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

该项目旨在开发针对多发性骨髓瘤（MM）的新型药理学药物，采用两种不同的策略：在基于热休克蛋白70（HSP 70）和热休克转录因子1（HSF 1）在MM中的病理生物学作用的最新发现的靶向方法中，开发了HSP 70和HSF 1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用于特定的抗MM活性，并进一步探索以发现新的靶点或作用机制。从HSP 70的结构信息开始，通过虚拟筛选鉴定了靶向核苷酸结合结构域和底物结合结构域之间界面的分子，得到了对MM细胞系有活性的五种化合物。使用最佳命中作为起始点，合成四氢异喹啉酮的聚焦文库，产生一系列针对MM细胞的活性分子，而对外周血单核细胞（PBMC）没有毒性。为了靶向HSF 1，通过Ugi-Heck合成法合成了两个苯基修饰的异喹啉化合物库。令人惊讶的是，只有开环的Ugi中间体显示出抑制作用，但与未知的作用模式。HSP 70的主要目标是计算和实验表征和优化的命中化合物。这就需要对HSP 70抑制剂的结合机制进行基于靶点的研究，并对HSP 70抑制剂的细胞内效应进行扩展的生物学测试。为了证明特异性HSP 70结合和变构阻断，我们将建立HSP 70测定法，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发展与实验相互作用的机制假设。这将指导缓蚀剂的优化和新化合物的合成。- 为了进一步开发HSF 1抑制剂，将使用先进的功能生物测定以及使用质谱的结构分析来揭示HSF 1和潜在抑制剂之间的相互作用模式。本研究的结果将为进一步研究HSF 1/HSP 70通路提供指导。由于具有很强的抗MM活性，我们合成了二醌B及其类似物。构效关系（SAR）研究表明，三个羟基中的每一个都是其活性所必需的，而其他取代基的影响尚不清楚。与二醌B相关的环氧化物和dioncophylline A，我们确定了两个进一步的代理商，是高毒性的MM细胞，但不是正常的PBMC。二醌B可能特异性地影响细胞凋亡和细胞周期调节，这从约一半的测试MM细胞系中的细胞凋亡细胞死亡中显而易见，而其余的细胞系表现出生长停滞或甚至缺乏任何生长抑制作用。对细胞死亡原因的研究揭示了半胱天冬酶依赖的过程，表明线粒体应激反应机制的参与。该项目现在将集中于SAR引导的进一步结构修饰和对二醌B、相关环氧化物和二醌茶碱A的作用模式的研究。它们将配备探针（如生物素），以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。将在MM小鼠模型中评价其对MM细胞生长的影响。

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.