The assembly of modular constructs for lipidation (lipo-TAGs), that is scaffolds incorporating one or two lipid chains, fluorescent labels for intracellular localization monitoring, and proper spacer/linker systems for bioconjugation, was the first task planned and accomplished during this project. The utilization of the Ugi-4CR, allowed me to build diverse lipo-TAGs in a highly efficient and modular way. These lipo-TAGs contain two lipid chains, either two palmitic or one palmitic and one myristic fatty-acid derived chains, spacers with different lengths and hydrophobic character, and eventually carboxyfluorescein, which was chosen as a fluorophore for internalization monitoring. Different linkers were either directly or post-Ugi-4CR introduced, i.e. containing the azide and dibenzocyclooctyne (DBCO) groups, allowing protein conjugation via a strain-promoted azide-alkyne cycloaddition (SPAAC), and maleimide, allowing site-specific cysteine modification via Michael addition.
As part of my endeavors using the Ugi-4CR for the assembly of the lipo-TAGs, I conceived the idea of developing a new multicomponent reaction based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi-4CR. This new multicomponent reaction allowed the synthesis of novel pyrazole amide derivatives and proved highly efficient for both the assembly of small molecules and the modification of peptides, including peptide stapling (Figure 2). The results of this work were recently published in a high-impact journal and presented at the 8th International Conference on Multicomponent Reactions and Related Chemistry, Burgos, Spain. The main significance of this part of the research lies in the growing demand for high diversity- and complexity-oriented processes, not only allowing the efficient assembly of modular systems such as the lipo-TAGs but also structurally complex and diverse libraries of molecules meeting different medicinal chemistry purposes.
The next task consisted of the synthesis of the lipobodies, that is the expression, purification, and modification of the different antibody formats. The main targets proposed in this research project were different oncogenic RAS proteins, but unfortunately, the expression of the intracellular domain antibodies targeting RAS was extremely challenging. Anticipating this type of risk, I decided to move forward with the proof of concept and selected other oncogenic targets such as the human epidermal growth factor receptor 2 (HER2) and mesothelin. As anti-HER2 formats, I chose two anti-HER2 affibodies with different cysteine mutation sites and the engineered antibody THIOMAB V205C. After efficiently achieving conjugation of these formats to the lipo-TAGs, the analytical and biophysical characterization of the obtained lipobodies allowed us to asses that neither the affibodies' structure nor their binding to the HER2 receptor was significantly affected in comparison with the parent affibodies. Thereafter, I modified these formats with fluorescently labeled lipo-TAGs, which allowed me to study their cell-internalization properties in comparison with the non-lipidated constructs.
The results of the current project were presented in internal group seminars as well as in the Halle-Glacier workshop “From Immunology to bioactive compounds". The most recent communication of the results of this project was at the Bayer Boundary Breaking Science Lecture on “Multicomponent reactions: exploring a wider chemical space toward medicinal and biomedical applications” which took place online on the 19th of October, 2023. Other dissemination forms include different interviews from the Bayer Science Foundation and the Women in Research #LINO23 blog.
