Nongenetic engineering of subcellular targeted therapeutic agents to facilitate precise cell activity manipulation

• Main Author: Do, Cong Thang
• Other Authors: Xing Bengang
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Chemistry
• Online Access: https://hdl.handle.net/10356/155657
• Institution: Nanyang Technological University

Precise cellular event manipulation is highly demanded in the current development of biomedical therapeutics. This direction requires highly interdisciplinary design systems that revolutionize cell fate regulation. To achieve precise cellular modulation, a thorough understanding of therapeutic agents entering the cells is required. The localization of such agents within the complex three-dimensional cellular structure has substantial implications in the precise activation of signal transduction and recruitment of functional components to regulate cell events and stress responses. Therefore, spatiotemporal modulation of organelle functions and signaling pathways offers ample opportunities in the understanding of disease procession. The above-mentioned methods can be utilized to expedite targeted theranostics with utmost efficacy. In chapter 2, we investigate photothermal nanotheranostics under the influence of nanoheaters’ subcellular distribution and their molecular mechanism on cellular heat response. We revealed the biological basis of divergent thermal effects at subcellular resolution by localizing photothermal nanoparticles into specific locations of cell sections. The cell surface conjugation and the lysosomal cellular uptake nanoheaters induced similar level of heat shock protein expression in live cells after photothermal excitation. However, heat stress from lysosome distribution nanoparticles promoted apoptosis through activation of Bid protein, whereas the heat from clusters of cell membrane localized nanoheater stimulated more membrane structure perturbation and necrotic cell death. This study stipulates intuitions underlying the different subcellular positions of nanoparticles for the selective thermal response, which provides valuable perspectives on optimal precision nanomedicine. In chapter 3, we developed furin cleavable peptide-substrate caged metabolic precursors that can be specifically cleaved in the cytoplasm of targeted tumor cells. Upon specific uptake and cleavage of the furin peptide moiety, the free azide sugar is released after self-immolation of the linker. The unnatural sugar subsequently undergoes a metabolic process to express clickable azide functional group on the cancer cell surface. The conjugated N3 can therefore 2 be easily labelled with DBCO-Cy3 which offers efficient, stable and selective conjugation of therapeutics agent independent of its uptake efficiency. In chapter 4, we introduce a strategy to degrade casein kinase 2α (CK2α) protein using PROTAC molecules. The heterobifunctional degraders were designed based on one warhead which specifically targets the CK2α protein, linked with a E3 ligase, CRBN. Upon incubation, the protein of interest was ubiquitinated and depleted by the 26S proteasome machinery. Our designed molecules exhibited concentration and time dependent degradation of CK2α in different cell lines and zebra fish. “Hook effect” was also observed in which high concentration beyond a certain threshold reduced its efficacy due to saturation of unfavorable binary complexes. Further studies are in progress to investigate the loss of protein function on the downstream effect of cellular regulations. In chapter 5, we propose a different type of small molecule protein degrader to deplete a protein of interest in specific type of cells. Unlike PROTAC molecules which require connection of two functional groups including protein ligand and the E3 ligase targeting moiety, molecule glues offer simpler chemical structure and are more applicable as clinical drugs. Lenalidomide and analogs of thalidomide is a CRBN E3 ligase targeting substrate that is known to degrade other proteins such as Ikaros and CK1α. Upon intracellular administration, this simple molecule showed uniquely high affinity to both proteins, triggering the proteolysis. In this chapter, a nitroreductase responsive moiety was introduced into the lenalidomide structure. Under hypoxic conditions, the caging moiety was removed, releasing the free lenalidomide to function as a CK1α protein degrader. This study paved new insights in the development of small molecule for protein degradation in selected cell type.