DNA INTERACTION AND MUTATIONAL ACTIVITY OF NOVEL BENZAZOLO[3,2-A]QUINOLINIUM SA

新型苯并[3,2-A]喹啉 SA 的 DNA 相互作用和突变活性

• 批准号: 8167845
• 负责人: Beatriz Zayas
• 金额: $ 12.9万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167845
• 关键词: Alkaloids; Antineoplastic Agents; Apoptotic; Area; Binding; Biological Assay; Biological Testing; Caspase; Cell Culture Techniques; Cells; Characteristics; Chemical Structure; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Adducts; DNA Binding; Drug toxicity; Environmental Carcinogens; Enzymes; Family; Funding; Genes; Goals; Grant; Guanosine; High Pressure Liquid Chromatography; Induced Mutation; Induction of Apoptosis; Institution; Ions; Lead; Literature; Mass Spectrum Analysis; Mentors; Methodology; Methods; Mitochondria; Molecular; Monitor; Mutation; Nature; Nucleosides; Pathway interactions; Research; Research Personnel; Resources; Ribose; Salts; Sodium Chloride; Solid Neoplasm; Somatic Mutation; Source; Supervision; Techniques; Testing; Tumor Cell Line; United States National Institutes of Health; Work; Xanthine Oxidase; adduct; analytical method; base; calf thymus DNA; cancer cell; environmental chemical; experience; graduate student; human tissue; ionization; killings; liquid chromatography mass spectrometry; neoplastic cell; novel

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的目的是表征四种新型生物补充抗癌药物的DNA结合能力和突变活性。这些新颖的化合物属于一个新的不自然阳离子生物碱家族，称为苯佐佐洛[3,2-A]喹啉盐（BQS）。在这项研究中，我们将测试四个含有BQS衍生物的新型硝基，即硝基苯唑[3,2-A]喹啉盐（NBQ'S）：NBQ-25，NBQ-38，NBQ-38，NBQ-59和NBQ-97。这些药物选择性毒性的基础取决于硝基群的还原及其与DNA共价结合的潜在能力。此外，这些NBQ的阳离子性质与室内的内部保留有关，从而可以选择性地杀死癌细胞。这两个特征使NBQ成为抗癌药物的强大候选人。 在这个项目中，我们将开发一种分析LCMS方法，该方法将使我们能够评估DNA与这些新型NBQ之间的相互作用。该项目的合作者和导师Gonzalez博士一直在研究这些新抗癌药物的合成和生物测试。在他们的研究中，他们表明NBQ-91可以在厌氧条件下和还原性酶低黄嘌呤/黄嘌呤氧化酶（HX/XO）结合在厌氧条件下与胸腺DNA结合。然而，直到现在尚未介绍其共价DNA加合物的结构鉴定。最近，Zayas博士开发了高性能液相色谱质谱法（HPLC-MS）方法，以表征一种新型奎诺林（NBQ91）的核苷相互作用能力。我们的初步研究通过LCMS分析清楚地表明了NBQ91-瓜氨酸加合物的形成。文献中支持的413.02 19i/z处突出的分子离子的形成与NBQ91-瓜氨酸加合物的化学结构一致，在D-ribose组丢失后已形成和碎片。除了DNA加合能力外，我们还将研究这些新型化合物的突变诱导能力。我们建议应用HPRT体细胞突变测定法以评估NBQ在实体瘤细胞培养系中诱导突变的能力。申请人的研究领域和长期研究目标的重点是研究环境化学物质和抗癌药与包括DNA在内的生物分子的相互作用。 Zayas博士为这个项目带来了她以前在HPLC-MS技术和突变诱导分析领域的经验。她既将此技术应用于新型抗癌药物的研究，并在人体组织和细胞中的环境致癌物中监测DNA加合物。 具体目的是： AIM 1：开发和验证一种可以应用的HPLC分析方法，可用于监测新型NBQ的脱氧核苷加合物的形成：NBQ25，NBQ38，NBQ95和NBQ2。 AIM 2：开发和验证电离电离质谱法（ESI-MS）方法，该方法将用于结构分析和鉴定形成的脱氧核苷-NBQ。 AIM 3：通过应用优化的分析条件（AIM 2）来表征NBQ与Calf Thymus DNA的相互作用，以酶消化的小牛胸腺DNA。 AIM 4：A431细胞上的石质毒性和用BQ处理的实体瘤细胞系上HPRT基因的突变诱导能力。 新目标5：确定用BQ处理的A431细胞上的线粒体损伤和凋亡诱导。最初没有提出这个目标，但是观察到的结果使我们评估了处理细胞对治疗细胞的凋亡诱导和线粒体损伤。 新目标6：NBQ-DNA加合物和前体的明确合成的合成方法：在原始文档中未提出此目标，但是，DG-NBQS加合物标准的合成将促进DG和DNA加合物的表征。这一新目标的完成是在合作者Osvaldo Cox博士和他的研究生Wigberto Hernandez的指导下完成的，后者也由Praabre资助。 新目标7：通过评估用BQ诱导的凋亡细胞激活胱天蛋白酶激活来确定凋亡诱导途径。