Natural products inhibitors targeting homology-directed DNA repair for cancer therapy

摘要 基因组不稳定是癌症的一个标志。DNA修复中的基因组不稳定缺陷使肿瘤发生 癌症进展将癌细胞推向依赖DNA修复和基因组的特定子集 维护因素。拟议计划的两个这样的目标是DNA修复蛋白RAD51和RAD52。 DNA修复网络的灵活性和适应性使癌细胞能够利用它们来实现快速 进化，遗传变异的逐渐积累，基因组的不稳定，侵袭性的获得， 药物和辐射抵抗力与疾病进展。缺乏同源重组的细胞 例如，肿瘤抑制基因BRCA1和BRCA2中的缺陷)需要RAD52才能存活，而过度表达 RAD51在癌症中的表达增加了放射和化疗的耐药性。这两种蛋白质都依赖于它们的单链DNA结合 来执行它们的细胞功能。通过干扰RAD52或RAD51结合抑制DNA修复，或 利用小分子包裹有毒的DNA修复中间体具有重要的治疗意义 潜力。识别每种蛋白质特有的单链DNA结合的干扰物代表着一种艰难的药物化学 这是一项挑战，但也是量身定制的低毒疗法的先决条件。天然产品在中国有着无与伦比的地位。 早期药物的发现和占据了蛋白质结合无与伦比的化学空间，这将是 用来阐明我们的目标蛋白的特定抑制的结构和化学方面。 我们已发表的和初步的研究建立了可靠的生物物理(基于FRET)分析来监测 人主要单链DNA结合蛋白RAD51、RAD52与复制蛋白A的结合 它的抑制很可能是有毒的。原则证明筛查证实，我们可以很容易地识别出 分子和天然产物专门抑制这三种蛋白质的每一种。我们还开发了一种 精确预测小分子与DNA结合之间相互作用的计算工作流程 RAD52的凹槽。在这里，我们建议开展一项筛选活动，以确定一系列天然产品 可发展成有效、特异和无毒的RAD52-ssDNA相互作用干扰物的支架 并建立发现RAD51天然产物抑制剂的框架。我们将放映一个独特的文库 天然产品，NCI天然产品发现计划(NPD)预提库，包括 32万个馏分，通过分离来自植物、海洋和 微生物。我们的高通量筛选和化合物验证(Aim1)，随后是In Silico 分析(AIM2)将产生一组天然产品支架，将告诉我们区域和结构 RAD52目标部位内的特征，可被靶向以实现特异性和高亲和力，并且在 潜在特定命中的化学特征。结构-活性关系和习得的特异性规则 然后可用于支架跳跃方法，以获得新的合成RAD52抑制剂。

ABSTRACT Genomic instability is a hallmark of cancer. Genome destabilizing defects in DNA repair enable tumorigenesis and cancer progression pushing cancer cells into dependence on a specific subset of DNA repair and genome maintenance factors. Two such targets of the proposed program are DNA repair proteins RAD51 and RAD52. The flexibility and adaptability of DNA repair networks allows cancer cells to employ them to enable rapid evolution, progressive accumulation of genetic variation, genomic instability, the acquisition of invasiveness, drug and radiation resistance and disease progression. Cells deficient in homologous recombination (due, for example to defects in tumor suppressors BRCA1 and BRCA2) require RAD52 for survival, while overexpression of RAD51 in cancers promotes radiation and chemotherapy resistance. Both proteins rely on their ssDNA binding to carry out their cellular functions. Inhibition of DNA repair by interfering with RAD52 or RAD51 binding, or entrapment of the toxic DNA repair intermediates with the help of small molecules has significant therapeutic potential. Identifying disruptors of ssDNA binding specific to each protein represents a tough medicinal chemistry challenge, but is a prerequisite for tailored low toxicity therapies. Natural products have an unrivaled place in early stage drug discovery and occupy an unparalleled chemical space for protein binding, which will be employed to elucidate the structural and chemical aspects of specific inhibition of our target proteins. Our published and preliminary studies established robust biophysical (FRET-based) assays to monitor the ssDNA binding by RAD51, RAD52 and Replication Protein A (RPA), a major human ssDNA binding protein whose inhibition is likely to be toxic. Proof-of-principle screens confirmed that we can readily identify small molecules and natural products specifically inhibiting each of the three proteins. We have also developed a computational workflow that accurately predicts interactions between small molecules and the DNA binding groove of RAD52. Here, we propose to carry out a screening campaign to identify a range of natural product scaffolds that can be developed into potent, specific and non-toxic disruptors of the RAD52-ssDNA interaction and to build a framework for discovery of RAD51 natural products inhibitors. We will screen a unique library of natural products, an NCI Program for Natural Product Discovery (NPNPD) Prefractionated Library consisting of 320,000 fractions, which were produced by separating the unique crude extracts derived from plant, marine, and microbial organisms. Our high throughput screening and compound validation (Aim1) followed by in silico analysis (Aim2) will yield sets of natural products scaffolds that will inform us on the regions and structural features within the target site of RAD52, that can be targeted to achieve specificity and high affinity, and on the chemical signature of the potential specific hits. Structure-activity relationships and rules for specificity learned from these campaigns can be then utilized in scaffold hopping approaches to new synthetic RAD52 inhibitors.