Search

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings.

Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-β and casein kinase-1α and consequently degraded by the proteasome via the SCFβTrCP ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKβ and cell motility controlled by Rap-integrinsignaling. © 2013 Elsevier Inc.

We developed a scalable synthesis of γ-thiolysine starting straight from lysine. The application of γ-thiolysine was compared to δ-thiolysine in the chemical synthesis of K48 and K33 linked diubiquitin conjugates. Both γ- and δ-thiolysine were found to perform equally efficiently as handles for non-enzymatic ubiquitination.

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 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.

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.

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-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.

The research presented here not only describes attempts to develop selective inhibitors for SENPs and PARK7 but also provides a chemical toolbox to study the function of PARK7. Both SENPs and PARK7 are attractive therapeutic targets for drug development. The results of the research and chemical tools described in this thesis will further aid in the development of more potent and selective inhibitors of SENPs and PARK7 for therapeutic purposes.

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 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.

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 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.

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 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.

Glycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe disease, but also in various cancers. The discovery of potent and selective glycosidase inhibitors for fundamental glycobiology studies and as leads for drug discovery requires access to suitable (glycomimetic) compound libraries, as well as easily applicable assays and screening formats. The research described in this Thesis was aimed to explore how covalent and irreversible glycosidase inhibitors can be applied in the screening of focused compound libraries on various glycosidases using fluorescence polarization activity-based protein profiling (FluoPol-ABPP).

The two branches of our highly advanced immune system work closely together to detect and eliminate pathological threats. The first line of defense is provided by the innate immune system via detection of pathogenic or tumor cell fragments. Adaptive immune cells, on the other hand, recognize pathogens or malignant cells more specifically by scanning peptides, small protein fragments, presented by MHC molecules on other cells in our body. Specialized white blood cells (cytotoxic or killer T cells) can distinguish self- from non-self-peptides and directly eliminate cells that display signs of infection or mutation.The work described in this dissertation highlights how adaptive immunity can be used to our advantage, either from a therapeutic or diagnostic perspective. Immunotherapies that induce or promote anti-tumor or anti-viral responses have proven efficacious against infection and cancer. One strategy described is the development of chemically-modified epitopes as peptide vaccines, but small-molecule chemical drugs are also playing an increasing role in the field of cancer immunotherapy.In addition, monitoring of immune status and response to treatment, as well as mapping of epitopes, can aid diagnosis and design of treatment plans. The second part describes a novel method and application to visualize and monitor cytotoxic T cells.

The research described in this thesis aimed focused on CHIKV replication and on the identification of much-needed inhibitors of CHIKV infection. Following the development of an in vitro assay to study CHIKV replication, this tool was used to characterize the mode of action (MoA) of antiviral compounds and suramin was identified as a potent inhibitor of viral RNA synthesis. However, we discovered that in cell culture, suramin’s antiviral activity was mainly due to inhibition of CHIKV binding/entry, and to a lesser extent of virus release. Suramin was also found to inhibit binding/entry and virion biogenesis of Zika virus (ZIKV), a recently emerged flavivirus. Due to its ability to form electrostatic interactions with positive charges on proteins, suramin may block the contact between virions and their (co)receptors, by interacting with either virus or receptor, or with both. Using radioactively-labelled suramin, it was clearly shown that the compound interacts with CHIKV particles, more specifically with their envelope proteins. Additionally, suramin could interfere with cell attachment and/or the structural changes required for fusion. Suramin-resistant CHIKV variants were selected, which contained mutations in the E2 envelope protein (involved in receptor interactions), supporting the idea that suramin blocks the early steps of the infectious cycle.

Malignant pleural mesothelioma (MPM) is an aggressive tumor of the mesothelial cells lining the pleura. The poor prognosis of patients indicates the importance to find more effective treatments. Therefore, this thesis focused on finding new treatment strategies for MPM. We present a method in which primary MPM cultures were chemically profiled to determine second-line treatment for patients. With this personalized treatment strategy we were able to predict individual patient responses to selected drugs. Based on the chemical profiles of all tumor cultures, these cultures could be divided in three groups. Transcriptomic analysis of these groups identified a unique genetic profile separating these groups and identifying the fibroblast growth factor receptor (FGFR) as a new target for the treatment of MPM. The cultures sensitive to FGFR inhibitors were dependent on FGFR3 mediated signaling which was regulated by BAP1, indicating BAP1 can serve as a biomarker for this treatment. Another target explored in this thesis was 5T4. We showed that 5T4 was only expressed in the tumor cells and that antibody-drug conjugates effectively kill high 5T4 expressing MPM cells. Altogether we present different promising novel strategies in the treatment of MPM, which could improve the survival outcome of these patients.

Chapter 2 describes a post-synthetic methodology to introduce a fluorescent label in highly lipophilic, Pam3Cys based conjugates. The fluorescent labels were appended to the peptide part of the conjugate with the aid of a strain promoted [3+2] azide-alkyne cycloaddition. In Chapter 3 a synthesis is discussed of a structurally simple human specific TLR-2 ligand with diminished lipophilicity, as compared to Pam3Cys. Conjugation of such moiety to peptide is studied and optimized to produce human specific analogues of the conjugates described in Chapter 2 with higher solubility and an equal propensity to activate TLR-2. The synthesis of a newly designed TLR-7 agonist is demonstrated in Chapter 4 as well as the synthesis of a selection of self-adjuvanting immunogenic peptides that contain a model MHC-I epitope (SIINFEKL). A biocompatible methodology to reduce an azide in a side chains of peptides is described in Chapter 5 with a particular focus on side reaction occurring during the reduction. A selection of phosphines is evaluated under biocompatible aqueous conditions. 6 describes the development of a convergent synthesis of the naturally occurring conjugate between the 5’- terminal fragment of genomic RNA from Coxsackie virus and the full-length viral genome-linked protein (VPg).

In this thesis we reflect on the effects differential DNA binding of the estrogen receptor α (ERα) can have on the behavior of breast cancer and which factors can contribute to this. ERα is a transcription factor than can drive tumor cell proliferation and approximately 70% off all breast tumors is thought to be dependent on the activity of this hormone-mediated transcription factor. After stimulation of ERα a wild variety of co-factors are recruited, leading to the assembly of a transcriptional complex. Although there are multiple ways of targeting the action of ERα and thereby inhibiting tumor growth, still a significant proportion of patients develop a recurrence. Cross-resistance between the different endocrine therapy options can occur, but a proportion of patients that relapse on one type of therapy can still benefit from a different treatment modality, illustrating the existence of multiple resistance mechanisms which can be treatment selective. A better understanding of ERα-biology and the development of endocrine therapy resistance, can lead the way to the discovery of novel biomarkers and potential drug targets, that can further increase patient survival.

Patients with advanced head and neck cancer are treated with a combination of chemotherapy (CT), radiotherapy (RT) and surgery. Despite these multimodal treatments, the prognosis of these patients is relatively poor due high local recurrence rates or development of a second primary tumor. The combination of CT (cisplatin, cetuximab) with concurrent RT (CRT) has resulted in minor survival benefits, but with substantial increase in toxicities (causing side effects), when compared to RT alone. This underscores the urgent need for novel radiosensitizers specifically targeting cancer cells and not normal tissues. Ideally, this will lead to improved survival with lower toxicities (side effects). To identify such radiosensitizers, we performed multiple drug screens in absence and presence of irradiation. One screen identified GSK635416A. This drug radiosensitizes cancer cells, thereby outperforming the radiosensitization of cisplatin and cetuximab, while exhibiting virtually no cytotoxicity in the absence of irradiation and in normal fibroblast cells. Screening the FDA-approved oncology drugs library identified rapamycin, raloxifene and tamoxifen as promising radiosensitizers for head and neck cancer.Another drawback in patients with advanced head and neck cancer is the variability in individual response to CRT. To this aim, we studied the sponge-supported culture assay for culturing fresh tumor biopsies, and optimized this culture method for its potential use as an individual preclinical treatment prediction model.

Traditional drug discovery approaches have been hampered by (in vitro) cell-culture models that poorly represent the situation in the human body. Principally, cells grow in the body in a three-dimensional (3D) environment that cannot generally be captured using cell culture methods. For this reason, cell-culture models have been developed where cells grow in a 3D-environment, which allows them to form structures that are more comparable to tissue in the body. However, the full complexity of these advanced cell-culture models can only be fully used for routine drug testing if the cell culture model can be used on a large scale (also termed high-throughput screening or HTS), and if the readout can capture all of the biological complexity reflected by the 3D-cultured cells (high-content screening or HCS). Due to these technological limitations, 3D cellular models are not yet routinely applied in drug and drug-target discovery. This thesis describes the development of fully-scalable 3D cell-culture screening platforms in the context of cancer and polycystic kidney disease.

The aim of my work was to identify novel proteins that are involved in different aspects of disease biology, in the hope that we can target these to treat the disease. Using large-scale screening technologies, I found a novel regulator of chemosensitivity to the anti-cancer drugs etoposide and doxorubicin. Furthermore, novel proteins and drugs that target MHCII antigen presentation were picked up. Interestingly, one of these drugs is currently used in the clinic to treat MHCII-dependent autoimmune diseases, but for which the mechanism of action is unknown. Lastly, these screens yielded enzymes that control the dynamics of endosomes in the cell, which can potentially be targeted in cancers that rely on overactivation of growth signaling receptors. Taken together, my work has provided new potential targets for the treatment or classification of several tumor types, as well as a potential mechanism of action for a widely used drug.

Immunotherapy of cancer has established itself in recent years as a promising novel approach to treat cancer patients. One of the experimental approaches is based on therapeutic vaccination. We have previously developed vaccines consisting of synthetic long peptides (SLP) which successfully eradicated premalignant lesions in 50% of patients. To further improve these vaccines, a Toll-like receptor ligand (TLR-L) was conjugated to SLP which enables targeting of the SLP to relevant antigen-presenting cells while concomitantly activating these cells. In fact, the research described in this thesis shows that TLR-L SLP conjugates induce enhanced antitumor immunity. Furthermore, optimization of the TLR-L led to even furher improved antitumor responses in mice. Using human cancer patient-derived lymph node cells, we show that lymph node-derived T cells are favorably activated by the TLR-L SLP conjugates. Finally, we combine multiple innate immune stimulatory agonists (TLR2-L and NOD2-L) in one molecule to establish synergistic immune activation. Overall, the research described in this thesis demonstrates the potency of TLR-L SLP conjugates as cancer vaccines, which could strongly contribute to the treatment of cancer patients.

Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion. The work described in this thesis has led to the identification of an activity-based probe (ABP) for DAGL-α. This ABP was instrumental for the identification of a new class of highly selective DAGL inhibitors. These novel inhibitors can be used to investigate the role of the diacylglycerol lipases in metabolic syndrome and neurodegenerative diseases such a Parkinson’s disease, Alzheimer’s disease and Multiple Sclerosis.

The work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and Staudinger‐Bertozzi reaction under native/denatured protein conditions Chapter 5 is focusing on the application of quantitative activity‐based proteasome profiling in the prognosis of cancer therapeutics. A combination of ABPP and global proteomics is performed to elucidate the bortezomib sensitivity and resistance mechanisms in leukemia and solid tumor cells. Chapter 6 describes the characterization of the newly discovered proteasome subunit β5t by ABPP and LC/MS proteomics. The subunit is proven to be catalytically active. A hydrophilic Thr residue on the P2 position of the proteasome inhibitor improves the inhibitory efficiency of β5t, which indicates it might prefer to cleave hydrophilic peptides. Chapter 7 describes the identification of O‐GlcNAcylation modifications on the ubiquitin receptor protein hHR23B and characterization of how the sugar moiety influences the conformation and functions of the protein.

Proteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP) and one or two 19S regulatory particles (RP). The constitutive proteasome core particle (cCP), which is expressed in all mammalian tissues, contains three catalytically active subunits, namely β1c, β2c and β5c. Lymphoid cells express another proteasome core particle known as the immunoproteasome (iCP). In iCPs, β1c, β2c and β5c are replaced by β1i, β2i and β5i. The research described in this thesis reports on the development of new subunit-selective inhibitors and activity-based probes, on the development of an assay to simultaneously monitor all cCP and iCP catalytic activities and on the development of a method that reports on CP catalytic active subunit composition. The tools that stem from the work described in this thesis can now be used to unravel the role of each individual catalytic subunit in a chemical genetics setting (selective and (near) complete inhibition of each subunit), and to clarify the role of mCPs, in, for instance, antigen presentation and cancer. Furthermore, these tools could possibly serve as leads in the discovery of agents for future treatment of cancer and autoimmune diseases.

The first part of this thesis describes the development of inhibitors of autotaxin (ATX or ENPP2), a phosphodiesterase that is responsible for the production of the lipid lysophosphatidic acid (LPA) in the circulation. ATX is implicated in several diseases including inflammation, fibrotic disease and cancer, making it an interesting potential drug target to study. ATX inhibitors are required in the validation process of ATX as a drug target. The second part of this thesis describes development of dual specificity phosphatases (DUSP) inhibitors that inhibit bacterial growth in human cells. These inhibitors act by inhibiting DUSP proteins in the host cell that are essential for bacterial growth. This approach can be used to control bacterial infection and could be a useful addition to the current treatment of bacterial infections that target solely the bacteria itself.

In cells, proteins are continuously synthesized and degraded to control protein levels and thereby regulate a wide variety of biochemical processes. The proteasome is the main cellular degradation machinery, responsible for the degradation of key proteins involved in the regulation of a wide range of cellular processes, including quality control, cell cycle progression, cell differentiation, signal transduction and apoptosis. Inhibition of the proteasome causes disruption of many regulatory processes, eventually leading to cell death. The observation that many cancer cells are more sensitive to proteasome inhibition than normal cells has led to the development of several proteasome inhibitors for the treatment of cancer. Proteasome activity in its broadest sense is the central theme of this thesis. The development and characterization of different chemical proteasome activity probes that can be used to study proteasome activity in a wide range of tissue types using a variety of assays are described. Subsequently, these probes are used to assess the effects of different proteasome inhibitors in preclinical and clinical studies and to purify proteasome and characterize its activity and composition. Finally, the molecular mechanism of proteasomal antigen splicing is studied.