Drug Development of Ribonucleotide Reductase Inhibitor

描述（由申请人提供）：本提案的总体目标是开发一类新的核糖核苷酸还原酶（RR）抑制剂，其比任何现有的小分子疗法更有效，并且可以克服对羟基脲（HU）的耐药性，这是FDA批准的唯一一种靶向RR型癌症的药物。此外，这类新的抑制剂将改善与3-氨基吡啶-2-甲醛缩氨基硫脲（3-AP，Triazoline.）相关的严重铁螯合副作用，一种目前正在临床试验中的化合物。通过修饰从筛选来自NCI开发治疗计划（DTP）的不同化合物文库中鉴定的命中物，开发了具有低<M范围抑制的先导化合物C 0 H 20。COH 20在9 μ M时对重组人RRM 2/RRM 1酶复合物的体外抑制活性为90.2%。此外，在小于10 μ M的范围内，观察到细胞培养物中人口咽癌KB细胞系、人白血病细胞系REH、MOLT-4和人前列腺癌LNCaP细胞系的生长抑制。重要的是，与3-AP相比，COH 20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，COH 20干扰亚基RRM 1和RRM 2之间的界面处的自由基转移途径，从而破坏RRM 1和RRM 2蛋白质-蛋白质相互作用。这代表了一种新的抑制机制，与现有的已知抑制剂完全不同。已进行Biacore结合分析、计算对接以及NMR饱和转移差（STD）研究以验证COH 20结合对RRM 2具有特异性。这些研究表明RRM 2与COH 20的V形结合口袋。提出了额外的构效关系（SAR）研究，这将指导未来的细化和优化。初步数据表明，COH 20代表了一类有前景的新型RR抑制剂，其优点是：（a）一种新型抑制机制和靶点特异性，可干扰RRM 1-RRM 2界面处的自由基转移途径，（B）效力远高于HU，IC 50在低-<M范围，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COH 20的性质，使其成为一种可行的候选抗癌药物，靶向过表达的基于RR的人类癌症。本提案的具体目标概述如下：体外测定COH 20对人癌细胞的细胞毒性。具体目标二。使用体内小鼠模型确认C 0 H 20的功效。具体目标三。使用计算机对接、Biacore分析、NMR分析和药物化学对COH 20的结合模式进行改进和验证。总之，我们提出了一个多学科的翻译计划，以开发一个新的和有前途的RRM 2抑制剂类从实验室到床边对人类癌症。目前的先导化合物COH 20是由我们的小组合成的，它可以避免RRM 2过表达引起的潜在耐药性问题，并避免目前正在进行的3-AP试验中出现的不良副作用。据信COH 20干扰RRM 1和RRM 2结合界面并中断自由基转移过程。双儿茶酚药效团也是新颖的，并代表了一种新的可能的淬灭机制，潜在的自由基抑制。总之，这些发现为RR抑制剂的未来发展确立了新的方向。

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.