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Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here.

Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [29]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [17]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [75]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [66]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [5, 24, 77].

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthase (FPGS), which normally promotes intracellular accumulation and activity of both natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. In this study, we characterize a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase (DHFR) and downstream one-carbon metabolism. Contrary to methotrexate, however, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folate concentrations for interaction with DHFR. Indeed, we show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1-induced growth inhibition, while FPGS-high CRC organoids are more sensitive to methotrexate. Our results thus argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, employing a vulnerability created by polyglutamylation deficiency.

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.

Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by corticotropin releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood. Using an array of cellular, endocrine and behavioral readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signaling to the generation of a stress response. The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243, and NAPE PLD inhibitor LEI401 all uniformly increased c-fos in the PVN, unmasked stress-linked behaviors, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress-like behavioral changes produced by disruption of eCB signaling. These data indicate that under resting conditions, constitutive eCB signaling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the principal enzyme that generates N-acylethanolamines (NAEs), a family of signaling lipids that includes the endocannabinoid anandamide. To investigate the biological function and biosynthesis of NAEs, we sought to develop potent NAPE-PLD inhibitors. To this aim, we utilized a high-throughput screening-compatible NAPE-PLD activity assay, which uses the fluorescence-quenched substrate PED6. This assay conveniently uses membrane fractions of NAPE-PLD overexpressing HEK293T cell lysates, thus avoiding the need for protein purification. Here, we give a detailed description of the NAPE-PLD PED6 fluorescence activity assay, which has increased throughput compared to previous radioactivity- or mass-spectrometry-based assays.

The endocannabinoids anandamide and 2-arachidonoylglycerol are not only metabolized by serine hydrolases, such as fatty acid amide hydrolase, monoacylglycerol lipase, and α,β-hydrolases 6 and 12, but they also serve as substrates for cyclooxygenases, cytochrome P450s, and lipoxygenases. These enzymes oxygenate the 1Z,4Z-pentadiene system of the arachidonic acid backbone of endocannabinoids, thereby giving rise to an entirely new array of bioactive lipids. Hereby, a protocol is provided for the enzymatic synthesis, purification, and characterization of various oxygenated metabolites of anandamide generated by lipoxygenases, which enables the biological study and detection of these metabolites.

N-acylethanolamines (NAEs), including N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), N-arachidonoylethanolamine (AEA, anandamide), N-docosahexaenoylethanolamine (DHEA, synaptamide) and their oxygenated metabolites are a lipid messenger family with numerous functions in health and disease, including inflammation, anxiety and energy metabolism. The NAEs exert their signaling role through activation of various G protein-coupled receptors (cannabinoid CB1 and CB2 receptors, GPR55, GPR110, GPR119), ion channels (TRPV1) and nuclear receptors (PPAR-α and PPAR-γ) in the brain and periphery. The biological role of the oxygenated NAEs, such as prostamides, hydroxylated anandamide and DHEA derivatives, are less studied. Evidence is accumulating that NAEs and their oxidative metabolites may be aberrantly regulated or are associated with disease severity in obesity, metabolic syndrome, cancer, neuroinflammation and liver cirrhosis. Here, we comprehensively review NAE biosynthesis and degradation, their metabolism by lipoxygenases, cyclooxygenases and cytochrome P450s and the biological functions of these signaling lipids. We discuss the latest findings and therapeutic potential of modulating endogenous NAE levels by inhibition of their degradation, which is currently under clinical evaluation for neuropsychiatric disorders. We also highlight NAE biosynthesis inhibition as an emerging topic with therapeutic opportunities in endocannabinoid and NAE signaling.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [Mock et al. Nat Chem. Biol., 2020, 16, 667−675]. Here, we describe the structure−activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

The most abundant endocannabinoid (eCB) in the brain, 2-arachidonoyl glycerol (2-AG), is hydrolyzed by α/β-hydrolase domain containing 6 (ABHD6); yet how ABHD6 controls stimuli-dependent increases in 2-AG production is unknown. To explore this question, we leveraged the recently developed 2-AG sensor, GRABeCB2.0, and found that stimulation of Neuro2a cells in culture with bradykinin (BK) acting at metabotropic B2K receptors and ATP acting at ionotropic P2X7 receptors led to differential increases in 2-AG levels. B2K triggered increases in 2-AG levels via diacylglycerol lipase (DAGL), and this mechanism was potentiated by increases in intracellular calcium and ABHD6 inhibition. By contrast, P2X7-triggered increases in 2-AG levels were dependent on DAGL and extracellular calcium but unaffected by ABHD6 inhibition. Thus, ABHD6 preferentially regulates metabotropic-dependent increases in 2-AG levels over ionotropic-dependent increases in 2-AG levels. Our study indicates that ABHD6 selectively controls stimuli-dependent increases in 2-AG production and emphasizes its specific role in eCB signaling.

Phenotypic screening is a powerful approach to identify novel antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) infection, but elucidation of the targets responsible for antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode-of-action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones, identified in a phenotypic screen to have high antibacterial potency against multidrug resistant S. aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as novel antibacterial chemotype with a polypharmacological mode-of-action, in which FabH, FphC and AdhE play a central role.

Repeated seizures result in a persistent maladaptation of endocannabinoid (eCB) signaling, mediated part by anandamide signaling deficiency in the basolateral amygdala (BLA) that manifests as aberrant synaptic function and altered emotional behavior. Here, we determined the effect of repeated seizures (kindling) on 2-arachidonoylglycerol (2-AG) signaling on GABA transmission by directly measuring tonic and phasic eCB-mediated retrograde signaling in anin vitroBLA slice preparation from male rats. We report that both activity-dependent and muscarinic acetylcholine receptor (mAChR)-mediated depression of GABA synaptic transmission was reduced following repeated seizure activity. These effects were recapitulated in sham rats by preincubating slices with the 2-AG synthesizing enzyme inhibitor DO34. Conversely, preincubating slices with the 2-AG degrading enzyme inhibitor KML29 rescued activity-dependent 2-AG signaling, but not mAChR-mediated synaptic depression, over GABA transmission in kindled rats. These effects were not attributable to a change in cannabinoid type 1 (CB1) receptor sensitivity or altered 2-AG tonic signaling since the application of the highly selective CB1 receptor agonist CP55,940 provoked a similar reduction in GABA synaptic activity in both sham and kindled rats, while no effect of either DO34 or of the CB1 inverse agonist AM251 was observed on frequency and amplitude of spontaneous IPSCs in either sham or kindled rats. Collectively, these data provide evidence that repeated amygdala seizures persistently alter phasic 2-AG-mediated retrograde signaling at BLA GABAergic synapses, probably by impairing stimulus-dependent 2-AG synthesis/release, which contributes to the enduring aberrant synaptic plasticity associated with seizure activity.SIGNIFICANCE STATEMENTThe plastic reorganization of endocannabinoid (eCB) signaling after seizures and during epileptogenesis may contribute to the negative neurobiological consequences associated with seizure activity. Therefore, a deeper understanding of the molecular basis underlying the pathologic long-term eCB signaling remodeling following seizure activity will be crucial to the development of novel therapies for epilepsy that not only target seizure activity, but, most importantly, the epileptogenesis and the comorbid conditions associated with epilepsy.

The clinical success of anti-IL-17 monoclonal antibodies (i.e., Cosentyx and Taltz) has validated Th17 pathway modulation for the treatment of autoimmune diseases. The nuclear hormone receptor RORγt is a master regulator of Th17 cells and affects the production of a host of cytokines, including IL-17A, IL-17F, IL-22, IL-26, and GM-CSF. Substantial interest has been spurred across both academia and industry to seek small molecules suitable for RORγt inhibition. A variety of RORγt inhibitors have been reported in the past few years, the majority of which are orthosteric binders. Here we disclose the discovery and optimization of a class of inhibitors, which bind differently to an allosteric binding pocket. Starting from a weakly active hit 1, a tool compound 14 was quickly identified that demonstrated superior potency, selectivity, and off-target profile. Further optimization focused on improving metabolic stability. Replacing the benzoic acid moiety with piperidinyl carboxylate, modifying the 4-aza-indazole core in 14 to 4-F-indazole, and incorporating a key hydroxyl group led to the discovery of 25, which possesses exquisite potency and selectivity, as well as an improved pharmacokinetic profile suitable for oral dosing.

Brevicidine and laterocidine are two recently discovered lipopeptide antibiotics with promising antibacterial activity. Possessing a macrocyclic core, multiple positive charges, and a lipidated N-terminus, these lipopeptides exhibit potent and selective activity against Gram-negative pathogens, including polymyxin-resistant isolates. Given the low amounts of brevicidine and laterocidine accessible by fermentation of the producing microorganisms, synthetic routes to these lipopeptides present an attractive alternative. We here report the convenient solid-phase syntheses of both brevicidine and laterocidine and confirm their potent anti-Gram-negative activities. The synthetic routes developed also provide convenient access to novel structural analogues of both brevicidine and laterocidine that display improved hydrolytic stability while maintaining potent antibacterial activity in both in vitro assays and in vivo infection models.

Bioorthogonal chemistry has allowed the study of biomolecules in living systems with minimal structural perturbations to the molecules under investigation. In the field of lipid research, this has allowed for the study of lipid uptake and processing, whilst minimising artefacts on their biology resulting from pendant detectable groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene containing oleic acid analogue, as a bioorthogonal probe. We here show that this lipid can be readily taken up by dendritic cells without toxic side-effects, and that it can subsequently be visualised in live cells using an inverse electron-demand Diels-Alder (IEDDA) reaction with quenched tetrazine fluorophores. Furthermore, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Brevicidine and laterocidine are two recently discovered lipopeptide antibiotics with promising antibacterial activity. Possessing a macrocyclic core, multiple positive charges, and a lipidated N-terminus, these lipopeptides exhibit potent and selective activity against Gram-negative pathogens, including polymyxin-resistant isolates. Given the low amounts of brevicidine and laterocidine accessible by fermentation of the producing microorganisms, synthetic routes to these lipopeptides present an attractive alternative. We here report the convenient solid-phase syntheses of both brevicidine and laterocidine and confirm their potent anti-Gram-negative activities. The synthetic routes developed also provide convenient access to novel structural analogues of both brevicidine and laterocidine that display improved hydrolytic stability while maintaining potent antibacterial activity in both in vitro assay and in vivo infection models.

Brevicidine and laterocidine are two recently discovered lipopeptide antibiotics with promising antibacterial activity. Possessing a macrocyclic core, multiple positive charges, and a lipidated N-terminus, these lipopeptides exhibit potent and selective activity against Gram-negative pathogens, including polymyxin-resistant isolates. Given the low amounts of brevicidine and laterocidine accessible by fermentation of the producing microorganisms, synthetic routes to these lipopeptides present an attractive alternative. We here report the convenient solid-phase syntheses of both brevicidine and laterocidine and confirm their potent anti-Gram-negative activities. The synthetic routes developed also provide convenient access to novel structural analogues of both brevicidine and laterocidine that display improved hydrolytic stability while maintaining potent antibacterial activity in both

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

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 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., LEI-945 is a first-in-class retinal-based probe that enables profiling aldehyde dehydrogenase activity in living cancer cells and maps the selectivity profile of ALDH inhibitors.

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation., European Journal of Medicinal Chemistry, 243, ISSN:0223-5234, ISSN:1768-3254

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 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., The authors introduce PharmacoSTORM single-molecule imaging that uses fluorescent ligands and immunolabeling for cellular and subcellular nanoscale molecular pharmacology. They demonstrate its capabilities by visualizing cariprazine binding to D3 dopamine receptors on Islands of Calleja granule cell axons.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

International audience; The European Federation for Medicinal chemistry and Chemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning chemical biology and medicinal chemistry. New ideas, tools, and technologies emerging from a wide array of scientific disciplines continuously energize this rapidly evolving area. Medicinal chemistry is the design, synthesis, and optimization of biologically active molecules aimed at discovering new drug candidatesa mission that in many ways overlaps with the scope of chemical biology. Chemical biology is by now a mature field of science for which a more precise definition of what it englobes, in the frame of EFMC, is timely. This article discusses chemical biology as currently understood by EFMC, including all activities dealing with the design and synthesis of biologically active chemical tools and their use to probe, characterize, or influence biological systems.

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [28]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [17]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [70]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [62]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [5, 23, 72].

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 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.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.