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

鉴定木犀草素作为 BRAF 降解分子，用于开发新的治疗药物

基本信息

批准号：
10356619
负责人：
Feng Liu-Smith
金额：
$ 21.6万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10356619
关键词：
Affinity Antioxidants BRAF gene Binding Biological Assay CRISPR/Cas technology CUL1 gene Cell Cycle Cell Cycle Arrest Cell Line Cell-Free System Cells Clinic Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Combined Modality Therapy Complex Crystallization Cutaneous Melanoma Cysteine Data Development Dimerization Docking Down-Regulation Drug Targeting Drug resistance Enzymes Exhibits F Box Domain FDA approved Flavonoids Gene Expression Profiling Genes Genetic Glycine Goals Growth Human Immunoprecipitation In Vitro Knock-out Ligase Luteolin MAP Kinase Gene MAP2K1 gene MEKs MG132 Malignant Neoplasms Mediating Melanoma Cell Methods Modeling Molecular Mutate Mutation Oncogenes Oxidative Stress Pathway interactions Patients Pharmaceutical Preparations Phosphotransferases Play Proteasome Inhibitor Proteins Proto-Oncogene Proteins B-raf Publishing Reactive Oxygen Species Regulation Reporting Role Route Signal Transduction Skin Cancer Structure System Therapeutic Agents Treatment outcome Western Blotting Xenograft procedure analog anaphase-promoting complex base cancer type cell growth combinatorial drug development drug discovery drug sensitivity experimental study gene function genome editing genome-wide in vitro Assay in vivo inhibitor knock-down melanoma multicatalytic endopeptidase complex mutant mutational status novel novel therapeutics overexpression oxidation protein degradation resistance mechanism response small hairpin RNA small molecule inhibitor targeted treatment transcriptome sequencing treatment group tumor tumor growth ubiquitin isopeptidase ubiquitin-protein ligase vector

项目摘要

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.

对于更有效、更持久的 BRAF 靶向治疗的临床需求仍未得到满足该探索性项目旨在进一步促进晚期黑色素瘤皮肤癌和其他 BRAF 突变癌症的治疗。 BRAF 的分子调控并开发此类新的治疗药物与目前的临床药物不同，长期目标是将木犀草素类似物开发为一类新的药物。该项目的短期目标是了解如何木犀草素可诱导 BRAF 降解，并在体外和体内抑制黑色素瘤细胞的生长。在所有检测到的木犀草素抑制 BRAF 的黑色素瘤细胞系中，与 Vemurafenib 具有显着的协同作用。无细胞系统中的激酶活性，并诱导黑色素瘤细胞中的 BRAF 降解，从而导致下调通过计算将木犀草素与 BRAF 晶体结构中的 Cys532 对接。因此，木犀草素主要通过直接结合抑制黑色素瘤生长受到阻碍。 BRAF 蛋白的可药物 Cys532 残基处，导致蛋白酶体介导的 BRAF-wt 降解。 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：确定 Cys532 在 BRAF 稳定性和激酶活性中的作用 Cys532 在 BRAF 功能中的作用很大程度上是。未知，但经计算预测为“可药物化”，为此目的，Cys532 将被基因组编辑。成为 BRAFwt/wt 和 BRAFV600E/V600E 背景中的甘氨酸 (C532G) 或酪氨酸 (C532Y)。将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 Cys532 的氧化在 BRAF 稳定性和活性中发挥作用。此外，还将检测药物敏感性（木犀草素、维罗非尼或木犀草素/维罗非尼组合）并比较这些遗传背景的体外和体内药物亲和力响应靶稳定性（DARTS）。测定将用于评估木犀草素与具有各种突变的 BRAF 的直接结合。确定木犀草素诱导 BRAF 降解的机制是木犀草素诱导 BRAF 降解。蛋白酶体依赖性但未鉴定出特定的 E3 连接酶和后期促进复合物 (APC)。据报道涉及 SKP1/CUL1/F-Box 复合物（以及抵消 SKP 活性的 USP28）在 BRAF 蛋白降解中，将使用针对 APC 或 SCF 的小分子抑制剂。确定最终将使用木犀草素介导的E3连接酶来破坏BRAF。通过敲除连接酶的核心成分（APC10 或 SKP1）来验证特定的 E3 连接酶复合物。与此同时，我们通过 RNA-Seq 确定了 USP35 和另外六种 E3 连接酶作为潜在的木犀草素靶向将检查酶对 BRAF 降解的影响，从而实现这些目标。我们启动一条可转化为临床试验的药物发现路线，无论是新药还是扩大 BRAFi/MEKi 治疗可能适用于 BRAF 野生型患者，甚至其他癌症类型。