Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is... Show moreMonoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents. Show less
Prokop, S.; Ábrányi-Balogh, P.; Barti, B.; Vámosi, M.; Zöldi, M.; Barna, L.; ... ; Katona, I. 2021
Immunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we... Show moreImmunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we introduce PharmacoSTORM super-resolution imaging that combines the complementary advantages of these approaches and enables cell-type- and compartment-specific nanoscale molecular measurements. We exploited rational chemical design for fluorophore-tagged high-affinity receptor ligands and an enzyme inhibitor; and demonstrated broad PharmacoSTORM applicability for three protein classes and for cariprazine, a clinically approved antipsychotic and antidepressant drug. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D3 dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-type- and subcellular context-dependent manner and within complex tissue preparations. Moreover, the results highlight the underappreciated neuropsychiatric significance of the Islands of Calleja in the ventral forebrain. Show less
Janssen, A.P.A.; Hengst, J.M.A. van; Béquignon, O.J.M.; Deng, H.; Westen, G.J.P. van; Stelt, M. van der 2019
Drug discovery programs of covalent irreversible, mechanism-based enzyme inhibitors often focus on optimization of potency as determined by IC50-values in biochemical assays. These assays do not... Show moreDrug discovery programs of covalent irreversible, mechanism-based enzyme inhibitors often focus on optimization of potency as determined by IC50-values in biochemical assays. These assays do not allow the characterization of the binding activity (Ki) and reactivity (kinact) as individual kinetic parameters of the covalent inhibitors. Here, we report the development of a kinetic substrate assay to study the influence of the acidity (pKa) of heterocyclic leaving group of triazole urea derivatives as diacylglycerol lipase (DAGL)-α inhibitors. Surprisingly, we found that the reactivity of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen atoms in the heterocyclic core determined to a large extent the binding activity of the inhibitor. This finding was confirmed and clarified by molecular dynamics simulations on the covalently bound Michaelis−Menten complex. A deeper understanding of the binding properties of covalent serine hydrolase inhibitors is expected to aid in the discovery and development of more selective covalent inhibitors. 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; 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
Esbroeck, A.C.M. van; Janssen, A.P.A.; Cognetta III, A.B.; Ogasawara, D.; Shpak, G.; Kroeg, M. van der; ... ; Stelt, M. van der 2017
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although... Show moreA recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system. Show less
2‐Arachidonoylglycerol (2‐AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1R and CB2R), thereby regulating a diverse range of physiological processes... Show more2‐Arachidonoylglycerol (2‐AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1R and CB2R), thereby regulating a diverse range of physiological processes including anxiety, appetite, inflammation, memory, pain sensation, and nociception. Diacylglycerol lipases (DAGLs) are the principle enzymes responsible for 2‐AG biosynthesis. Recently, the (patho)physiological functions of DAGLs have been explored by both genetic methods and chemical tools. This review will focus on the recent efforts to develop highly selective and in vivo active DAGLs inhibitors using activity‐based protein profiling. Show less
Deng, H.; Wel, T. van der; Berg, R.J.B.H.N. van den; Nieuwendijk, A.M.C.H. van den; Janssen, F.J.; Baggelaar, M.P.; ... ; Stelt, M. van der 2017
The thesis describes the design, synthesis and application of chemical tools to study the physiological roles of DAGLa/b and MAGL in vitro and in vivo. Chapter2 reports on the design, synthesis... Show moreThe thesis describes the design, synthesis and application of chemical tools to study the physiological roles of DAGLa/b and MAGL in vitro and in vivo. Chapter2 reports on the design, synthesis and in vitro characterization of DH376 as a new dual DAGL inhibitors. In Chapter 3 the discovery of DH379 as a tailor-made activity-based probe for DAGLa/b and the effects of acute pharmacological blockade of DAGLa/b by DH376 in healthy and lipopolysaccharide-treated mice on brain lipid networks and neuroinflammation is described. Chapter 4 reports the efficacy of DH376 in refeeding behavior of fasted mice. In Chapter 5 the development of the first DAGL PET ligand [18F]DH439 is disclosed. The structure-activity relationship of disubstituted piperidinyl ureas as DAGL inhibitors is reported in Chapter 6. The design, synthesis and application of a highly selective tailor-made activity-based imaging probe for MAGL is discussed in Chapter 7. Show less
Deng, H.; Kooijman, S.; Nieuwendijk, A.M.C.H. van den; Ogasawara, D.; Wel, T. van der; Dalen, F. van; ... ; Steltt, M. van der 2017
Neocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here,... Show moreNeocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here, the authors report the discovery and characterization of the biosynthetic pathway of NZS through genome mining and gene inactivation. The in vitro assays characterized two enzymes: NzsA is a P 450 hydroxylase and NzsG is a new phytoene-synthase-like prenyltransferase (PTase). This is the first reported native PTase that specifically acts on the carbazole nucleus. Finally, the authors' in vitro reconstituted expt. demonstrated a coupled reaction catalyzed by NzsG and NzsA tailoring the NZS biosynthesis. [on SciFinder(R)] Show less
