Inhibition and Catalytic Degradation of Promutagenic DNA Deaminases

项目总结 这项提议旨在开发第一个有效和特定的APOBEC促突变效应的拮抗剂 细胞中的DNA脱氨酶。获得全基因组序列有助于揭示常见的突变 各种癌症的签名。其中一个突出的突变特征，称为SBS2，包括 在同一条链上含有高密度C到T/G替换的超突变簇，这是一种现象 被称为kataegis。这些特征，除了5‘-TC基序的突变丰富外，还指向 APOBEC3(A3)家族酶的致病作用。虽然这些酶通常会发生突变并限制 逆转录病毒或逆转录元件，研究证实了两个家族成员，APOBEC3A(A3A)和 APOBEC3B(A3B)在针对宿主基因组的病理性突变中具有重要作用。相对的 每种酶的贡献仍然是一个激烈辩论的问题，因为遗传方法是专门针对 A3A或A3B因其在基因组上的高度同源性和并列而受到限制。此外， 目前还没有分子工具可以破坏A3的功能。因此，迫切需要分子 可以抑制或耗尽细胞中的A3酶的探针。这一建议建立在一个假设的基础上 对A3酶的机制和底物选择性的洞察可以被用来设计有效的和 特定的拮抗剂。具体地说，我们已经证明了基于机理的抑制剂部分可以是 以首选的二级结构呈现，并被工程化为抗核酸外切酶的DNA分子以产生 有效的A3A纳米分子抑制剂。这些分子提供了方便的官能化的机会，这 可用于将经典的缓蚀剂转化为能够诱导催化降解的分子 通过前所未有的核酸抑制剂和蛋白酶体的组合，靶向细胞中的APOBEC酶 目标(PROTAC)技术。综上所述，这项提议旨在通过引入 干扰APOBEC细胞功能的工具，以揭示其潜在的生物学意义，并为 潜在的治疗方法。

PROJECT SUMMARY This proposal aims to develop the first potent and specific antagonists of the pro-mutagenic effects of APOBEC DNA deaminase enzymes in cells. Access to whole genome sequences has helped reveal common mutational signatures across various cancers. One such prominent mutational signature, termed SBS2, includes hypermutated clusters containing a high density of C to T/G substitutions on the same strand, a phenomenon known as kataegis. These features, in addition to the enrichment of the mutations in 5’-TC motifs, point to a causative role for APOBEC3 (A3) family enzymes. While these enzymes normally mutate and restrict retroviruses or retroelements, studies have confirmed that two family members, APOBEC3A (A3A) and APOBEC3B (A3B), have a prominent role in pathological mutagenesis targeting the host genome. The relative contributions of each enzyme remain a matter of vigorous debate, as genetic approaches specifically targeting A3A or A3B are limited by their high homology to one another and juxtaposition on the genome. Furthermore, no molecular tools currently exist that can disrupt A3 function. There is therefore a pressing need for molecular probes that can either inhibit or deplete A3 enzymes from cells. This proposal builds on the hypothesis that insights into the mechanism and substrate selectivity of A3 enzymes can be leveraged to design potent and specific antagonists. Specifically, we have demonstrated that mechanism-based inhibitor moieties can be presented in preferred secondary structures and engineered into exonuclease-resistant DNA molecules to yield potent nanomolar inhibitors of A3A. These molecules present the opportunity for facile functionalization, which can be utilized to convert classical inhibitors into molecules capable of inducing the catalytic degradation of the target APOBEC enzymes in cells, via an unprecedented combination of nucleic acid inhibitors and proteasome targeting (PROTAC) technology. Taken together, this proposal aims to fill a critical gap in the field by introducing tools to perturb APOBEC function in cells in order to reveal their underlying biology and offer a roadmap for potential therapeutics.