From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson’s disease. From a ...high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited β-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.
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-phenylethoxycarbonyl}amino)-1,2-oxazol-5-ylphenyl}phenyl)cyclopropane-1-carboxylic acid (BMS-986202) and 2-{4-methoxy-3-2-(3-methylphenyl)ethoxybenzamido}-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)benzoylamino}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.
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-dihydropyrido3,4-bpyrazin-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.
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+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors.
Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1β, an effect not seen in THIK-1-depleted microglia.
In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1β from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.
•THIK-1 mRNA and protein are highly enriched in microglia in human cortical tissue.•THIK-1 mRNA is increased in microglial from AD brains compared to non-AD controls.•Disclosure of C101248, the first selective small-molecule to block THIK-1 currents.•THIK-1 inhibition prevented the NLRP3-dependent release of IL-1β from microglia.
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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.
The low affinity metabotropic glutamate receptor mGluR7 has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the ...functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR7 agonists. Of particular interest is the chromane CVN636, a potent (EC50 7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR7 compared to not only other mGluRs, but also a broad range of targets. CVN636 demonstrated CNS penetrance and efficacy in an in vivo rodent model of alcohol use disorder. CVN636 thus has potential to progress as a drug candidate in CNS disorders involving mGluR7 and glutamatergic dysfunction.
The low affinity metabotropic glutamate receptor mGluR
has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the ...functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR
agonists. Of particular interest is the chromane
, a potent (EC
7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR
compared to not only other mGluRs, but also a broad range of targets.
demonstrated CNS penetrance and efficacy in an
rodent model of alcohol use disorder.
thus has potential to progress as a drug candidate in CNS disorders involving mGluR
and glutamatergic dysfunction.
The Lysophosphatidic Acid 1 Receptor (LPA
receptor) has been linked to the initiation and progression of a variety of poorly treated fibrotic conditions. Several compounds that have been described as ...LPA
receptor antagonists have progressed into clinical trials: 1-(4-{4-3-methyl-4-({(1R)-1-phenylethoxycarbonyl}amino)-1,2-oxazol-5-ylphenyl}phenyl)cyclopropane-1-carboxylic acid (BMS-986202) and 2-{4-methoxy-3-2-(3-methylphenyl)ethoxybenzamido}-2,3-dihydro-1H-indene-2-carboxylic acid (SAR-100842). We considered that as LPA
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)benzoylamino}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 LPA
receptor. Surprisingly we find that SAR-100842 also behaves like a NAM. BMS-986202 on the other hand behaves like an orthosteric antagonist.
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could ...potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
Background & Aims Little is known on the gender-specific effect and potential role of non-linear associations between metabolic syndrome (MetS) components and liver cancer risk. We evaluated these ...associations based on the UK Biobank cohort. Methods We included 474,929 individuals without previous cancer based on the UK Biobank cohort. Gender-specific hazard ratios (HRs) and 95% confidence interval (CIs) were calculated by Cox proportional hazards regression, adjusting for potential confounders. Non-linear associations for individual MetS components were assessed by the restricted cubic spline method. Results Over a median follow-up of 6.6 years, we observed 276 cases of liver cancer (175 men, 101 women). MetS HR 1.48, 95% CI 1.27-1.72 and central obesity HR 1.65, 95% CI 1.18-2.31 were associated with higher risk of liver cancer in men but not in women. Participants with hyperglycaemia has higher risk of liver cancer. High waist circumference and blood glucose were dose-dependently associated with increased liver cancer risk in both genders. For high density lipoprotein (HDL) cholesterol (both genders) and blood pressure (women), U-shaped associations were observed. Low HDL cholesterol (< 1.35 mmol/L) in men and high HDL cholesterol in women (> 1.52 mmol/L) were associated with increased liver cancer risk. Conclusions MetS components showed gender-specific linear or U- shaped associations with the risk of liver cancer. Our study might provide evidence for individualized management of MetS for preventing liver cancer. Keywords: Metabolic syndrome, Liver cancer, Gender-specific effect, Non-linear association, UK Biobank
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