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

点击翻译按钮获取中文摘要

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.