Targeting APOBEC3A's genomic mutagenic activity with functionalized DNA dumbbells

利用功能化 DNA 哑铃靶向 APOBEC3A 的基因组诱变活性

• 批准号: 10066574
• 负责人: Juan Carlos Serrano
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066574
• 关键词: Affinity; Base Pairing; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biology; Cancer Biology; Catalogs; Cell Line; Cell model; Cells; Chemicals; Clinic; Clinical; Complex; Coupled; Cyclization; Cytidine Deaminase; Cytosine; Cytosine deaminase; DNA; DNA Damage; DNA Double Strand Break; DNA Markers; DNA Repair; DNA biosynthesis; Deamination; Detection; Development; Disease; Doxycycline; Elements; Enzymes; Event; Exonuclease; Family; Family member; Flow Cytometry; Foundations; Frequencies; Genetic study; Genome; Genomic DNA; Genomics; High Pressure Liquid Chromatography; Host Defense; Human; In Vitro; Kinetics; Lead; Ligands; Link; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Modality; Modeling; Molecular; Molecular Conformation; Molecular Probes; Mutagenesis; Mutation; Nucleic Acids; Pathologic; Patients; Peptides; Periodicity; Play; Positioning Attribute; Process; Protac; Protein Inhibition; Proteins; Research; Resistance; Retroelements; Retroviridae; Role; Route; Scientific Advances and Accomplishments; Single-Stranded DNA; Somatic Mutation; Source; Structure; Structure-Activity Relationship; Technology; Testing; Therapeutic; Tissues; Translating; Ubiquitination; Up-Regulation; Uracil; Validation; Work; Yeasts; Zebularine; amino group; analog; base; cancer genome; cancer type; cellular targeting; chemotherapeutic agent; clinical development; cytosine analog; design; genome integrity; in vivo; in vivo evaluation; inhibitor/antagonist; innovation; insight; interest; member; nanomolar; novel; outcome forecast; overexpression; preference; protein degradation; recruit; response; scaffold; small molecule; stem; therapeutic development; therapeutic target; tool; tumor; ubiquitin-protein ligase

Project Summary Kataegic mutational signatures are localized, hypermutated clusters found across the genomes of multiple cancer types. These mutational marks have been associated with tumor development and adaptation, and ongoing research is aimed at deciphering their wider role in patient prognosis and resistance to chemotherapeutic agents. In efforts to ascertain the source of kataegis, sequencing studies have revealed the majority of these mutations are C to T/G substitutions enriched within 5’-TCN sequence contexts. Identification of this feature led to the suspicion and subsequent validation that members of the APOBEC3 cytidine deaminase family are the source of genomic mutation in kataegis. The APOBEC3 (A3) family plays a crucial role in defense against retroviruses and retrotransposable elements by deaminating C to U in single-stranded DNA (ssDNA) intermediates. However, misregulation of A3A and A3B can lead to pathologic deamination of the host genome. During events where genomic DNA becomes single-stranded, such as in DNA replication or repair, cytosine bases become prone to deamination, leading to targeted mutations or promotion of double-stranded DNA breaks. Targeting of these genomic mutators thus presents an attractive therapeutic strategy for evading APOBEC- driven kataegis in cancer. However, we currently lack molecular probes that can modulate APOBEC activity in the lab or strategies for development of clinical therapeutics. APOBEC3A has recently been shown to prefer ssDNA substrates in a stem-loop conformation, with the target cytosine placed in the 3’ end of the loop, a finding verified in both biochemical studies and genetic studies where a predominant number of the APOBEC-driven mutations in tumors are in this mesoscale structural context. In this proposal, we seek to exploit this substrate preference to develop more potent inhibitors of A3A and translate them towards cellular targeting of A3A’s genomic mutagenetic activity. We have already shown that placing methylzebularine, an inhibitory base towards cytidine deaminases, within cyclized DNA dumbbells, a scaffold mimicking A3A’s preferred substrate structure, results in subnanomolar-level inhibition of A3A in vitro. In aim 1, we will identify the mode of inhibition of these DNA dumbbells and perform structure-activity relationship studies on a panel of structurally diverse dumbbells to determine which features translate to more potent inhibitors. In aim 2, we will advance these results towards cellular studies on U2OS cells with inducible A3A overexpression and evaluate whether these inhibitors can block A3A-mediated genomic DNA damage and increased mutational load. Finally, in aim 3, we will exploit the stem portion of the dumbbell to modulate the mode of inhibition towards active protein degradation in a manner analogous to proteolysis-targeting chimeras (PROTACs). To do this, we will conjugate an mZ dumbbell to VH032, an E3 ligase recruiting ligand, and assess its ability to degrade A3A and block its genomic mutagenic activity. Completion of this proposal will advance rationally designed nucleic-acid based inhibitors of A3A, providing two novel routes to perturb APOBEC-driven kataegis in cancer through classic inhibition or protein degradation.

项目摘要 Kataegic突变特征是在基因组中发现的局部化，高度突变的簇， 多种癌症类型。这些突变标记与肿瘤的发展和适应有关， 目前正在进行的研究旨在破译它们在患者预后和耐药性方面的更广泛作用。 化疗剂。为了确定kataegis的来源，测序研究揭示了 这些突变中的大多数是在5 '-TCN序列背景内富集的C至T/G取代。识别 这一特征导致了怀疑和随后的验证，APOBEC 3胞苷脱氨酶的成员， 家族是山慈菇基因组突变的来源。APOBEC 3（A3）家族在防御中起着至关重要的作用 通过将单链DNA（ssDNA）中的C脱氨基为U来对抗逆转录病毒和逆转录转座因子 中间体的然而，A3 A和A3 B的错误调节可导致宿主基因组的病理性脱氨。 在基因组DNA变成单链的事件期间，例如在DNA复制或修复中，胞嘧啶 碱基变得易于脱氨基，导致靶向突变或促进双链DNA断裂。 因此，靶向这些基因组突变体提供了一种有吸引力的治疗策略，用于逃避APOBEC-1。 在癌症中被驱动的kataegis然而，我们目前缺乏可以调节APOBEC活性的分子探针， 实验室或临床治疗的发展策略。APOBEC 3A最近被证明更喜欢 ssDNA底物呈茎环构象，靶胞嘧啶位于环的3'端，这是一个发现， 在生物化学研究和遗传学研究中得到证实，其中绝大多数APOBEC驱动的 肿瘤中的突变是在这种中尺度结构背景下。在这个提议中，我们试图利用这种基质 优先开发更有效的A3 A抑制剂，并将其转化为A3 A的细胞靶向 基因组诱变活性。我们已经证明，将甲基zebularine，一种抑制碱， 胞苷脱氨酶，在环化DNA哑铃内，模拟A3 A的优选底物结构的支架， 导致体外亚纳摩尔水平的A3 A抑制。在目标1中，我们将确定这些抑制的模式， DNA哑铃，并对一组结构多样的哑铃进行结构-活性关系研究 以确定哪些特征转化为更有效的抑制剂。在目标2中，我们将把这些成果推向 对具有诱导型A3 A过表达的U2 OS细胞进行细胞研究，并评估这些抑制剂是否可以 阻断A3 A介导的基因组DNA损伤和增加的突变负荷。最后，在目标3中，我们将利用 以调节对活性蛋白质降解的抑制模式， 类似于蛋白水解靶向嵌合体（PROTAC）。为此，我们将mZ哑铃形物与VH 032共轭， E3连接酶募集配体，并评估其降解A3 A和阻断其基因组诱变活性的能力。 该提案的完成将推进合理设计的基于核酸的A3 A抑制剂，提供两个 通过经典抑制或蛋白质降解干扰APOBEC驱动的kataegis的新途径。