Drug candidates with a covalent binding mode have gained interest since the approval of multiple covalent anticancer drugs, but were long avoided due to concerns regarding promiscuous reactivity... Show moreDrug candidates with a covalent binding mode have gained interest since the approval of multiple covalent anticancer drugs, but were long avoided due to concerns regarding promiscuous reactivity with off-target proteins. In this dissertation, the scope and versatility of the newly discovered in situ thiol–alkyne reaction is evaluated: the nonactivated alkynes exhibit an unprecedented target reactivity with excellent thiol selectivity, thus potentially outperforming currently used cysteine-reactive warheads. Chapter 1 starts with the history of (ir)reversible covalent inhibition, the reactivity of (non)activated alkynes, and the serendipitous discovery of the thiol–alkyne reaction. Established technologies for direct detection of covalent protein–drug adducts are reviewed in Chapter 2, and in Chapter 3 a detailed guide for the evaluation of (ir)reversible covalent inhibitors to obtain relevant kinetic parameters is provided, accompanied by kinetic simulations and step-wise protocols for enzymatic activity assays. In Chapter 4, the nitrile warhead in reversible CatK inhibitor odanacatib (ODN) is replaced with alkyne warheads to investigate whether it an irreversible covalent adduct is formed with cysteine protease cathepsin K (CatK) despite having a small recognition element. In Chapter 5, we evaluate if nonactivated alkynes can target noncatalytic cysteine residues by replacing the irreversible covalent acrylamide warhead in EGFR/HER2 inhibitor neratinib by an alkyne warhead. In Chapter 6, the impact of substituents on the alkyne warhead is explored using a panel of ubiquitin-based ABPs bearing substituents on the propargylamide warhead. Finally, the most important findings are summarized in Chapter 7, and placed in the context of covalent drug discovery. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
This work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full... Show moreThis work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full-length proteins that are able to recognize them in a linkage-specific manner. Chapter 2 describes the development of a high-throughput (HT) fluorescence polarization assay used to discover alpha-helical ubiquitin binding domains. This leads to validation of known interacting binding domains and the discovery of a novel set of K6-linkage specific UBDs. In Chapter 3, all eight diubiquitin proteins are prepared using click chemistry that renders the diubiquitin proteins immune to proteolysis by deubiquitinases (DUBs). This allowed for pulldown efforts in different cell lysates and identification of novel linkage-specific interacting proteins using tandem mass spectrometry. The procedure of this effort is outlined in Chapter 4. The study in Chapter 5 details the interaction of one of the major hits found in Chapter 3: the linkage-specific interaction of K27 diubiquitin and UCHL3. The development of a panel of specific assay reagents combined with X-ray crystallography and kinetic modelling lead to a proposed model in which the consequences of the interaction between K27Ub2 and UCHL3 are explained. In Chapter 6 future directions are offered. Show less
In this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO... Show moreIn this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO modifications with examples of substrates where the precise site of modification is important, and substrates where the modification site seems interchangeable.We show that the deubiquitinating enzymes regulate a separate subset of the ubiquitinome than the proteasome. The PARylating enzyme PARP1 show increased activity after ubiquitination, which is regulated by deubiquitinating enzymes.Furthermore, we developed an in-vivo tool to study SUMO dependent interaction using proximity labeling which can be used for microscopy or identification by mass spectrometry.Finally, we investigate binders of Ubiquitin and SUMO chains with specific linkages and elucidate a preference for some chain linkages based on biological pathway of the binder. We also explore possible binding domains and predict the structure of some chain binding complexes using protein docking. Show less
Proteins play an essential role in almost all the processes of a living organism, and post-translational modifications (PTM) can regulate their structure, location, function, and fate. Ubiquitin... Show moreProteins play an essential role in almost all the processes of a living organism, and post-translational modifications (PTM) can regulate their structure, location, function, and fate. Ubiquitin and ISG15 are two of the most versatile and common PTM modifiers in mammalian cells. Aberrant modification of Ubiquitin and ISG15 in cells results in various diseases, such as cancers and microbial infections. This dissertation introduces the development of small-molecule inhibitors against ISG15 deconjugating enzyme USP18 and ubiquitin deconjugating enzyme OTUB2 separately, and the identification of USP16 as a dual deconjugating enzyme that cleaves Ubiquitin and ISG15 from substrates. These research achievements are supposed to deepen the understanding of biology, regulation, and biochemical mechanisms of Ubiquitin and ISG15 deconjugating enzymes, thus paving the way for deconjugating enzymes-targeted therapeutics development in the future. Show less
Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin... Show moreUbiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signalling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research. Show less
This thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an... Show moreThis thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an understudied modification—UFM1. While the first chapter describes the attempt to introduce an unnatural amino acid into proteins to enable covalent substrate capture, the subsequent chapter reports on a unique cascading activity-based probe capable of being relayed through the enzyme cascade. The repertoire of activity-based probes was later expanded to include the Ubiquitin-like modifiers SUMO and UFM1. Additionally, the enigmatic role of UFM1 was deciphered by adapting a proteomics method previously used for SUMO, to uncover UFM1-modifed substrates. This approach enabled the dissection of a relevant pathway governed by UFMylation—ribosomal function during SRP-mediated protein translocation. Show less
The capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is... Show moreThe capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is therefore tightly controlled by regulatory mechanisms. Post-translational modifications (PTMs) are able to directly change the function of a protein and thereby provide a quick functional switch. This thesis focusses on the roles of small ubiquitin-like modifiers (SUMOs) and their crosstalk with other post-translational modifications during cell division, at the proteome-wide level as well as the single target protein level. Show less
