Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel... Show moreGlobal healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo. Show less
In the last decades, activity-based protein profiling (ABPP) has emerged as a powerful chemical tool that may aid the ever-challenging drug discovery process. In this thesis ABPP is explored as a... Show moreIn the last decades, activity-based protein profiling (ABPP) has emerged as a powerful chemical tool that may aid the ever-challenging drug discovery process. In this thesis ABPP is explored as a versatile tool in drug discovery and cell biology.ABPP enabled rapid assessment of clinical samples from patients suffering from cardiac ischemia, thereby giving insight into the serine hydrolase activity profile of these patients. The identification of molecular role players may lead to the discovery of novel therapeutic targets or biomarkers. In addition, ABPP can provide insight in a drug’s interaction landscape, by enabling target engagement studies and inhibitor selectivity profiling. This was demonstrated by the identification of multiple off targets of the experimental drug BIA 10-2474 that caused severe neurological symptoms in a phase I clinical trial. In zebrafish larvae, the ABPP methodology enabled in vivo selectivity profiling and in addition served as a powerful tool to map the kinase and serine hydrolase landscape throughout embryonic development. Lastly, combining ABPP with other biochemical techniques including CRISPR/Cas9 technology and lipidomics, can provide new insights in cellular biology, which was showcased by the identification of ABHD6 as a diacylglycerol-lipase in a cellular model of neuronal differentiation. Show less
The disease acute myeloid leukemia (AML) is characterized by fast progression and low survival rates. Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation... Show moreThe disease acute myeloid leukemia (AML) is characterized by fast progression and low survival rates. Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. This thesis describes the discovery and evaluation of new chemical series of FLT3 inhibitors and furthermore employs label-free chemical proteomics techniques to evaluate new and existing FLT3 inhibitors for their cellular kinase off-target profile. Show less
Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal... Show more Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal storage disorders (LSDs) are caused by inborn metabolic errors due to deficiency in specific lysosomal enzymes, most commonly GHs. Diagnosis and treatment of LSDs require regular quantification of the active lysosomal enzymes in patient tissues. Activity-based protein profiling (ABPP) has emerged in the past decades as a powerful technique to study enzyme families in cell extracts and living tissues. Originally developed for serine hydrolases and cysteine proteases, various enzyme classes can be studied by means of ABPP today, including retaining GHs. The research described in this thesis focused on expanding the field of activity-based glycosidase profiling through the development of functional and configurational analogues of cyclophellitol aziridine as activity-based probes (ABPs) for various retaining glycoside hydrolases (GHs), namely α-L-fucosidases, β-glucosidases, α-glucosidases and β-glucuronidases. Attention is focused on the design and synthesis of the cyclophellitol aziridine derivatives and their application in chemical biology studies of various retaining GHs. Show less
This Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are... Show moreThis Thesis aims at the development of novel subunit selective inhibitors of the proteasome. -Three vinyl sulfone analogues of three epoxyketone containing inhibitors described in literature are synthesised and characterised. The nature of the electrophile determines subunit selectivity of the inhibitor. -Ten Michael acceptors are coupled to three peptoid tails yielding thirty peptide-like compounds. None of the peptoid Michael acceptors are efficient proteasome inhibitors. Using the Staudinger-Bertozzi ligation followed by affinity purification, tryptic digestion of the isolated proteins and LC/MS/MS identification, the targets of two compounds in HEK293T and RAW264.7 cells are determined. -Syringolins are potent proteasome inhibitors found in nature. A syringolin structural motif was built in peptoid epoxyketones and vinyl sulfones yielding a 16-membered library of proteasome inhibitors. The distance between the urea and electrophile dictates subunit selectivity. -Three cyclooctynes are compared to the Staudinger-Bertozzi two-step labelling strategy. Cyclooctynes quantitatively convert azide labelled proteasomes at a lower concentration than the Staudinger-Bertozzi phosphane but cyclooctyne two-step labelling is associated with tremendous background labelling. -A library of peptoid vinyl sulfones with basic amino acid side chains is synthesised. A compound with two benzylamine residues is a potent and _2 selective proteasome inhibitor. Attachment of a BODIPY fluorophore yielded a _2 selective probe. Show less
