Drug Development of Ribonucleotide Reductase Inhibitor

描述(由申请人提供)：这项提案的首要目标是开发一种新的核糖核苷酸还原酶(RR)抑制剂，这种抑制剂比任何现有的小分子疗法都更有效，并可以克服对羟基脲(HU)的耐药性，羟基脲(HU)是FDA批准的唯一针对RR癌症的药物。此外，这种新型抑制剂将改善与3-氨基吡啶-2-甲醛缩氨基硫脲(3-AP，Triapine)相关的严重铁络合副作用，该化合物目前正在进行临床试验。通过对从NCI开发治疗计划(DTP)筛选的多种化合物文库中鉴定的HIT进行修饰，开发出具有低-M范围抑制作用的先导化合物COH20。COH20显示重组人RRM2/RRM1酶复合体在体外9M时的抑制率为90.2%。此外，在小于10&L；M的范围内，观察到人口咽癌KB细胞系、人白血病细胞系REH、MOLT-4和人前列腺癌LNCaP细胞系在细胞培养中的生长抑制。重要的是，与3-AP相比，COH20对人正常成纤维细胞的毒性显著降低。计算模型和生物物理测量的结果表明，COH20干扰了RRM1和RRM2亚基之间界面的自由基转移途径，从而破坏了RRM1和RRM2蛋白-蛋白质之间的相互作用。这代表了一种与现有已知抑制剂完全不同的新的抑制机制。已经进行了Biaccore结合分析、计算对接以及核磁共振饱和转移差(STD)研究，以验证COH20结合是RRM2特有的。这些研究表明RRM2与COH20有一个V形的结合口袋。提出了更多的结构活性关系(SAR)研究，这些研究将指导未来的精制和优化。初步数据表明，COH20代表了一类有希望的新型RR抑制剂，具有以下优点：(A)一种新的抑制机制和靶向特异性，干扰RRM1-RRM2界面的自由基转移途径，(B)比HU更有效，IC50在低-1M范围内，以及(C)改善铁螯合相关的副作用，如3-AP所见。因此，我们的目标是优化COH20的性质，使其成为一种可行的候选药物，用于靶向过度表达的基于RR的人类癌症。本提案的具体目标概述如下：具体目标一.测定COH20对体外培养的人癌细胞的细胞毒性。特定目的II.确认COH20在活体小鼠模型中的疗效。具体目标III.使用计算机对接、生物核心分析、核磁共振分析和药物化学来改进和验证COH20的结合模式。综上所述，我们提出了一个多学科的转化计划，以开发从长凳到床边的一类新的、有前景的RRM2抑制剂来对抗人类癌症。目前由我们团队合成的先导化合物COH20可能会避开RRM2过表达导致的潜在耐药性问题，并避免目前正在进行的3-AP试验中出现的不良副作用。COH20被认为干扰RRM1和RRM2结合界面并中断自由基转移过程。双儿茶酚药效团也是新的，代表了一种新的可能的自由基抑制猝灭机制。综上所述，这些发现为RR抑制剂的未来发展奠定了新的方向。

DESCRIPTION (provided by applicant): The overarching goal of this proposal is to develop a new class of Ribonucleotide Reductase (RR) inhibitors that are more effective than any of the existing small molecule therapies, and can overcome resistance to Hydroxyurea (HU), the only FDA approved drug for targeting RR- based cancers. In addition, this new class of inhibitors will ameliorate the severe iron chelation side effects associated with 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine.), a compound that is currently being tested in clinical trials. A lead compound, COH20, with low-<M range inhibition was developed through modification of a hit identified from screening a diverse compound library from NCI Developmental Therapeutics Program (DTP). COH20 demonstrated 90.2% inhibitory activity of recombinant human RRM2/RRM1 enzyme complex at 9<M in vitro. Moreover, at less than 10<M range, growth inhibitions of human oropharyngeal cancer KB cell line, human leukemia cell line REH, MOLT-4, and human prostate cancer LNCaP cell line in cell culture were observed. Importantly, COH20 shows significantly less toxicity to human normal fibroblast cells when compared to 3-AP. Results from computational modeling and biophysical measurements suggest that COH20 interferes with the radical transfer pathway at the interface between subunits RRM1 and RRM2, hence disrupting RRM1 and RRM2 protein-protein interaction. This represents a novel inhibition mechanism that is completely different from existing known inhibitors. Biacore binding analysis, computational docking, as well as NMR Saturation Transfer Difference (STD) studies has been performed to verify COH20 binding is specific to RRM2. These studies suggest a V-shaped binding pocket of RRM2 with COH20. Additional Structure Activity Relation (SAR) studies are proposed which will guide future refinement and optimization. Preliminary data suggest that COH20 represents a promising new class of RR inhibitors with the advantages of (a) a novel inhibition mechanism and target specificity that interferes with the radical transfer pathway at the RRM1-RRM2 interface, (b) much greater potency than HU with an IC50 at the low-<M range, and (c) ameliorating iron chelation-related side effects as seen with 3-AP. Therefore, our objectives are to optimize the properties of COH20 into a viable candidate as an anticancer drug targeting overexpressed RR-based human cancers. The specific aims for this proposal are outlined as follows: Specific Aim I. Determine the Cytotoxicity of COH20 in Human Cancer Cells In Vitro. Specific Aim II. Confirm the Efficacy of COH20 using In Vivo Mouse Models. Specific Aim III. Refinement and Validation of the Binding Mode of COH20 Using Computer Docking, Biacore Analysis, NMR Analysis and Medicinal Chemistry. In summary, we propose a multidisciplinary translational program to develop a new and promising class of RRM2 inhibitors from bench to bedside against human cancers. The current lead compound, COH20, which was synthesized by our group, may circumvent potential drug resistance problems caused by the overexpression of RRM2 as well as avoid undesirable side effects seen from the currently undergoing trials of 3-AP. COH20 is believed to interfere at the RRM1 and RRM2 binding interface and interrupt with the radical transfer process. The bis- catechol pharmacophore is also novel and represents a new possible quenching mechanism underlying free radical inhibition. Taken together, these findings establish a new direction for the future development of RR inhibitors.