Chemical Interrogation of Human DNA Cytosine Deaminases

 描述(申请人提供)：APOBEC3(A3)酶是DNA胞嘧啶到尿嘧啶脱氨酶，目标是破坏外来DNA，作为先天免疫的一部分。A3酶在多种人类疾病中也发挥着不可或缺的作用。APOBEC3G(A3G)在复制过程中亚致死性地使HIV-1cDNA脱氨基，提供了一个基因组突变的来源，有助于病毒的进化、适应和耐药性。APOBEC3B(A3B)在至少6种人类癌症中过表达，导致高水平的C-U基因组突变，推动肿瘤进化、异质性和耐药性。因此，A3G和A3B的小分子抑制剂最终可能产生应用于传染病和癌症的新疗法。该项目的长期目标是开发A3G和A3B的小分子抑制剂作为临床药物。这项应用的目标是开发A3G和A3B的有效化学探针，以实现对这两种酶的机制研究。高通量筛选、化学合成和生化测试以前曾被用于鉴定A3G的共价抑制剂。这一应用的中心假设是：1)最近发表的A3G共价抑制剂已经确定了酶中对小分子调制敏感的区域，可以在探针设计中利用这一区域；2)最近发现的和未发表的A3G和A3B的非共价抑制剂可以通过基于计算的方法进行优化，以产生有效和选择性的化学探针。开发A3G和A3B小分子探针的基本原理是分别对它们在HIV-1限制和癌症进化中的作用进行机制研究。此外，小分子A3G和A3B探针将作为未来药物发现工作的启动点。本申请的具体目的是：1)开发双功能可逆的共价A3G抑制剂；2)开发A3G和A3B的非共价抑制剂。在第一个目标中，现有的共价靶向A3G的小分子将被结构修饰，使其也与A3G的锌原子结合，产生预计比现有化合物更有效的双功能抑制剂。此外，现有的A3G共价抑制剂将经过修改，加入可以可逆地与A3G Cys321结合的化学部分，产生的分子预计比现有的共价抑制剂交叉反应少，因此将在细胞裂解物和细胞中抑制A3G。在第二个目标中，基于未发表的非共价A3G和A3B抑制剂的计算模型将被用于指导A3G和A3B的更具选择性和更有效的非共价抑制剂的开发。这种方法是创新的，因为本提案中描述的A3G和A3B的小分子抑制剂代表了已知的针对这些酶的一流分子。这项工作也是创新的，因为独特的跨学科科学家团队，他们都拥有突变研究的专业知识，专注于提供A3脱氨酶的新型化学探针。这项拟议的研究具有重要意义，因为它有望提供有关A3抑制的基本化学知识，这些知识将成为未来药物的基础。

 DESCRIPTION (provided by applicant): APOBEC3 (A3) enzymes are DNA cytosine-to-uracil deaminases that target foreign DNA for destruction as part of innate immunity. A3 enzymes also play integral roles in multiple human diseases. APOBEC3G (A3G) sub-lethally deaminates HIV-1 cDNA during replication, providing a source of genomic mutation that contributes to viral evolution, adaptation, and drug resistance. APOBEC3B (A3B) is overexpressed in at least 6 human cancers, resulting in high levels of C-to-U genomic mutations that drive tumor evolution, heterogeneity, and drug resistance. Therefore, small molecule inhibitors of A3G and A3B may ultimately yield novel therapeutics for applications in infectious disease and cancer. The long-term goal of this project is to develop small molecule inhibitors of A3G and A3B as clinical drugs. The objective in this application is to develop potent chemical probes of A3G and A3B to enable mechanistic studies of both enzymes. High-throughput screening, chemical synthesis, and biochemical testing have been previously employed to identify covalent inhibitors of A3G. The central hypotheses of this application are: 1) Recently published A3G covalent inhibitors have identified a region of the enzyme that is susceptible to small molecule modulation, which can be exploited in probe design, and 2) Recently identified and unpublished non-covalent inhibitors of A3G and A3B can be optimized by computation-based methods to yield potent and selective chemical probes. The rationale for developing A3G and A3B small molecule probes is to enable mechanistic studies of their roles in HIV-1 restriction and cancer evolution, respectively. Furthermore, small molecule A3G and A3B probes will serve as launch points for future drug discovery efforts. The specific aims of this application are: 1) To develop bifunctiona and reversible covalent A3G inhibitors and 2) To develop non-covalent inhibitors of A3G and A3B. In the first aim, existing small molecules that covalently target A3G will be structurally modified to also engage the zinc atom of A3G, yielding bifunctional inhibitors that are predicted to be more potent than existing compounds. Additionally, existing covalent inhibitors of A3G will be modified to incorporate chemical moieties that can reversibly engage A3G Cys321, yielding molecules that are predicted to be less cross-reactive than the established covalent inhibitors, and therefore, will inhibit A3G in cell lysate and cells. In the second aim, computational modeling based on unpublished non-covalent A3G and A3B inhibitors will be used to guide the development of more selective and more potent non-covalent inhibitors of A3G and A3B. This approach is innovative because the small molecule inhibitors of A3G and A3B described in this proposal represent the first- in-class molecules known to target these enzymes. This work is also innovative due to the unique cross- disciplinary team of scientists, all with expertise in mutation research, that are focused on delivering novel chemical probes of A3 deaminases. The proposed research is significant because it expected to deliver fundamental chemical knowledge of A3 inhibition that will serve as the underpinning for future drugs.