Use of styrylbenzothiazole photoswitches in drugs for spatiotemporal control over microtubule-dependent biology

Gao, Li (2021): Use of styrylbenzothiazole photoswitches in drugs for spatiotemporal control over microtubule-dependent biology. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Thousands of biological processes take place within a single cell, every second of its existence. These processes are meticulously orchestrated and highly regulated in order to guarantee proper functioning and homeostasis within the organism. Multiple proteins assemble to form highly sophisticated molecular machines and structures to carry out certain tasks at exact timepoints, such as the microtubule-based mitotic spindle apparatus, which separates the chromosomes during mitosis; or the replisome that performs DNA replication with an unprecedented level of precision and reliability. The visualization of cells, their biological processes, and our understanding of the biomolecular function of proteins, have grown rapidly with the invention of groundbreaking new technologies and continuous improvement of existing instruments. Cryo electron microscopy, high-speed or super-resolution microscopy have considerably pushed the limits of temporal and spatial resolution past the micron spatial scale and millisecond time scale, opening new frontiers in modern biology. Small molecule drugs which can modulate, inhibit or amplify these machineries and their functions, have been a key factor to explore the molecular dynamics of proteins and cellular systems. Consequently, while selective small molecule inhibitors for certain protein targets have garnered much interest in drug discovery, as promising drug candidates to treat diseases, they have also become crucial research tools in order to perturb and study protein and network function. However, the technological leap has created a discrepancy between modern technology and classic drugs when used as research tools. With high-precision instruments able to observe highly dynamic cellular systems, it is the current set of molecular tools available to researchers that lack the spatiotemporal precision to control these dynamic cellular systems on a submicron and millisecond scale. Influencing or controlling biological processes with classic small molecule inhibitors without spatiotemporal specificity is a poor method to investigate a finely tuned machine. Part One of this work will focus on the introduction of innovative molecular tools that allow simple control over a highly dynamic cellular system: the microtubule cytoskeleton. This is intended to optimally utilize, and in return further develop, state of the art imaging techniques and to expand biologists arsenal of molecular tools that can help to answer key questions in neuroscience, embryology, and cytoskeleton research, by using light as a non-invasive, high-precision, bioorthogonal regulator for biological application. I introduce the styrylbenzothiazole (SBT) photoswitch as a research tool in cell biology, with initial application to reliably enable fast and reversible in situ optical control over the microtubule cytoskeleton. To situate this application I will discuss the problems of current state-of-the-art photoswitchable microtubule destabilizers, in particular azobenzene based PST-1, and showcase the SBTubs as an SBT-based alternative. We confirm their biological utility as photoswitchable tubulin inhibitors, compare their (photo)chemical & metabolic robustness, and test their compatibility to common fluorescent imaging tags over the azobenzene scaffold. Part Two will focus on consolidating the newly introduced SBTs as a powerful alternative photoswitch scaffold with complementary features to azobenzenes. I perform an SAR study and identify two lead compounds SBTub2M and SBTubA4P as low nanomolar and water-soluble photoswitchable antimitotics that enable photocontrol over microtubule dynamics and structure in 3D systems and animal models (D. rerio, X. tropicalis), thus bringing in vivo photopharmacology one step closer to realization. Furthermore, I report the first photopharmacology study of a styrylthiazole (ST) photoswitch. In the final part of my thesis I will apply this ST photoswitch scaffold instead to address the taxane binding site, showing its applicability and general features. I design and synthesize the first ever reported photoswitchable epothilones (STEpos) and offer proof of principle of their light-dependent stabilization of microtubules. Taken together, this research makes a contribution towards (a) spatiotemporal control of microtubule research, helping antimitotic photopharmaceuticals advance beyond 2D cell culture and (b) reshaping the field of photopharmacology by introducing a focus on photoswitches like the SBTs which respond to biologists’ technological needs & capacities, which will contribute to new SBT-based photopharmaceuticals both in microtubule research and beyond.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Photopharmacology, Photoswitch, Azobenzene, Microtubules, Chemical Optogenetics
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. Oktober 2021
• 1. Bericht­erstatter:in: Thorn-Seshold, Oliver