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Dimeric ligands for G-protein coupled receptors that are involved in human reproduction, namely the gonadotropin releasing hormone receptor, the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were synthesized and biologically evaluated.

In this thesis, solid phase peptide synthesis (SPPS) and chemical protein synthesis are used to synthesize novel tools such as assay-reagents and activity-based probes based on Ubiquitin-like proteins⁠. These probes are used to explore the function and specificity of deconjugation enzymes⁠.

Contains fulltext : 292991.pdf (Publisher’s version ) (Open Access) Radboud University, 13 juni 2023 Promotor : Kraan, P.M. van der Co-promotores : Bonger, K.M., Bosch, M.H.J. van den 163 p.

The inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative bioorthogonal chemistry. The research described in this Thesis aims to develop synthetic strategies which enable the IEDDA pyridazine elimination to be applied as a versatile toolbox in chemical biology studies. More specifically, it entails modification of antigenic (MHC-I) peptides and (CD1d) glycolipids with a trans-cyclooctene (TCO) moiety to allow chemical control over the recognition of these biomolecules by immune cells. Synthetic advances which encompass the entire scope of the IEDDA pyridazine elimination are additionally described.

The inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative bioorthogonal chemistry. The research described in this Thesis aims to develop synthetic strategies which enable the IEDDA pyridazine elimination to be applied as a versatile toolbox in chemical biology studies. More specifically, it entails modification of antigenic (MHC-I) peptides and (CD1d) glycolipids with a trans-cyclooctene (TCO) moiety to allow chemical control over the recognition of these biomolecules by immune cells. Synthetic advances which encompass the entire scope of the IEDDA pyridazine elimination are additionally described.

Cytotoxic T-cells (CTLs) are involved in the clearance of viruses and killing of tumor cells. The capacity of these killer cells to clear viruses and kill tumor cells can be harbored by the creation of vaccines. Improving understanding of T-cell activation and the possibility to understand, influence or even have control over this process might lead to better anti-cancer vaccines. Chemical tools can be a good addition to fill in the gaps of knowledge about T-cell activation.

Large parts of the research described in this Thesis aims at the development of oligopeptide-masked toxins and their in situ immunoproteasome-mediated activation. Chapter 2 focuses on the development of selective fluorogenic substrates for each of the iCP and cCP proteasome subunits. Chapter 3 describes the utilization of proteasome cleavable linkers in order to release a toxic entity in immunoproteasome expressing cells. By selecting oligopeptide sequences of previously described immunoproteasome-selective fluorogenic substrates and inhibitors, and linking these to doxorubicin via a cleavable linker, new constructs were obtained. Chapter 4 describes an immunoproteasome inhibitor-doxorubicin conjugate that targets multiple myeloma derived cells and releases doxorubicin upon low-dose photon irradiation. Chapter 5 describes how a trans-cyclooctene (TCO) is utilized for the release of a toxic payload via a click to release reaction in conjunction with selective proteasome targeting. TCOs were used in order to facilitate toxin activation via an inverse electron demanding Diels-Alder reaction with tetrazine as a dienophile. Chapter 6 gives insight in the development of pan-immuno subunit selective proteasome inhibitors based on P3 and P4 scaffold exchanges. Finally, the research described in this thesis is summarized in Chapter 7, and future prospects based the presented results are discussed.

Phosphorylation affects all four major biomolecules – proteins, lipids, carbohydrates and nucleic acids – and plays a pivotal role in the most fundamental cellular functions. A prime example of a PTM that caught a lot of scientific interest is adenosine diphosphate ribosylation (ADP-ribosylation), carried out by the PARP family of enzymes. ADP-ribosylation is an indirect form of (pyro)phosphorylation, where a monomer or polymer of ADPr is attached to an acceptor protein. The process of ADP-ribosylation has been linked to DNA damage repair, telomere maintenance and regulation of apoptosis, highlighting it of biomedical importance, most notably in relation to cancer therapy and inflammatory disorders. Well-defined phosphorylated molecular tools have already proven to be effective at elucidating the molecular mechanisms behind both normal and pathological cell function. However, the natural pyrophosphate group is inherently susceptibility to hydrolysis, trans-esterification and enzymatic cleavage. This property limits their use through possible premature degradation or restricted synthetic accessibility. The research described in this thesis focuses on the development of new molecular tools, primarily intended to facilitate the study of adenosine diphosphate ribosylation. In this context new reagents and methodologies have been developed for the synthesis of stabilized pyrophosphorylated bioisosteres.

The studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a degree of ambiguity, this approach allows to iteratively refine and validate the model and interpret its meaning for the molecular basis of protein function. Often all three points at the same time. This dynamic nature makes the use of structural models in the design of therapeutic compounds especially useful since the inhibition of a certain protein function might not require a structure to be accurate down to the last atom but rather highlight key interactions or structural features that can be addressed in context of small molecule or peptide inhibitors. Presented are the use of strucutral biochemistry techniques to investigate the mechanism of how the ubiquitine ligase PSIP1 obtains its target specificity. Furthermore, another epigenetic effector protein PSIP1 is investigated with the aim to develop a workflow for the design of potential peptide-based inhibitors.

Contains fulltext : 202990.pdf (Publisher’s version ) (Open Access) Radboud University, 08 mei 2019 Promotores : Pruijn, G.J.M., Rutjes, F.P.J.T. Co-promotor : Bonger, K.M. 181 p.

Contains fulltext : 204181.pdf (Publisher’s version ) (Open Access) Radboud University, 23 mei 2019 Promotor : Pruijn, G.J.M. Co-promotor : Bonger, K.M. 191 p.

Contains fulltext : 197532.pdf (Publisher’s version ) (Open Access) Radboud University, 20 november 2018 Promotor : Rutjes, F.P.J.T. Co-promotor : Bonger, K.M. 189 p.