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
This thesis describes the synthesis and biochemical evaluation of a variety of cyclophellitol based activity-based probes and inhibitors targeting various endo- and exo-acting retaining... Show moreThis thesis describes the synthesis and biochemical evaluation of a variety of cyclophellitol based activity-based probes and inhibitors targeting various endo- and exo-acting retaining glycosidases. In the last two decades a variety of probes and inhibitors for (hemi)cellulose degrading enzymes have been developed. However, at the onset of the work described in this thesis the focus has been mainly on cellulases, xylanases, xyloglucanases and glycanases. Chapter 2 describes the design and synthesis of a first generation of inhibitors and probes for β-mannanases. The general scaffold is composed of a β-1,4-linked mannobioside featuring either an epoxide or an aziridine warhead. All synthesized probes feature a linker attached to the nonreducing end and are condensed with either biotin or Cy5. Biochemical evaluation in an Aspergillus niger secretome revealed the utility of these compounds but also the limitations. The probes react efficiently with some endo-β-mannanases yet lack reactivity with others. Chapter 3 describes the design and synthesis of a second generation of β-mannanase probes and inhibitors designed to overcome the limitations of the first generation by changing the general scaffold. In this chapter two conceptually distinct ABP scaffolds are presented: a β-1,4-linked mannotriose and a β-1,4-linked manno-gluco configured scaffold. The second generation Cy5 probes are evaluated in Aspergillus niger secretomes and compared to the first generation. Chapter 4 describes studies towards the design and synthesis of inhibitors and ABPS for both α-N-acetylglucosaminidase and α-Nacetylgalactosaminidase. The chapter introduces an orthogonally protected ribose building block as common intermediate from which both mannoseand talose configured cyclohexenes are synthesized. Furthermore it describes a late state stereoselective C-2 inversion with an azide to install a 20 nitrogen atom at C-2. Chapter 5 presents a summary and suggestions forfuture research. Show less
Cells express a large array of membrane receptors on their surface that function as a communication channel between the extra- and intracellular environment of the cell. Ligands for these receptors... Show moreCells express a large array of membrane receptors on their surface that function as a communication channel between the extra- and intracellular environment of the cell. Ligands for these receptors span a wide range of biomolecules, from proteins to carbohydrates to small molecules. Some receptors are continuously recycling between the membrane and the inside of a cell, whereas others are in a steady-state at the membrane and need ligand binding for their activation and subsequent internalization. Synthetic molecules that bind to these membrane receptors can be used to either modulate their function, or to target a reporter group (i.e. a fluorescent dye) and/or a bio-active compound (drug, protein) to cells that express this receptor, ensuring delivery to a specific cell-type. The research described in this Thesis combines synthetic and biochemical methodologies to create ligands that interact selectively with membrane receptors of the GPCR and lectin-binding families. Attachment of synthetic probes, proteins or cytostatic molecules to these ligands by a variety of chemical and enzymatic methods ensured their uptake exclusively into cells that expressed the receptor of interest. Visualization of this process was enabled by the incorporation of a fluorescent dye into the final constructs. Show less
