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

对于更有效和更持久的针对BRAF的靶向治疗，仍有临床需求未得到满足 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，开发新的治疗药物 不同于目前的临床药物。我们的长期目标是开发木犀草素类似物作为一种新的 MAPK依赖型癌症的治疗剂。该项目的短期目标是了解如何 木犀草素诱导BRAF降解。木犀草素在体内外对黑色素瘤细胞生长的抑制作用 在所研究的所有黑色素瘤细胞系中，与维莫拉非尼有显著的协同作用。木犀草素抑制BRAF 无细胞系统中的激酶活性，并诱导黑色素瘤细胞中BRAF的降解，从而导致BRAF表达下调。 MAPK通路的调控。木犀草素通过计算与BRAF晶体结构中的Cys532对接 对接分析。因此推测木犀草素主要通过直接结合抑制黑色素瘤的生长。 在可药物的Cys532残基上与BRAF蛋白结合，导致蛋白酶体介导的BRAF降解。BRAF-重量 而BRAF-MT可能使用不同的机制。提出了两个具体目标：具体目标1：确定 Cys532在BRAF稳定性和激酶活性中的作用Cys532在BRAF功能中的作用很大 未知，但经计算预测为“可用药”。为此，将对Cys532进行基因组编辑，以 在BRAFwt/wt和BRAFV600E/V600E背景中成为甘氨酸(C532G)或酪氨酸(C532Y)。BRAF蛋白 在不同编辑的遗传背景下，将通过Western blotts分析稳定性和激酶活性。 木犀草素诱导的ROS是否通过氧化Cys532对BRAF的稳定性和活性起作用。 此外，还将检测药物敏感性(木犀草素、维莫拉非尼或木犀草素/维莫拉非尼的组合)，并 在体外和体内对这些遗传背景进行了比较。药物亲和力反应靶点稳定性(DARTS) 实验将用于评估木犀草素与具有各种突变的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。木犀草素诱导的BRAF降解 蛋白酶体依赖，但没有鉴定出一种特定的E3连接酶。后期促进复合体(APC)和 据报道参与了Skp1/CUL1/F-Box复合体(以及中和SKP活性的USP28) 在BRAF蛋白的降解中。在这一目标中，将使用针对APC或SCF的小分子抑制剂来 测定木犀草素介导的E3连接酶对BRAF的破坏作用。慢病毒shRNA方法将最终用于 通过敲除连接酶的核心成分(APC10或Skp1)来验证特定的E3连接酶复合体。 同时，我们通过RNA-Seq确定了USP35和另外6个E3连接酶作为潜在的木犀草素靶标 将检测其对BRAF降解的影响的酶。完成这些目标将使 美国将启动一条可转化为临床试验的药物发现路线，要么是新药，要么是扩大 BRAFi/Meki治疗可能适用于BRAF野生型患者，甚至其他癌症类型。

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.