Authors:	Schwickert, Marvin
Title:	Development of covalent enzyme inhibitors: RNA methyltransferases and proteases as targets
Online publication date:	14-Aug-2023
Year of first publication:	2023
Language:	english
Abstract:	Covalent inhibitors were usually avoided in drug development due to concerns of possible toxic side effects caused by off-target modification. Now they are increasingly emerging not only as therapeutic drugs but also as biochemical tools. Compared to non-covalent inhibitors, they have several advantages such as higher affinity, more efficient competition with endogenous substrates, prolonged residence times, and less susceptibility to drug resistance. The aim of this work was the development and analysis of potential covalent inhibitors for three different enzyme classes. The RNA methyltransferases (MTases) DNMT2, NSUN2, and NSUN6 are involved in various physiological processes and have also been found to be associated to diseases such as cancer, yet their biological roles are largely unknown. Therefore, they represent promising targets for drug discovery and for the development of activity-based probes (ABP) that can be used to improve understanding of these enzymes and their RNA modifications. Based on the natural product inhibitor S-adenosyl-L-homocysteine (SAH), additional side chains (alkyl, aromatic, warhead-decorated) were attached to the scaffold to explore the affinity and selectivity profile for DNMT2, NSUN2, and NSUN6. The first generation of SAH-based inhibitors revealed an alkyne derivative as a potent inhibitor (IC50 = 12.9 ± 1.9 μM) of DNMT2. Subsequently, electron-deficient aromatic side chains were analyzed representing the second generation of SAH-based inhibitors, leading to not only the most potent but also the first covalent DNMT2 inhibitor (IC50 = 1.2 ± 0.1 μM) that is selective over NSUN2 and NSUN6. These findings represent a promising basis for the development of ABPs for future studies. Additionally, NSUN6 selective inhibitors were identified that can be further optimized to increase affinity. Schistosoma mansoni is a parasitic flatworm occurring in 53 countries that causes schistosomiasis. An in-house inhibitor library was tested for anti-schistosomal activity and inhibitory effect on S. mansoni cathepsin B1 (SmCB1), the most abundant cysteine protease in the parasite’s gut. Based on a peptidomimetic inhibitor that exhibited the highest anti-schistosomal activity, more lipophilic substructures as well as alternative warheads were introduced into the scaffold, resulting in an increase in cell permeability and efficacy against S. mansoni. However, a correlation to SmCB1 inhibition could not be observed since the structural class in focus showed only weak inhibition of this enzyme. Both peptidomimetic sequence and warhead affect the affinity and selectivity of protease inhibitors, but the exact impact of each part is unclear. To analyze this, a comprehensive study was conducted combining established peptidomimetic sequences of inhibitors of five proteases with seven different types of warheads. Cross-testing revealed that the peptidomimetic sequence mainly causes selectivity, but the combination with a suitable warhead is crucial to achieve activity against a protease of interest in the first place as well as to avoid off-target reactivity. This work describes the development of bortezomib congeners as potential proteasome inhibitors (one out of five inhibitor sets) for this study.
DDC:	540 Chemie 540 Chemistry and allied sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-9296
URN:	urn:nbn:de:hebis:77-openscience-9ea1e907-1d51-4939-8ce5-aa652db177f05
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	XX, 305 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen