A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

In vivo catecholaminergic metabolism in the medial prefrontal cortex of ENU2 mice: an investigation of the cortical dopamine deficit in phenylketonuria.

OBJECTIVE: Phenylketonuria (PKU) is an inherited metabolic disease characterized by plasma hyperphenylalaninemia and several neurological symptoms that can be controlled by rigorous dietetic treatment. The cellular mechanisms underlying impaired brain functions are still unclear. It has been proposed, however, that phenylalanine interference in cognitive functions depends on impaired dopamine (DA) transmission in the prefrontal cortical area due to reduced availability of the precursor tyrosine. Here, using Pah(enu2) (ENU2) mice, the genetic murine model of PKU, we investigated all metabolic steps of catecholamine neurotransmission within the medial preFrontal Cortex (mpFC), availability of the precursor tyrosine, synthesis and release, to find an easy way to reinstate normal cortical DA neurotransmission. METHODS AND RESULTS: Analysis of blood and brain levels of tyrosine showed reduced plasma and cerebral levels of tyrosine in ENU2 mice. Western blot analysis demonstrated deficient tyrosine hydroxylase (TH) protein levels in mpFC of ENU2 mice. Cortical TH activity, determined in vivo by measuring the accumulation of l-3,4-dihydroxyphenylalanine (L-DOPA) in mpFC after inhibition of L-aromatic acid decarboxylase with NSD-1015, was reduced in ENU2 mice. Finally, a very low dose of L-DOPA, which bypasses the phenylalanine-inhibited metabolic steps, restored DA prefrontal transmission to levels found in healthy mice. CONCLUSION: The data suggests that a strategy of using tyrosine supplementation to treat PKU is unlikely to be effective, whereas small dose L-DOPA administration is likely to have a positive therapeutic effect.