodologies of the unequivocal synthesis of NBQ-DNA adducts and precursors: This objective was not proposed in the original document however, the synthesis of a dG-NBQs adduct standard will facilitate the characterization of the dG and DNA adducts. The completion of this new objective is under the supervision of the collaborator Dr. Osvaldo Cox and his graduate student Wigberto Hernandez who has been funded by PRAABRE as well. New Objective 7: Determine the apoptotic induction pathway by evaluating caspases activation on apoptotic cells induced with BQ's.
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