Cardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol... Show moreCardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol and lipid accumulation. Despite available effective cholesterol lowering medication, considerable risk for recurrent vascular events remains. This residual risk is at least in part explained by high blood lipid levels. The research described in this thesis revealed novel therapeutic strategies that improve lipid metabolism and reduce atherosclerosis development in mice. Inhibition of the endocannabinoid system was found to be an effective strategy, as well as concomitant activation of two incretin hormone receptors, namely those for GIP and GLP1. For combined GIP/GLP1 receptor agonism we additionally showed strongly attenuated hepatic steatosis. We were also able to identify additional targets to attenuate hyperlipidemia by studying the mechanisms underlying the strong day-night rhythm of brown adipose tissue, which is a lipid combusting tissue. In this thesis, I also stress the importance of the choice in animal model when studying lipid-modifying interventions, and describe the development of the software tool RandoMice which can be used to improve the quality of preclinical studies by creating well-balanced experimental groups. Show less
This thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be... Show moreThis thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be responsible for the calcium-dependent formation of N-acylphosphatidylethanolamines (NAPEs) in cells. NAPEs are low-abundant lipid species that play roles in membrane stabilization, cell signaling and homeostasis. They are well-known as precursors to the signaling lipids of the N-acylethanolamine (NAE) class, but their own biological functions remain relatively poorly understood. To find inhibitors of PLA2G4E, a focused compound library was screened in a newly developed activity-based protein profiling (ABPP) assay. Hits were identified and optimized by building structure-activity relationships (SARs) through organic synthesis and activity assays. WEN091 was identified as potent inhibitor of PLA2G4E that was able to reduce cellular NAPE levels. Cellular target engagement was confirmed by use of a tailored activity-based probe. Using these molecular tools, the relevance of NAPEs and PLA2G4E in cellular processes and disease may be elucidated. Show less
Mohr, F.; Hurrle, T.; Burggraaff, L.; Langer, L.; Bemelmans, M.P.; Knab, M.; ... ; Bräse, S. 2021
We report the development and extensive structure-activity relationship evaluation of a series ofmodified coumarins as cannabinoid receptor ligands. In radioligand, and [35S]GTPgS binding assays... Show moreWe report the development and extensive structure-activity relationship evaluation of a series ofmodified coumarins as cannabinoid receptor ligands. In radioligand, and [35S]GTPgS binding assays theCB receptor binding affinities and efficacies of the new ligands were determined. Furthermore, we used aligand-based docking approach to validate the empirical observed results. In conclusion, several crucialstructural requirements were identified. The most potent coumarins like 3-butyl-7-(1-butylcyclopentyl)-5-hydroxy-2H-chromen-2-one (36b, Ki CB2 13.7 nM, EC50 18 nM), 7-(1-butylcyclohexyl)-5-hydroxy-3-propyl-2H-chromen-2-one (39b, Ki CB2 6.5 nM, EC50 4.51 nM) showed a CB2 selective agonistic profilewith low nanomolar affinities. Show less
In this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the... Show moreIn this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the role of the ECS in regulating locomotion and anxiety, and its interaction with the hypothalamic-pituitary-interrenal (HPI) axis, or stress axis. This study has provided us with an interesting animal model which allows for pharmacological screening of Cnr1 agonists, and their involvement in the CNS, as shown by a change in locomotion, anxiety-like behavior and HPI axis activity. The zebrafish larval model can be used as a complementary model to the existing rodent animal models, to study the ECS. The zebrafish larval model brings several interesting features, such as optical transparency and possibilities for high-throughput screening. Furthermore, a complete ECS is present, there is lack of endogenous activity, allowing for exogenous compound screening, and zebrafish data is generally in line with rodent literature. Since the ECS is involved in many diseases, more research of this system may result in the discovery of novel drugs and drug targets. 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
Less than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better... Show moreLess than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better understanding of the role of selectivity and a better insight in the off-target activities of drug candidates could greatly aid in preventing candidates to fail for these reasons. This thesis has tried to address some aspects in this challenging part of drug discovery. The use of activity-based protein profiling as presented in Chapters 2 and 3 in drug discovery and hit-to-lead optimization, and in Chapter 5 and 6 for the interaction profiling of a drug candidate, highlights the versatility and importance of this chemical biology technique. Combined with knowledge derived from biochemical assays, such as that developed in Chapter 4, ABPP can greatly aid the medicinal chemist. The recent surge in popularity of machine learning algorithms, backed by exponential growth of the amount of biological data available, holds great promise for drug discovery. Chapters 7 and 8 showed the applicability of one such algorithm, which was able to quite reliably predict interaction profiles. The challenges in finding, determining and predicting selectivity are far from solved, but, by incrementally expanding our understanding of the binding of small molecules to their (off-)targets, truly selective inhibitors might at some point become a reality or their necessity might be mitigated. Show less
This thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse... Show moreThis thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse tissue is described. This method is used to compare a Niemann-Pick Type C mouse model to healthy mice. Additionally, several novel activity-based probes for the enzymes diacylglycerol lipase alpha and a/b-hydrolase domain containing protein 6 are described. Show less
In this thesis, a system pharmacology approach, integrating metabolomics, pharmacology and chemical biology, was applied to understand and modulate the endocannabinoid system across different model... Show moreIn this thesis, a system pharmacology approach, integrating metabolomics, pharmacology and chemical biology, was applied to understand and modulate the endocannabinoid system across different model systems (cells, zebrafish, mice and humans). The endocannabinoid system (ECS) and its function in the brain was discovered after identifying THC: the active ingredient of cannabis (marijuana) plant. It is perhaps the most important physiological signalling system involved in establishing and maintaining human health. In disease conditions the ECS system is dysregulated and becomes either highly active or less active. Due to the widespread effects throughout the body, targeting ECS system is believed to hold promise as a treatment target in the field of medicine. In this thesis, the functional role of the ECS was studied in healthy and diseased conditions. In addition, the ECS was modulated using enzyme inhibitors to gain a better understanding in different signalling pathways. Furthermore, to understand the drug pharmacology and whether such new compound inhibitors may modify disease biochemistry; this includes the study of target engagement, downstream effects and the off-target effects inhibitors. Overall, we are convinced that by applying system pharmacology approach to study ECS can lead to develop novel therapeutic biomarkers to treat metabolic or neurological diseases. Show less
