Design, characterization and optimization of HSP70 inhibitors, HSF-1 inhibitors and anti-cancer naphthoquinones and naphthylisoquinoline for the treatment of multiple myeloma

本项目旨在开发针对多发性骨髓瘤（MM）的新型药物，采用两种不同的策略：基于热休克蛋白70 （HSP70）和热休克转录因子1 （HSF1）在MM中的病理生物学作用的最新发现，以靶向方法开发了HSP70和HSF1的抑制剂。在一种互补的方法中，从自然界中选择的化合物被优化为特定的抗mm活性，并进一步探索发现新的靶点或作用机制。从HSP70的结构信息出发，通过虚拟筛选确定了靶向核苷酸结合域和底物结合域界面的分子，得到了5种对MM细胞系有活性的化合物。以最佳靶点为起始点，合成了四氢异喹啉类药物的重点文库，得到了一系列对MM细胞有活性的分子，对外周血单核细胞（PBMCs）无毒性。以HSF1为靶点，通过Ugi-Heck合成了两个苯基修饰异喹啉化合物文库。令人惊讶的是，只有开环的Ugi中间体表现出抑制作用，但作用方式未知。HSP70的主要目标是计算和实验表征和优化命中化合物。这需要对HSP70抑制剂的结合机制进行基于靶标的研究，并对其细胞内效应进行广泛的生物学测试。为了证明HSP70的特异性结合和变构阻断，我们将建立HSP70实验，进行结晶实验，并进行生物物理结合研究。将进行分子动力学模拟以探索结合机制，并在与实验的相互作用中发展机制假设。这将指导抑制剂的优化和新化合物的合成。为了进一步开发HSF1抑制剂，将使用先进的功能生物学分析和质谱结构分析来揭示HSF1与潜在抑制剂之间的相互作用模式。该结果将指导改进HSF1抑制剂的开发，作为阐明HSF1/HSP70通路的工具。由于其具有较强的抗mm活性，我们合成了二醌B及其修饰类似物。构效关系（SAR）研究表明，这三个羟基对它们的活性都是必不可少的，而其他取代基的影响尚不清楚。使用二氯醌b相关的环氧化物和二酚碱a，我们确定了另外两种对MM细胞具有高毒性但对正常pbmc没有毒性的药物。Dioncoquinone B可能特异性影响凋亡和细胞周期调节，这从大约一半的MM细胞系的凋亡细胞死亡中可以明显看出，而其余的细胞系表现出生长停滞甚至没有任何生长抑制作用。对细胞死亡原因的研究揭示了一个半胱天冬酶依赖的过程，表明线粒体应激反应机制的参与。该项目现在将侧重于sar引导下的进一步结构修饰和对二恶英醌B、相关环氧化物和二恶英酚a的作用模式的研究。它们将配备探针（如生物素）来研究它们与细胞靶标的相互作用。机制研究计划分析线粒体过程，细胞凋亡和生长停滞特异性调节的分子效应。它们对MM细胞生长的影响将在MM小鼠模型中进行评估。

This project aims to develop novel pharmacological agents against multiple myeloma (MM) pursuing two different strategies: In a targeted approach based on recent findings on the pathobiological role of heat shock protein 70 (HSP70) and on the heat shock transcription factor 1 (HSF1) in MM inhibitors of HSP70 and HSF1 are developed. In a complementary approach, selected compounds from nature are optimized for specific anti-MM activity, and further explored to discover novel targets or mechanisms of action. Starting from structural information about HSP70, molecules targeting the interface between the nucleotide-binding and the substrate-binding domains were identified by virtual screening, resulting in five compounds active against an MM cell line. Using the best hit as starting point, a focused library of tetrahydroisoquinolinones was synthesized, yielding a series of active molecules against MM cells without toxicity on peripheral blood mononuclear cells (PBMCs). To target HSF1, two libraries of phenyl-decorated isoquinoline compounds were synthesized via the Ugi-Heck synthesis. Surprisingly, only ring-open Ugi intermediates showed inhibition, yet with unknown mode of action.The major goal for HSP70 is the computational and experimental characterization and optimization of the hit compounds. This requires target-based investigation of the binding mechanism and extended biological testing of the intracellular effects of HSP70 inhibitors. To prove specific HSP70 binding and allostery blockade, we will establish an HSP70 assay, conduct crystallization experiments, and perform biophysical binding studies. Molecular- dynamics simulations will be performed to probe the binding mechanism and develop mechanistic hypotheses in interplay with the experiments. This will guide the inhibitor optimization and the synthesis of new compounds. - For the further development of HSF1 inhibitors advanced functional biological assays as well as structural analyses using mass spectrometry will be used to reveal the mode of interaction between HSF1 and potential inhibitors. The results will guide the development of improved HSF1 inhibitors as tools for the elucidation of the HSF1/HSP70 pathway.Due to their strong anti-MM activities, we synthesized dioncoquinone B and modified analogs. Structure-activity relationship (SAR) studies revealed each of the three OH groups to be essential for their activities, while the influence of the other substituents is still unclear. With a dioncoquinone B-related epoxide and dioncophylline A we identified two further agents that are highly toxic to MM cells but not to normal PBMCs. Dioncoquinone B might specifically affect apoptosis and cell cycle regulation as obvious from apoptotic cell death in about half of the tested MM cell lines, whereas the rest of the cell lines exhibited growth arrest or even lacked any growth inhibitory effect. Studies on the cause of cell death revealed a caspase-dependent process indicating involvement of mitochondrial stress response mechanisms. The project will now focus on SAR-guided further structural modifications and on studies on the mode of action of dioncoquinone B, related epoxides, and dioncophylline A. They will be equipped with probes (like biotin) to study their interactions with cellular target(s). Mechanistic studies are planned to analyze molecular effects on mitochondrial processes, apoptosis, and growth-arrest-specific regulation. Their effects on MM cell growth will be evaluated in an MM mouse model.