Identification of luteolin as a BRAF-degrading molecule for developing new therapeutic agents

对于BRAF的更有效和持久的靶向治疗， 晚期黑色素瘤皮肤癌和其他BRAF突变癌症。这一探索性项目旨在进一步 了解BRAF的分子调控，并开发这种新的治疗药物， 不同于目前的临床药物。长期目标是开发毛地黄黄酮类似物作为一类新的 MAPK依赖性癌症的治疗剂。这个项目的短期目标是了解如何 毛地黄黄酮诱导BRAF降解。木犀草素在体外和体内均能抑制黑色素瘤细胞的生长， 在所有检查的黑素瘤细胞系中与维罗非尼具有显著的协同作用。木犀草素抑制BRAF 激酶活性，并诱导黑色素瘤细胞中的BRAF降解，这导致黑色素瘤细胞中的BRAF水平下降。 MAPK通路的调节。通过计算机模拟，将木犀草素对接到BRAF晶体结构中的Cys 532上。 对接分析因此推测毛地黄黄酮主要通过直接结合抑制黑色素瘤生长 BRAF蛋白的Cys 532残基，导致蛋白酶体介导的BRAF降解。BRAF-wt 和BRAF-mt可以使用不同的机制。提出了两个具体目标：具体目标1： Cys 532在BRAF稳定性和激酶活性中的作用。Cys 532在BRAF功能中的作用主要是 未知，但计算预测为“可用药”。为此，Cys 532将被基因组编辑， 在BRAFwt/wt和BRAFV 600 E/V600 E背景中，变成甘氨酸（C532 G）或Tyr（C532 Y）。BRAF蛋白 在各种编辑的遗传背景中通过蛋白质印迹分析稳定性和激酶活性。 木犀草素诱导的ROS是否通过氧化Cys 532在BRAF稳定性和活性中起作用。 此外，将测定药物敏感性（毛地黄黄酮、维罗非尼或毛地黄黄酮/维罗非尼组合）， 在体外和体内进行这些遗传背景的比较。药物亲和反应靶点稳定性（DARTS） 测定将用于评估毛地黄黄酮与具有各种突变的BRAF的直接结合。具体目标2： 确定木犀草素诱导BRAF降解的机制。叶黄素诱导的BRAF降解是 蛋白酶体依赖性的，但一个特定的E3连接酶没有确定。后期促进复合物（APC）和 据报道，SKP 1/CUL 1/F-Box复合物（以及抵消SKP活性的USP 28）参与了 在BRAF蛋白降解中的作用。在该目的中，将使用对APC或SCF特异性的小分子抑制剂， 测定木犀草素介导的E3连接酶对BRAF的破坏。慢病毒shRNA方法将最终用于 通过敲除连接酶的核心组分（APC 10或SKP 1）来验证特异性E3连接酶复合物。 同时，我们通过RNA-Seq鉴定了USP 35和另外六种E3连接酶作为潜在的毛地黄黄酮靶向酶。 将检测其对BRAF降解的影响的酶。实现这些目标将使 我们开始一个药物发现路线，这是转化为临床试验，无论是新药，或扩大 BRAFi/MEKi治疗可能是BRAF野生型患者，甚至是其他癌症类型。

There are still unmet clinic needs for more effective and long-lasting targeted therapy against BRAF in advanced melanoma skin cancer and other BRAF-mutated cancers. This exploratory project aims to further understand molecular regulation of BRAF and to develop such new therapy drugs that are mechanistically different than the current clinic drugs. The long-term goal is to develop luteolin analogues as a new class of therapeutic agents on MAPK-dependent cancers. The short-term goal of this project is to understand how luteolin induces BRAF degradation. Luteolin inhibited melanoma cell growth in vitro and in vivo, and exhibited substantial synergistic effect with Vemurafenib in all melanoma cell lines examined. Luteolin inhibited BRAF kinase activity in a cell-free system, and induced BRAF degradation in melanoma cells, which led to down- regulation of the MAPK pathway. Luteolin was docked to Cys532 in BRAF crystal structure by a computational docking analysis. It is thus hypothesized that luteolin inhibits melanoma tumor growth primarily via direct binding to BRAF protein at the druggable Cys532 residue, resulting in proteasome-mediated BRAF degradation. BRAF-wt and BRAF-mt may use different mechanisms. Two specific aims are proposed: Specific Aim 1: To identify the role of Cys532 in BRAF stability and kinase activity. The role of Cys532 in BRAF function is largely unknown but was computationally predicted as “druggable”. In this aim Cys532 will be genome-edited to become a glycine (C532G) or a Tyr (C532Y) in BRAFwt/wt and BRAFV600E/V600E backgrounds. BRAF protein stability and kinase activity will be analyzed by western blots in the various edited genetic backgrounds. Whether luteolin-induced ROS play a role in BRAF stability and activity through oxidation of Cys532. Additionally, drug sensitivity (luteolin, Vemurafenib or combination of luteolin/Vemurafenib) will be assayed and compared in these genetic backgrounds in vitro and in vivo. Drug Affinity Responsive Target Stability (DARTS) assay will be used to evaluate direct binding of luteolin to BRAF with various mutations. Specific Aim 2: To identify the mechanism of luteolin-induced BRAF degradation. Luteolin-induced BRAF degradation is proteasome-dependent but a specific E3 ligase is not identified. Anaphase-Promoting Complex (APC) and the SKP1/CUL1/F-Box complexes (as well as USP28 which counteracts SKP activity) were reported to be involved in BRAF protein degradation. In this aim small molecule inhibitors specific for APC or for SCF will be used to determine luteolin-mediated E3 ligase for BRAF destruction. Lentiviral shRNA approach will be used at last to validate the specific E3 ligase complex by knocking down the core component of the ligases (APC10 or SKP1). In parallel, we identified USP35 and six additional E3 ligases through RNA-Seq as potential luteolin targeted enzymes which will be examined for their impact on BRAF degradation. Completion of these aims will enable us to initiate a drug discovery route which is translatable into clinical trials, either for new drugs, or to expand BRAFi/MEKi treatment to perhaps BRAF-wild type patients, or even other cancer types.