Development of a new class of BLVRB-targeted redox therapeutics in breast cancer

总结 在活跃增殖的癌细胞中固有的代谢和线粒体活性的增强产生了一种抑制癌细胞增殖的机制。 过量的活性氧（ROS），与细胞内氧化还原失衡有关， 为了在慢性氧化应激中生存，癌细胞进化为激活清除/抗氧化剂 酶来恢复氧化还原平衡。与正常细胞相比， 为新的细胞靶点提供了一个治疗窗口。此外，化疗和放疗（在 部分）归因于氧化应激，导致不可逆的氧化损伤和细胞死亡，以及活化 氧化还原调节途径的改变被认为会促进对这些疗法的抵抗。强制性的依赖 癌细胞的抗氧化防御途径作为一个基本的促生存机制表明，广泛的 它们在乳腺癌中的靶向转化效用。氧化还原适应机制的调节代表了 这是根除癌细胞和/或恢复对常规疗法的化学敏感性的可行策略。 我们首次鉴定了血红素（Fe ~（2+）-原卟啉IX）分解代谢酶BLVRB（胆绿素 IXβ还原酶）作为乳腺癌新的细胞靶点。我们证明了必要的和非冗余的亲- 乳腺癌细胞中BLVRB的生存抗氧化功能，与治疗抗性和不良结局相关 在乳腺癌患者中。本申请的主要假设是BLVRB在氧化还原过程中起作用。 乳腺癌细胞中抗氧化处理和细胞保护的调节途径。次要假设 BLVRB-选择性抑制剂可被开发为用于乳腺癌新的和潜在的无毒策略 在正常细胞中具有最小预测脱靶效应的癌症治疗。使用（1）BLVRB/抑制剂共晶体 结构，（2）SAR的计算RMSD矩阵，以及（3）广泛的ADME/T和PK研究，我们确定了 两种具有良好生物利用度和口服PK特征的先导化合物，选择性阻断BLVRB氧化还原 偶合器. 本提案的目的是（1）扩展BLVRB临床前靶点的初步原理验证研究 使用体内乳腺癌模型进行验证，以及（2）表征用于治疗乳腺癌的一流BLVRB选择性抑制剂。 体外和体内功效。研究设计：我们将应用体内遗传模型进行靶点验证， 通过使用BLVRB+/+的基因互补研究同时解决氧化还原依赖性机制， BLVRB-/-乳腺癌等基因系：（1）确认在肿瘤生长和转移负荷中的必要功能; (2)以建立氧化还原依赖性表型（目的1）。目的2将验证电极导线的临床前有效性 在体外和原位乳腺癌移植中使用良好建立的表型读出的化合物 模型我们还将讨论合成的致死性BLVRB抑制剂与标准的护理化疗在体内。 影响：如果成功，拟议的工作将是乳腺氧化还原抑制剂的一流临床前验证 癌症，代表了癌症治疗的潜在范式转变。

SUMMARY Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells generates an excessive amount of reactive oxygen species (ROS), associated with intracellular redox imbalance that impacts cellular viability.To survive chronic oxidative stress, cancer cells evolve to activate scavenging/anti-oxidant enzymes to restore redox balance. This differential activation of antioxidant pathways compared to normal cells provides a therapeutic window for novel cellular targets. Moreover, the effects of chemo- and radiotherapy (in part) are attributed to oxidative stress that causes irreversible oxidative damage and cell death, and activation of redox-regulating pathways is thought to promote resistance to such therapies. The obligatory dependence of cancer cells on antioxidant defense pathways as a fundamental pro-survival mechanism suggests the broad translational utility of their targeting in breast cancer. Modulation of redox-adaptation mechanisms represents a feasible strategy to eradicate cancer cells and/or restore chemosensitivity to conventional therapies. For the first time, we identified the heme (Fe2+-protoporphyrin IX) catabolic enzyme BLVRB (biliverdin IXβ reductase) as a new cellular target in breast cancer. We demonstrated the requisite and non-redundant pro- survival antioxidant function of BLVRB in breast cancer cells, coupled with therapy resistance and poor outcomes in breast cancer patients. The primary hypothesis of this application is that BLVRB functions in a redox- regulated pathway of antioxidant handling and cytoprotection in breast cancer cells. The secondary hypothesis is that BLVRB-selective inhibitor(s) may be developed as a novel and potentially non-toxic strategy for breast cancer treatment with minimal predicted off-target effects in normal cells. Using (1) BLVRB/inhibitor co-crystal structures, (2) computational RMSD matrices for SARs, and (3) extensive ADME/T and PK studies, we identified two lead compounds with excellent bioavailability and oral PK characteristics that selectively block BLVRB redox coupling. The objectives of this proposal are (1) to extend initial proof-of-principle studies for BLVRB pre-clinical target validation using in vivo breast cancer models, and (2) to characterize first-in-class BLVRB-selective inhibitors for in vitro and in vivo efficacy. Study Design: We will apply in vivo genetic models for target validation, simultaneously addressing redox-dependent mechanisms by gene complementation studies using BLVRB+/+ and BLVRB-/- breast cancer isogenic lines: (1) to confirm requisite functions in tumor growth and metastatic burden; (2) to establish redox-dependent phenotype (Aim 1). Aim 2 will validate the pre-clinical efficacy of lead compounds using well-established phenotypic read-outs in vitro and in orthotopic breast cancer implantation models. We will also address synthetic lethality BLVRB inhibitors with standard-of-care chemotherapy in vivo. Impact: If successful, the proposed work would be first-in-class pre-clinical validation of redox inhibitors in breast cancer, representing a potential paradigm shift for cancer therapeutics.