Structure-Activity Relationship Studies of Aryl Sulfoxides as Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is the key enzyme for the hydrolysis of endocannabinoid 2-arachidonoylglycerol (2-AG). The central role of MAGL in the metabolism of 2-AG makes it an attractive therapeutic target for a variety of disorders, including inflammation-induced tissue injury, pain, multiple sclerosis, and cancer. Previously, we reported LEI-515, an aryl sulfoxide, as a peripherally restricted, covalent reversible MAGL inhibitor that reduced neuropathic pain and inflammation in preclinical models. Here, we describe the structure-activity relationship (SAR) of aryl sulfoxides as MAGL inhibitors that led to the identification of LEI-515. Optimization of the potency of high-throughput screening (HTS) hit 1 yielded compound ±43. However, ±43 was not metabolically stable due to its ester moiety. Replacing the ester group with α-CF2 ketone led to the identification of compound ±73 (LEI-515) as a metabolically stable MAGL inhibitor with subnanomolar potency. LEI-515 is a promising compound to harness the therapeutic potential of MAGL inhibition.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol 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.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a ß-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 µM) and C11 (EC50 = 11.4 µM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 µM) and 27 (LN6023, EC50 = 3.5 µM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Hypoxia ameliorates maternal diet-induced insulin resistance during pregnancy while having a detrimental effect on the placenta.

Maternal overweight/obesity contributes significantly to the development of gestational diabetes, which causes risks to both mother and fetus and is increasing sharply in prevalence worldwide. Since hypoxia reprograms energy metabolism and can alleviate weight gain, adiposity, insulin resistance (IR), and dyslipidemia, we set out to study the potential of sustained reduced ambient oxygen tension (15% O2 ) during pregnancy for alleviating the detrimental effects of diet-induced IR in C57Bl/6N mice, taking normal chow-fed and normoxia (21% O2 ) groups as controls. Our data show that hypoxic intervention reduced maternal weight gain, adiposity, and adipose tissue inflammation, and ameliorated maternal glucose metabolism and IR during gestation in diet-induced IR relative to normoxia. Where diet-induced IR reduced maternal hemoglobin and increased serum erythropoietin levels, hypoxic intervention compensated for these changes. Diet-induced IR reduced fetal growth in normoxia, and even more in hypoxia. Hypoxic intervention reduced liver weight gain during pregnancy in the dams with diet-induced IR, maternal liver weight being positively associated with embryo number. In case of diet-induced IR, the hypoxic intervention compromised placental energy metabolism and vascularization and increased end-pregnancy placental necrosis. Altogether, these data show that although hypoxic intervention mediates several beneficial effects on maternal metabolism, the combination of it with diet-induced IR is even more detrimental to the placental and fetal outcome than diet-induced IR alone.

Synthesis and SAR evaluation of coumarin derivatives as potent cannabinoid receptor agonists.

We report the development and extensive structure-activity relationship evaluation of a series of modified coumarins as cannabinoid receptor ligands. In radioligand, and [35S]GTPγS binding assays the CB receptor binding affinities and efficacies of the new ligands were determined. Furthermore, we used a ligand-based docking approach to validate the empirical observed results. In conclusion, several crucial structural requirements were identified. The most potent coumarins like 3-butyl-7-(1-butylcyclopentyl)-5-hydroxy-2H-chromen-2-one (36b, Ki CB2 13.7 nM, EC50 18 nM), 7-(1-butylcyclohexyl)-5-hydroxy-3-propyl-2H-chromen-2-one (39b, Ki CB2 6.5 nM, EC50 4.51 nM) showed a CB2 selective agonistic profile with low nanomolar affinities.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family.

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the α-ketoamide series using activity-based protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family members. LEI-301 reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, LEI-301 may help to dissect the physiological role of the PLAATs.

Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells.

BACKGROUND: Neuroinflammation plays a vital role in Alzheimer's disease and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system, and they play essential roles in the maintenance of homeostasis and responses to neuroinflammation. The orphan G-protein-coupled receptor 55 (GPR55) has been reported to modulate inflammation and is expressed in immune cells such as monocytes and microglia. However, its effects on neuroinflammation, mainly on the production of members of the arachidonic acid pathway in activated microglia, have not been elucidated in detail. METHODS: In this present study, a series of coumarin derivatives, that exhibit GPR55 antagonism properties, were designed. The effects of these compounds on members of the arachidonic acid cascade were studied in lipopolysaccharide (LPS)-treated primary rat microglia using Western blot, qPCR, and ELISA. RESULTS: We demonstrate here that the various compounds with GPR55 antagonistic activities significantly inhibited the release of PGE2 in primary microglia. The inhibition of LPS-induced PGE2 release by the most potent candidate KIT 17 was partially dependent on reduced protein synthesis of mPGES-1 and COX-2. KIT 17 did not affect any key enzyme involved on the endocannabinoid system. We furthermore show that microglia expressed GPR55 and that a synthetic antagonist of the GPR receptor (ML193) demonstrated the same effect of the KIT 17 on the inhibition of PGE2. CONCLUSIONS: Our results suggest that KIT 17 is acting as an inverse agonist on GPR55 independent of the endocannabinoid system. Targeting GPR55 might be a new therapeutic option to treat neurodegenerative diseases with a neuroinflammatory background such as Alzheimer's disease, Parkinson, and multiple sclerosis (MS).

Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels.

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gßγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides.

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic ß cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P3 > PI(3,5)P2 > PI(4,5)P2 ≈ PI(3,4)P2 >> PI(4)P. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C-coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.

Properties and functions of TRPM1 channels in the dendritic tips of retinal ON-bipolar cells.

An increase in light intensity induces a depolarization in retinal ON-bipolar cells via a reduced glutamate release from presynaptic photoreceptor cells. The underlying transduction cascade in the dendritic tips of ON-bipolar cells involves mGluR6 glutamate receptors signaling to TRPM1 proteins that are an indispensable part of the transduction channel. Several other proteins are recognized to participate in the transduction machinery. Deficiency in many of these leads to congenital stationary night blindness, because rod bipolar cells, a subgroup of ON-bipolar cells, constitute the main route for sensory information under scotopic conditions. Here, we review the current knowledge about TRPM1 ion channels and how their activity is regulated within the postsynaptic compartment of ON-bipolar cells. The functional properties of TRPM1 channels in the dendritic compartment are not well understood as they differ substantially from those of recombinant TRPM1 channels. Critical evaluation of possible explanations of these discrepancies indicates that some key components of this transduction pathway might still not be known. The continued exploration of this pathway will yield further clinically useful insights.

Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo.

Transient receptor potential melastatin 3 (TRPM3) is a calcium-permeable nonselective cation channel that is expressed in a subset of dorsal root (DRG) and trigeminal ganglia sensory neurons. TRPM3 can be activated by the neurosteroid pregnenolone sulfate (PregS) and heat. TRPM3⁻/⁻ mice display an impaired sensation of noxious heat and thermal hyperalgesia. We have previously shown that TRPM3 is blocked by the citrus fruit flavanones hesperetin, naringenin, and eriodictyol as well as by ononetin, a deoxybenzoin from Ononis spinosa. To further improve the tolerability, potency, and selectivity of TRPM3 blockers, we conducted a hit optimization procedure by rescreening a focused library that was composed of chemically related compounds. Within newly identified TRPM3 blockers, isosakuranetin and liquiritigenin displayed favorable properties with respect to their inhibitory potency and a selective mode of action. Isosakuranetin, a flavanone whose glycoside is contained in blood oranges and grapefruits, displayed an IC50 of 50 nM and is to our knowledge the most potent inhibitor of TRPM3 identified so far. Both compounds exhibited a marked specificity for TRPM3 compared with other sensory TRP channels, and blocked PregS-induced intracellular free Ca²âº concentration signals and ionic currents in freshly isolated DRG neurons. Furthermore, isosakuranetin and previously identified hesperetin significantly reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Because the physiologic functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo.

TRPM3 channels provide a regulated influx pathway for zinc in pancreatic beta cells.

Zinc is stored in insulin-containing dense core vesicles of pancreatic beta-cells where it forms crystals together with insulin and calcium ions. Zinc ions are therefore released together with insulin upon exocytosis of these vesicles. Consequently, pancreatic beta-cells need to take up large amounts of zinc from the extracellular space across their plasma membrane. The pathways for zinc uptake are only partially understood. TRPM3 channels are present in pancreatic beta-cells and can be activated by the endogenous steroid pregnenolone sulfate. We demonstrate here that recombinant TRPM3 channels are highly permeable for many divalent cations, in particular also for zinc ions. Importantly, TRPM3 channels endogenously expressed in pancreatic beta-cells are also highly permeable for zinc ions. Using FluoZin3 to image changes of the intracellular zinc concentration, we show that pancreatic beta-cells take up zinc through TRPM3 channels even when extracellular zinc concentrations are low and physiological levels of calcium and magnesium are present. Activation of TRPM3 channels also leads to depolarization of beta-cells and to additional zinc influx through voltage-gated calcium channels. Our data establish that TRPM3 channels constitute a regulated entry pathway for zinc ions in pancreatic beta-cells.