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Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K

We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.

GPR6 is an orphan G-protein–coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

The Lysophosphatidic Acid 1 Receptor (LPA1 receptor) has been linked to the initiation and progression of a variety of poorly treated fibrotic conditions. Several compounds that have been described as LPA1 receptor antagonists have progressed into clinical trials: 1-(4-{4-[3-methyl-4-({[(1R)-1-phenylethoxy]carbonyl}amino)-1,2-oxazol-5-yl]phenyl}phenyl)cyclopropane-1-carboxylic acid (BMS-986202) and 2-{4-methoxy-3-[2-(3-methylphenyl)ethoxy]benzamido}-2,3-dihydro-1H-indene-2-carboxylic acid (SAR-100842). We considered that as LPA1 receptor function is involved in many normal physiological processes, inhibition of specific signalling pathways associated with fibrosis may be therapeutically advantageous. We compared the binding and functional effects of a novel compound; 4-({(Cyclopropylmethyl)[4-(2-fluorophenoxy)benzoyl]amino}methyl}benzoic acid (TAK-615) with BMS-986202 and SAR-100842. Back-scattering interferometry (BSI) was used to show that the apparent affinity of TAK-615 was enhanced in the presence of LPA. The binding signal for BMS-986202 was not detected in the presence of LPA suggesting competition but interestingly the apparent affinity of SAR-100842 was also enhanced in the presence of LPA. Only BMS-986202 was able to fully inhibit the response to LPA in calcium mobilisation, β-arrestin, cAMP, GTPγS and RhoA functional assays. TAK-615 and SAR-100842 showed different inhibitory profiles in the same functional assays. Further binding studies indicated that TAK-615 is not competitive with either SAR-100842 or BMS-986202, suggesting a different site of binding. The results generated with this set of experiments demonstrate that TAK-615 acts as a negative allosteric modulator (NAM) of the LPA1 receptor. Surprisingly we find that SAR-100842 also behaves like a NAM. BMS-986202 on the other hand behaves like an orthosteric antagonist.

The Lysophosphatidic Acid 1 Receptor (LPA

GPR81, which exhibits a high degree of homology with GPR109a, has been recently identified as a lactate receptor. Similar to GPR109a, the activation of GPR81 by lactate suppresses lipolysis, suggesting that GPR81 may be a potential drug target for treating dyslipidemia. In addition, the fact that GPR81 is expressed only in adipocytes, whereas GPR109a is expressed in various tissues and cells, including Langerhans cells, which are considered responsible for flushing, indicates that targeting GPR81 could lead to the development of antidyslipidemia agents with a reduced risk of this side effect. However, the pharmacological role of GPR81 remains largely unclear, mainly because of the lack of potent and selective surrogate GPR81 agonists suitable for in vivo studies. In the present study, we showed that lactate-induced suppression of lipolysis in explants of white adipose tissue (WAT) depends on the presence of GPR81. We also performed high-throughput screening (HTS) and identified four novel chemical clusters as GPR81 agonists. Chemical optimization of aminothiazole derivatives led to the discovery of a lead compound with improved potency. The compound inhibited lipolysis in differentiated 3T3-L1 adipocytes. Finally, intraperitoneal administration of this compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. This is the first description of a 50nM GPR81 selective agonist with in vivo efficacy, without the side effect, i.e., flushing. These results suggest that GPR81 is an attractive drug target for treating dyslipidemia without the risk of flushing.

VIP/PACAP receptor activation stimulates the production of [cAMP]i and [Ca2+]i by coupling to independent G-protein subunits, although agonist potencies for the different transduction pathways appear to differ. Using CHO-K1 cells stably expressing the human VIP/PACAP receptors (hVPAC1R, hVPAC2R, and hPAC1R), functional assays ([cAMP]i and [Ca2+]i) were established and the receptor pharmacology was characterized with five peptide agonists (VIP, PACAP-27, PACAP-38, [Ala(11,22,28)]VIP, and R3P65). The rank order of potency (ROP) was consistent between assays for the individual receptor subtypes, however, higher agonist concentrations (approximately 100-fold) were required for stimulating [Ca2+]i when compared to [cAMP]i.

The 5-HT(2A) receptor (5-HT(2A)R) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT(2A)R gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT(2A)R expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT(2A)R tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT(2A)Rs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT(2A)R displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT(2A)R.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF–PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC1, VPAC2 and PAC1R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.

Summary All chronic open wounds (pressure sores - superficial and deep - and leg ulcers) were inspected over a two day period in a Department of Health Care of the Elderly (DHCE). A high but similar pressure sore prevalence rate was observed in acute/ rehabilitation wards and long stay wards (19.8% and 20.8% respectively). Sub-analysis of these figures revealed that the majority of pressure sores had occurred prior to admission to the DHCE. The distribution of pressure relieving devices was not uniform and treatment regimes differed. The cost of pressure sores in both human and financial terms is substantial and, as the majority of pressure sores are preventable, unwarranted. Education of doctors and nurses concerning chronic wounds (as well as patient awareness programmes) must be developed and prevention strategies implemented.