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

摘要 活跃增殖的癌细胞固有的代谢和线粒体活性增强会产生 过量的活性氧(ROS)，与细胞内氧化还原失衡相关，从而影响 细胞存活率。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 恢复氧化还原平衡的酶。与正常细胞相比，这种抗氧化剂途径的差异激活 为新的细胞靶点提供了一个治疗窗口。此外，化疗和放射治疗的效果(in 部分)归因于导致不可逆转的氧化损伤和细胞死亡的氧化应激，以及激活 氧化还原调节通路的变化被认为会增加对此类疗法的抵抗力。对…的强制性依赖 癌细胞在抗氧化防御途径上作为促进生存的基本机制提示了广泛的 它们的靶向在乳腺癌中的翻译效用。氧化还原适应机制的调制代表着一种 根除癌细胞和/或恢复对传统疗法的化疗敏感性的可行战略。 我们首次鉴定了血红素(Fe2-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。我们展示了必要且非冗余的PRO- BLVRB在乳腺癌细胞中的生存抗氧化功能，加上治疗抵抗和不良结局 在乳腺癌患者中。这一应用的主要假设是BLVRB在氧化还原中起作用- 乳腺癌细胞中抗氧化剂处理和细胞保护的调节途径。二次假说 牛血清白蛋白选择性抑制剂(S)可能被开发为一种新的、潜在无毒的乳房治疗策略 在正常细胞中预测的最小偏离靶点效应的癌症治疗。使用(1)BLVRB/缓蚀剂共晶 结构，(2)SARS的计算RMSD矩阵，以及(3)广泛的ADME/T和PK研究，我们确定 两种具有良好生物利用度和口服PK特性的先导化合物选择性地阻断BLVRB氧化还原 耦合。 这项建议的目标是(1)扩展BLVRB临床前目标的初步原则证明研究 使用体内乳腺癌模型进行验证，以及(2)表征一流的BLVRB选择性抑制剂 体外和体内疗效。研究设计：我们将应用体内遗传模型进行靶向验证， 利用BLVRB/和基因互补研究同时解决氧化还原依赖机制 BLVRB-/-乳腺癌等基因系：(1)确定在肿瘤生长和转移负荷中所必需的功能； (2)建立氧化还原依赖表型(目标1)。Aim 2将验证铅的临床前疗效 使用成熟的表型读数的化合物在体外和乳腺癌原位植入中的应用 模特们。我们还将在体内用标准护理化疗来解决合成致命性BLVRB抑制剂。 影响：如果成功，这项拟议的工作将成为乳房氧化还原抑制剂临床前验证的第一步 癌症，代表着癌症治疗学潜在的范式转变。

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.