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Paracetamol, one of the most widely used pain-relieving drugs, is deacetylated to 4-aminophenol (4-AP) that undergoes fatty acid amide hydrolase (FAAH)-dependent biotransformation into N-arachidonoylphenolamine (AM404), which mediates TRPV1-dependent antinociception in the brain of rodents. However, paracetamol is also converted to the liver-toxic metabolite N-acetyl-p-benzoquinone imine already at therapeutic doses, urging for safer paracetamol analogues. Primary amine analogues with chemical structures similar to paracetamol were evaluated for their propensity to undergo FAAH-dependent N-arachidonoyl conjugation into TRPV1 activators both in vitro and in vivo in rodents. The antinociceptive and antipyretic activity of paracetamol and primary amine analogues was examined with regard to FAAH and TRPV1 as well as if these analogues produced acute liver toxicity. 5-Amino-2-methoxyphenol (2) and 5-aminoindazole (3) displayed efficient target protein interactions with a dose-dependent antinociceptive effect in the mice formalin test, which in the second phase was dependent on FAAH and TRPV1. No hepatotoxicity of the FAAH substrates transformed into TRPV1 activators was observed. While paracetamol attenuates pyrexia via inhibition of brain cyclooxygenase, its antinociceptive FAAH substrate 4-AP was not antipyretic, suggesting separate mechanisms for the antipyretic and antinociceptive effect of paracetamol. Furthermore, compound 3 reduced fever without a brain cyclooxygenase inhibitory action. The data support our view that analgesics and antipyretics without liver toxicity can be derived from paracetamol. Thus, research into the molecular actions of paracetamol could pave the way for the discovery of analgesics and antipyretics with a better benefit-to-risk ratio. (C) 2020 The Author(s). Published by Elsevier Masson SAS., Funding Agencies|Medical Faculty of Lund University [ALFSKANE-450751]; AFA Forsakring [140376]; Hjarnfonden [FO2019-0316]; Stiftelsen Olle Engkvist ByggmastareSwedish Research Council [189-290]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [07879]; Royal Physiographic Society, Lund Sweden; Clermont Auvergne University, INSERM; Agence Nationale de la Recherche of the French government through the program "Investissements dAvenir (I-Site CAP 20-25)French National Research Agency (ANR)

The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Delta 1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1., Funding Agencies|Swedish Research CouncilSwedish Research Council [2014-3801, 2010-5787, 2007-6110]; FormasSwedish Research Council Formas [2007-718]; Research School of Pharmaceutical Sciences FLAK; Medical Faculty at Lund University [ALFSKANE-451751]

The mode of action of paracetamol (acetaminophen), which is widely used for treating pain and fever, has remained obscure, but may involve several distinct mechanisms, including cyclooxygenase inhibition and transient receptor potential ankyrin 1 (TRPA1) channel activation, the latter being recently associated with paracetamol?s propensity to elicit hypothermia at higher doses. Here, we examined whether the antipyretic effect of paracetamol was due to TRPA1 activation or cyclooxygenase inhibition. Treatment of wild-type and TRPA1 knockout mice rendered febrile by immune challenge with LPS with a dose of paracetamol that did not produce hypothermia (150 mg/kg) but is known to be analgetic, abolished fever in both genotypes. Paracetamol completely suppressed the LPS-induced elevation of prostaglandin E2 in the brain and also reduced the levels of several other prostanoids. The hypothermia induced by paracetamol was abolished in mice treated with the electrophile-scavenger N-acetyl cysteine. We conclude that paracetamol?s antipyretic effect in mice is dependent on inhibition of cyclooxygenase activity, including the formation of pyrogenic prostaglandin E2, whereas paracetamol-induced hypothermia likely is mediated by the activation of TRPA1 by electrophilic metabolites of paracetamol, similar to its analgesic effect in some experimental paradigms.?Mirrasekhian, E., Nilsson, J. L. Å., Shionoya, K., Blomgren, A., Zygmunt, P. M., Engblom, D., Högestätt, E. D., Blomqvist, A. The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1?mediated hypothermia and is associated with prostaglandin inhibition in the brain., Funding agencies: Swedish Medical Research Council [20725, 07879, 2014-3801]; European Research Council (ERC) Starting Grant; Knut and Alice Wallenberg Foundation; Swedish Brain Foundation; Swedish Cancer Foundation [16/0572]; County Council of Ostergotland; Medical Facult

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H-1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H-1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage'' compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentration

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations.