Structure-Guided Discovery of cis-Hexahydro-pyrido-oxazinones as Reversible, Drug-like Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is a key enzyme involved in the metabolism of the endogenous signaling ligand 2-arachidonoylglycerol, a neuroprotective endocannabinoid intimately linked to central nervous system (CNS) disorders associated with neuroinflammation. In the quest for novel MAGL inhibitors, a focused screening approach on a Roche library subset provided a reversible benzoxazinone hit exhibiting high ligand efficiency. The subsequent design of the three-dimensional cis-hexahydro-pyrido-oxazinone (cis-HHPO) moiety as benzoxazinone replacement enabled the combination of high MAGL potency with favorable ADME properties. Through enzymatic resolution an efficient synthetic route of the privileged cis-(4R,8S) HHPO headgroup was established, providing access to the highly potent and selective MAGL inhibitor 7o. Candidate molecule 7o matches the target compound profile of CNS drugs as it achieves high CSF exposures after systemic administration in rodents. It engages with the target in the brain and modulates neuroinflammatory processes, thus holding great promise for the treatment of CNS disorders.

Comparative targeted lipidomics between serum and cerebrospinal fluid of multiple sclerosis patients shows sex and age-specific differences of endocannabinoids and glucocorticoids.

Multiple sclerosis (MS) is a complex chronic neuroinflammatory disease characterized by demyelination leading to neuronal dysfunction and neurodegeneration manifested by various neurological impairments. The endocannabinoid system (ECS) is a lipid signalling network, which plays multiple roles in the central nervous system and the periphery, including synaptic signal transmission and modulation of inflammation. The ECS has been identified as a potential target for the development of novel therapeutic interventions in MS patients. It remains unclear whether ECS-associated metabolites are changed in MS and could serve as biomarkers in blood or cerebrospinal fluid (CSF). In this retrospective study we applied targeted lipidomics to matching CSF and serum samples of 74 MS and 80 non-neuroinflammatory control patients. We found that MS-associated lipidomic changes overall did not coincide between CSF and serum. While glucocorticoids correlated positively, only the endocannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) showed a weak positive correlation (r = 0.3, p < 0.05) between CSF and serum. Peptide endocannabinoids could be quantified for the first time in CSF but did not differ between MS and controls. MS patients showed elevated levels of prostaglandin E2 and steaorylethanolamide in serum, and 2-oleoylglycerol and cortisol in CSF. Sex-specific differences were found in CSF of MS patients showing increased levels of 2-AG and glucocorticoids in males only. Overall, arachidonic acid was elevated in CSF of males. Interestingly, CSF eCBs correlated positively with age only in the control patients due to the increased levels of eCBs in young relapsing-remitting MS patients. Our findings reveal significant discrepancies between CSF and serum, underscoring that measuring eCBs in blood matrices is not optimal for detecting MS-associated changes in the central nervous system. The identified sex and age-specific changes of analytes of the stress axis and ECS specifically in the CSF of MS patients supports the role of the ECS in MS and may be relevant for drug development strategies.

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

Euphane and Tirucallane Triterpenes with Trypanocidal Activity from Euphorbia desmondii.

The phytochemical investigation of Euphorbia desmondii resulted in the isolation of 15 previously undescribed triterpenoids (desmondiins A, C-P) and 8 already described compounds. The structures of the isolated compounds were determined by extensive spectroscopic analyses. The compounds were identified as tirucallane and euphane triterpenes based on 7-keto-8-ene, 11-keto-8-ene, or 7,11-diketo-8-ene skeletons. Additionally, the selective trypanocidal activities of these compounds against Trypanosoma cruzi were evaluated. Desmondiins A, C, D, F, H, and M exhibited IC50 values in the range of 3-5 µM, and selectivity indices between 5-9, against T. cruzi epimastigotes over the host cell (RAW264.7 macrophages). Furthermore, desmondiin A efficiently inhibited amastigote replication in host cells (IC50 = 2.5 ± 0.3 µM), which was comparable to that of the positive control, benznidazole (3.6 ± 0.4 µM). Overall, the isolated euphane and tirucallane triterpenoids could act as antichagasic lead scaffolds.

A Highly Potent, Orally Bioavailable Pyrazole-Derived Cannabinoid CB2 Receptor- Selective Full Agonist for In Vivo Studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein-mediated efflux from the brain. 3H and 14C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.

Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.

Hypersensitive quantification of major astringency markers in food and wine by substoichiometric quenching of silicon-rhodamine conjugates.

Tannins are chemically diverse polyphenols in plant-derived products that not only show diverse biological activities but also play a crucial role in determining the sensory attributes of food and beverages. Therefore, their accurate and cost-effective quantification is essential. Here, we identified a novel fluorescence quenching mechanism of different synthetic rhodamine fluorophores, with a high selectivity towards tannic acid (TA) and catechin-3-gallate (C3G) compared to a structurally diverse panel of tannins and polyphenols. Specific chemical conjugates of silicon-rhodamine with alkyl linkers attached to bulky apolar moieties had a limit of detection near 500 pM and a linear range spanning 5-100 nM for TA. We validated the assay on 18 distinct red wine samples, which showed high linearity (R2 = 0.92) with methylcellulose precipitation with no interference from anthocyanins. In conclusion, a novel assay was developed and validated that allows the sensitive and selective quantification of major astringency markers abundant in food and beverages.

CNS Drug Discovery in Academia: Where Basic Research Meets Innovation.

The involvement of academic research in drug discovery is consistently growing. However, academic projects seldom advance to clinical trials. Here, we assess the landscape of drug discovery within the National Centre of Competence in Research (NCCR) TransCure launched by the Swiss National Science Foundation to foster basic research and early-stage drug discovery on membrane transporters. This included transporters in central nervous system (CNS) disorders, which represent a huge unmet medical need. While idea championship, sustainable funding, collaborations between disciplines at the interface of academia and industry are important for translational research, Popperian falsifiability, strong intellectual property and a motivated startup team are key elements for innovation. This is exemplified by the NCCR TransCure spin-off company Synendos Therapeutics, a clinical stage biotech company developing the first selective endocannabinoid reuptake inhibitors (SERIs) as novel treatment for neuropsychiatric disorders. We provide a perspective on the challenges related to entering an uncharted druggable space and bridging the often mentioned "valley of death". The high attrition rate of drug discovery projects in the CNS field within academia is often due to the lack of meaningful animal models that can provide pharmacological proof-of-concept for potentially disruptive technologies at the earliest stages, and the absence of solid intellectual property.

A highly potent, orally bioavailable pyrazole-derived cannabinoid CB2 receptor-selective full agonist for in vivo studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC50 < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.

Endocannabinoids and related lipids linked to social exclusion in individuals with chronic non-medical prescription opioid use.

Opioid-related overdose deaths are still on the rise in North America, emphasizing the need to better understand the underlying neurobiological mechanisms regarding the development of opioid use disorder (OUD). Recent evidence from preclinical and clinical studies indicate that the endocannabinoid system (ECS) may play a crucial role in stress and reward, both involved in the development and maintenance of substance use disorders. Animal models demonstrate a specific crosstalk between the ECS and the endogenous opioid system. However, translational studies in humans are scarce. Here, we investigated basal plasma levels of the endocannabinoids anandamide (AEA) and 2-arachidonoyglycerol (2-AG), and eight endocannabinoid-related lipids, including oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), as well as whole blood fatty acid amide hydrolase (FAAH) activity in chronic non-medical prescription opioid users (NMPOU; n = 21) compared to opioid-naïve healthy controls (n = 29) considering age, sex, and cannabis use as potential confounders. Additionally, the association of endocannabinoids and related lipids with the participants' response to experimentally induced social exclusion was examined. We found significantly elevated basal AEA, OEA, and PEA levels in NMPOU compared to controls, but no differences in FAAH activity, 2-AG, or other endocannabinoid-related lipids. Within NMPOU, higher AEA levels were associated with lower perception of social exclusion. Robust positive correlations within N-acylethanolamines (i.e., AEA, OEA, and PEA) indicate strong metabolic associations. Together with our recent findings of elevated basal 2-AG levels in dependent cocaine users, present results indicate substance-specific alterations of the ECS that may have implications in the search for novel therapeutic interventions for these populations.

Discovery and development of macrocyclic peptide modulators of the cannabinoid 2 receptor.

The cannabinoid type 2 receptor (CB2R), a G protein-coupled receptor, is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small molecules, including endo-, phyto-, and synthetic cannabinoids, peptides-owing to their size-may offer a different interaction space to facilitate differential interactions with the receptor. Here, we explore plant-derived cyclic cystine-knot peptides as ligands of the CB2R. Cyclotides are known for their exceptional biochemical stability. Recently, they gained attention as G protein-coupled receptor modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics, we identified the cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1 µM and a potency (EC50) of 8 µM. Leveraging deep learning networks, we verified the structural topology of vodo-C1 and modeled its molecular volume in comparison to the CB2R ligand binding pocket. In a fragment-based approach, we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB2R. This study introduces a macrocyclic peptide phytocannabinoid, which served as a template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.

LAT1-dependent placental methionine uptake is a key player in fetal programming of metabolic disease.

The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.

The Concise Guide to PHARMACOLOGY 2023/24: Enzymes.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is 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 almost 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/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes 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-2023, and supersedes data presented in the 2021/22, 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.

Bioactivity-guided isolation of trypanocidal coumarins and dihydro-pyranochromones from selected Apiaceae plant species.

Bioactivity-guided isolation of natural products from plant matrices is widely used in drug discovery. Here, this strategy was applied to identify trypanocidal coumarins effective against the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease (American trypanosomiasis). Previously, phylogenetic relationships of trypanocidal activity revealed a coumarin-associated antichagasic hotspot in the Apiaceae. In continuation, a total of 35 ethyl acetate extracts of different Apiaceae species were profiled for selective cytotoxicity against T. cruzi epimastigotes over host CHO-K1 and RAW264.7 cells at 10 µg/mL. A flow cytometry-based T. cruzi trypomastigote cellular infection assay was employed to measure toxicity against the intracellular amastigote stage. Among the tested extracts, Seseli andronakii aerial parts, Portenschlagiella ramosissima and Angelica archangelica subsp. litoralis roots exhibited selective trypanocidal activity and were subjected to bioactivity-guided fractionation and isolation by countercurrent chromatography. The khellactone ester isosamidin isolated from the aerial parts of S. andronakii emerged as a selective trypanocidal molecule (selectivity index ∼9) and inhibited amastigote replication in CHO-K1 cells, though it was significantly less potent than benznidazole. The khellactone ester praeruptorin B and the linear dihydropyranochromones 3'-O-acetylhamaudol and ledebouriellol isolated from the roots of P. ramosissima were more potent and efficiently inhibited the intracellular amastigote replication at < 10 µM. The furanocoumarins imperatorin, isoimperatorin and phellopterin from A. archangelica inhibited T. cruzi replication in host cells only in combination, indicative of superadditive effects, while alloimperatorin was more active in fractions. Our study reports preliminary structure-activity relationships of trypanocidal coumarins and shows that pyranocoumarins and dihydropyranochromones are potential chemical scaffolds for antichagasic drug discovery.

Pharmacometabolic Effects of Pteryxin and Valproate on Pentylenetetrazole-Induced Seizures in Zebrafish Reveal Vagus Nerve Stimulation.

Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in the lack of knowledge about the bioavailability and pharmacodynamic effects of bioactive compounds in this model organism. Here, we employed a combined methodology of LC-ESI-MS/MS analytics and targeted metabolomics with behavioral experiments to evaluate the anticonvulsant and potentially toxic effects of the angular dihydropyranocoumarin pteryxin (PTX) in comparison to the antiepileptic drug sodium valproate (VPN) in zebrafish larvae. PTX occurs in different Apiaceae plants traditionally used in Europe to treat epilepsy but has not been investigated so far. To compare potency and efficacy, the uptake of PTX and VPN into zebrafish larvae was quantified as larvae whole-body concentrations together with amino acids and neurotransmitters as proxy pharmacodynamic readout. The convulsant agent pentylenetetrazole (PTZ) acutely reduced the levels of most metabolites, including acetylcholine and serotonin. Conversely, PTX strongly reduced neutral essential amino acids in a LAT1 (SLCA5)-independent manner, but, similarly to VPN specifically increased the levels of serotonin, acetylcholine, and choline, but also ethanolamine. PTX dose and time-dependent manner inhibited PTZ-induced seizure-like movements resulting in a ~70% efficacy after 1 h at 20 µM (the equivalent of 4.28 ± 0.28 µg/g in larvae whole-body). VPN treated for 1 h with 5 mM (the equivalent of 18.17 ± 0.40 µg/g in larvae whole-body) showed a ~80% efficacy. Unexpectedly, PTX (1-20 µM) showed significantly higher bioavailability than VPN (0.1-5 mM) in immersed zebrafish larvae, possibly because VPN in the medium dissociated partially to the readily bioavailable valproic acid. The anticonvulsive effect of PTX was confirmed by local field potential (LFP) recordings. Noteworthy, both substances specifically increased and restored whole-body acetylcholine, choline, and serotonin levels in control and PTZ-treated zebrafish larvae, indicative of vagus nerve stimulation (VNS), which is an adjunctive therapeutic strategy to treat refractory epilepsy in humans. Our study demonstrates the utility of targeted metabolomics in zebrafish assays and shows that VPN and PTX pharmacologically act on the autonomous nervous system by activating parasympathetic neurotransmitters.

Goods and Bads of the Endocannabinoid System as a Therapeutic Target: Lessons Learned after 30 Years.

The cannabis derivative marijuana is the most widely used recreational drug in the Western world and is consumed by an estimated 83 million individuals (∼3% of the world population). In recent years, there has been a marked transformation in society regarding the risk perception of cannabis, driven by its legalization and medical use in many states in the United States and worldwide. Compelling research evidence and the Food and Drug Administration cannabis-derived cannabidiol approval for severe childhood epilepsy have confirmed the large therapeutic potential of cannabidiol itself, Δ9-tetrahydrocannabinol and other plant-derived cannabinoids (phytocannabinoids). Of note, our body has a complex endocannabinoid system (ECS)-made of receptors, metabolic enzymes, and transporters-that is also regulated by phytocannabinoids. The first endocannabinoid to be discovered 30 years ago was anandamide (N-arachidonoyl-ethanolamine); since then, distinct elements of the ECS have been the target of drug design programs aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here a critical review of our knowledge of the goods and bads of the ECS as a therapeutic target is presented to define the benefits of ECS-active phytocannabinoids and ECS-oriented synthetic drugs for human health. SIGNIFICANCE STATEMENT: The endocannabinoid system plays important roles virtually everywhere in our body and is either involved in mediating key processes of central and peripheral diseases or represents a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of the components of this complex system, and in particular of key receptors (like cannabinoid receptors 1 and 2) and metabolic enzymes (like fatty acid amide hydrolase and monoacylglycerol lipase), will advance our understanding of endocannabinoid signaling and activity at molecular, cellular, and system levels, providing new opportunities to treat patients.

Assay of Endocannabinoid Uptake.

Endocannabinoids at nanomolar physiological concentrations cross cellular membranes by facilitated diffusion, a process that can be studied by measuring transport kinetics and endocannabinoid trafficking employing radioligands and mass spectrometry. Here, we describe radiosubstrate-based assays using arachidonoyl[1-3H]ethanolamine and 2-arachidonoyl[1,2,3-3H]glycerol to measure cellular endocannabinoid uptake in a three-phase assay with human U937 cells. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS)-based lipidomics was used to interrogate the roles of serum and albumin for endocannabinoid trafficking in U937 cells.

The Evaluation of l-Tryptophan Derivatives as Inhibitors of the l-Type Amino Acid Transporter LAT1 (SLC7A5).

A series of derivatives of the substrate amino acid l-tryptophan have been investigated for inhibition of the L-type amino acid transporter LAT1 (SLC7A5), which is an emerging target in anticancer drug discovery. Of the four isomeric 4-, 5-, 6-, or 7-benzyloxy-l-tryptophans, the 5-substituted derivative was the most potent, with an IC50 of 19 µM for inhibition of [3 H]-l-leucine uptake into HT-29 human colon carcinoma cells. The replacement of the carboxy group in 5-benzyloxy-l-tryptophan by a bioisosteric tetrazole moiety led to a complete loss in potency. Likewise, the corresponding tetrazolide derived from l-tryptophan itself was found to be neither a substrate nor an inhibitor of the transporter. Increasing the steric bulk at the 5-position, while reasonably well tolerated in some cases, did not result in an improvement in potency. At the same time, none of these derivatives was found to be a substrate for LAT1-mediated transport.