Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of... Show moreRetinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts. Show less
Acute myocardial infarction and subsequent post-infarction heart failure are among the leading causes of mortality worldwide. The endocannabinoid system has emerged as an important modulator of... Show moreAcute myocardial infarction and subsequent post-infarction heart failure are among the leading causes of mortality worldwide. The endocannabinoid system has emerged as an important modulator of cardiovascular disease, however the role of endocannabinoid metabolic enzymes in heart failure is still elusive. Herein, we investigated the endocannabinoids and their metabolic enzymes in ischemic end-stage failing human hearts and non-failing controls.\nQuantitative real-time PCR, targeted lipidomics, and activity-based protein profiling (ABPP) enabled assessment of the endocannabinoids and their metabolic enzymes in ischemic end-stage failing human hearts and non-failing controls. Based on lipidomic analysis, two subgroups were identified within the ischemic heart failure group; the first similar to control hearts and the second with decreased levels of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) and drastically increased levels of the endocannabinoid anandamide (AEA), other N-acylethanolamines (NAEs) and free fatty acids. The altered lipid profile was accompanied by strong reductions in the activity of 13 hydrolases, including the 2-AG hydrolytic enzyme monoacylglycerol lipase (MGLL).\nOur findings suggest the presence of different biological states within the ischemic heart failure group, based on alterations in the lipid and hydrolase activity profiles. In addition, this study demonstrates that ABPP is a valuable tool to rapidly analyze enzyme activity in clinical samples with potential for novel drug and biomarker discovery.\nAIM\nMETHODS AND RESULTS\nCONCLUSIONS Show less
Soethoudt, M.; Alachouzos, G.; Rooden, E.J. van; Moya Garzón, M.D.; Berg, R.J.B.H.N. van den; Heitman, L.H.; Stelt, M. van der 2018
Introduction: Δ9-Tetrahydrocannabinol (THC), the principle psychoactive ingredient in Cannabis, is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous... Show moreIntroduction: Δ9-Tetrahydrocannabinol (THC), the principle psychoactive ingredient in Cannabis, is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous neurological side effects. The cannabinoid receptors (CBRs) are responsible to a large extent for these, but not all biological responses are mediated via the CBRs.Objectives: The identification of additional target proteins of THC to enable a better understanding of the (adverse) physiological effects of THC.Methods: In this study, a chemical proteomics approach using a two-step photoaffinity probe is applied to identify potential proteins that may interact with THC.Results: Photoaffinity probe 1, containing a diazirine as a photocrosslinker, and a terminal alkyne as a ligation handle, was synthesized in 14 steps. It demonstrated high affinity for both CBRs. Subsequently, two-step photoaffinity labeling in neuroblastoma cells led to identification of four potential novel protein targets of THC. The identification of these putative protein hits is a first step towards a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC. 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
Rooden, E.J. van; Kreekel, R.; Hansen, T.; Janssen, A.P.A.; Esbroeck, A.C.M. van; Dulk, H. den; ... ; Stelt, M. van der 2018
Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired.... Show moreDiacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels–Alder ligation in living cells. Show less
Rooden, E.J. van; Esbroeck, A.C.M. van; Baggelaar, M.P.; Deng, H.; Florea, B.I.; Rosa Alcalde Marques, A.; ... ; Stelt, M. van der 2018
The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the... Show moreThe endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies. Show less
Rooden, E.J. van; Kohsiek, M.J.J.; Kreekel, R.; Esbroeck, A.C.M. van; Nieuwendijk, A.M.C.H. van den; Janssen, A.P.A.; ... ; Stelt, M. van der 2018
Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2‐arachidonoylglycerol. The fluorescent activity‐based probes DH379 and HT‐01 have been previously shown to... Show moreDiacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2‐arachidonoylglycerol. The fluorescent activity‐based probes DH379 and HT‐01 have been previously shown to label DAGLs and to cross‐react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity‐based probes 1 and 2, the design of which was based on the structures of DH379 and HT‐01, respectively. Probe 1 contains a BODIPY‐FL and a 2,4‐dinitroaniline moiety as a fluorophore–quencher pair, whereas probe 2 employs a Cy5‐fluorophore and a cAB40‐quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity‐based protein profiling assays using human cell‐ and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability. Show less
Rooden, E.J. van; Florea, B.I.; Deng, H.; Baggelaar, M.P.; Esbroeck, A.C.M. van; Zhou, J.; ... ; Stelt, M. van der 2018
Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off... Show moreActivity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples. Show less
Soethoudt, M.; Stolze, S.C.; Westphal, M.V.; Stralen, L. van; Martella, A.; Rooden, E.J. van; ... ; Stelt, M. van der 2018
Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as... Show moreChemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photo-click chemistry and hold promise as biomarkers in translational drug discovery. Show less
Rooden, E.J. van; Bakker, A.T.; Overkleeft, H.S.; Stelt, M. van der 2018
Activity‐based protein profiling is a method to study a subset of the enzymatically active proteome. This method uses chemical probes that covalently react with active enzymes. These labelled... Show moreActivity‐based protein profiling is a method to study a subset of the enzymatically active proteome. This method uses chemical probes that covalently react with active enzymes. These labelled proteins can subsequently be analysed by means of a detection tag on the probe. A diverse set of probes has been developed for many enzyme classes, such as serine hydrolases, proteases, glycosidases and kinases. Different analytical techniques are currently available to visualise, identify and quantify probe‐labelled proteins with high efficiency. Activity‐based protein profiling has well‐developed applications in discovering new drug targets and in profiling inhibitors for potency and selectivity. Activity‐based protein profiling will, therefore, continue to aid research both in fundamental biology and drug discovery. Show less
In this paper, a new synthetic route toward 6-hydroxysphingosine and α-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a... Show moreIn this paper, a new synthetic route toward 6-hydroxysphingosine and α-hydroxy ceramide is described. The synthesis employs a cross-metathesis to unite a sphingosine head allylic alcohol with a long-chain fatty acid alkene that also bears an allylic alcohol group. To allow for a productive CM coupling, the sphingosine head allylic alcohol was protected with a cyclic carbonate moiety and a reactive CM catalyst system, consisting of Grubbs II catalyst and CuI, was employed. Show less
