Discovery of α-Amidobenzylboronates as Highly Potent Covalent Inhibitors of Plasma Kallikrein.

Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound 20 IC50 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that 20 showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (23), a known nonselective covalent PKa inhibitor.

PET ligands [18F]LSN3316612 and [11C]LSN3316612 quantify O-linked-ß-N-acetyl-glucosamine hydrolase in the brain.

We aimed to develop effective radioligands for quantifying brain O-linked-ß-N-acetyl-glucosamine (O-GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O-GlcNAc through the enzyme pair OGA and O-GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O-GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC50) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [3H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [18F]LSN3316612 and [11C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [18F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (VT) values by 110 min of scanning. Overall, [18F]LSN3316612 is preferred over [11C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.

Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors.

BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography.

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.

Current status of muscarinic M1 and M4 receptors as drug targets for neurodegenerative diseases.

The cholinergic signalling system has been an attractive pathway to seek targets for modulation of arousal, cognition, and attention which are compromised in neurodegenerative and neuropsychiatric diseases. The acetylcholine muscarinic receptor M1 and M4 subtypes which are highly expressed in the central nervous system, in cortex, hippocampus and striatum, key areas of cognitive and neuropsychiatric control, have received particular attention. Historical muscarinic drug development yielded first generation agonists with modest selectivity for these two receptor targets over M2 and M3 receptors, the major peripheral sub-types hypothesised to underlie the dose-limiting clinical side effects. More recent compound screening and medicinal chemistry optimization of orthosteric and allosteric agonists, and positive allosteric modulators binding to sites distinct from the highly homologous acetylcholine binding pocket have yielded a collection of highly selective tool compounds for preclinical validation studies. Several M1 selective ligands have progressed to early clinical development and in time will hopefully lead to useful therapeutics for treating symptoms of Alzheimer's disease and related disorders. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

TRPV3 in Drug Development.

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP (Transient Receptor Potential) super-family. It is a relatively underexplored member of the thermo-TRP sub-family (Figure 1), however, genetic mutations and use of gene knock-outs and selective pharmacological tools are helping to provide insights into its role and therapeutic potential. TRPV3 is highly expressed in skin, where it is implicated in skin physiology and pathophysiology, thermo-sensing and nociception. Gain of function TRPV3 mutations in rodent and man have enabled the role of TRPV3 in skin health and disease to be particularly well defined. Pre-clinical studies provide some rationale to support development of TRPV3 antagonists for therapeutic application for the treatment of inflammatory skin conditions, itch and pain. However, to date, only one compound directed towards block of the TRPV3 receptor (GRC15300) has progressed into clinical trials. Currently, there are no known clinical trials in progress employing a TRPV3 antagonist.

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus.

Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans.

123I-iododexetimide preferentially binds to the muscarinic receptor subtype M1 in vivo.

UNLABELLED: The muscarinic M1 receptor (M1R) is highly involved in cognition, and selective M1 agonists have procognitive properties. Loss of M1R has been found in postmortem brain tissue for several neuropsychiatric disorders and may be related to symptoms of cognitive dysfunction. (123)I-iododexetimide is used for imaging muscarinic acetylcholine receptors (mAchRs). Considering its high brain uptake and intense binding in M1R-rich brain areas, (123)I-iododexetimide may be an attractive radiopharmaceutical to image M1R. To date, the binding affinity and selectivity of (123)I-iododexetimide for the mAchR subtypes has not been characterized, nor has its brain distribution been studied intensively. Therefore, this study aimed to address these topics. METHODS: The in vitro affinity and selectivity of (127)I-iododexetimide (cold-labeled iododexetimide), as well as its functional antagonist properties (guanosine 5'-[γ-(35)S-thio]triphosphate [GTPγ(35)S] assay), were assessed on recombinant human M1R-M5R. Distributions of (127)I-iododexetimide and (123)I-iododexetimide in the brain were evaluated using liquid chromatography-mass spectrometry and storage phosphor imaging, respectively, ex vivo in rats, wild-type mice, and M1-M5 knock-out (KO) mice. Inhibition of (127)I-iododexetimide and (123)I-iododexetimide binding in M1R-rich brain areas by the M1R/M4R agonist xanomeline, or the antipsychotics olanzapine (M1R antagonist) and haloperidol (low M1R affinity), was assessed in rats ex vivo. RESULTS: In vitro, (127)I-iododexetimide displayed high affinity for M1R (pM range), with modest selectivity over other mAchRs. In biodistribution studies on rats, ex vivo (127)I-iododexetimide binding was much higher in M1R-rich brain areas, such as the cortex and striatum, than in cerebellum (devoid of M1Rs). In M1 KO mice, but not M2-M5 KO mice, (127)I-iododexetimide binding was strongly reduced in the frontal cortex compared with wild-type mice. Finally, acute administration of both an M1R/M4R agonist xanomeline and the M1R antagonist olanzapine was able to inhibit (123)I-iododexetimide ex vivo, and (123)I-iododexetimide binding in M1-rich brain areas in rats, whereas administration of haloperidol had no effect. CONCLUSION: The current results suggest that (123)I-iododexetimide preferentially binds to M1R in vivo and can be displaced by M1R ligands. (123)I-iododexetimide may therefore be a useful imaging tool as a way to further evaluate M1R changes in neuropsychiatric disorders, as a potential stratifying biomarker, or as a clinical target engagement biomarker to assess M1R.

Activation of transient receptor potential ankyrin 1 induces CGRP release from spinal cord synaptosomes.

Transient receptor potential ankyrin 1 (TRPA1) is a sensor of nociceptive stimuli, expressed predominantly in a subpopulation of peptidergic sensory neurons which co-express the noxious heat-sensor transient receptor potential vanilloid 1. In this study, we describe a spinal cord synaptosome-calcitonin gene-related peptide (CGRP) release assay for examining activation of TRPA1 natively expressed on the central terminals of dorsal root ganglion neurons. We have shown for the first time that activation of TRPA1 channels expressed on spinal cord synaptosomes by a selection of agonists evokes a concentration-dependent release of CGRP which is inhibited by TRPA1 antagonists. In addition, our results demonstrate that depolarization of spinal cord synaptosomes by a high concentration of KCl induces CGRP release via a T-type calcium channel-dependent mechanism whilst TRPA1-induced CGRP release functions independently of voltage-gated calcium channel activation. Finally, we have shown that pre-treatment of synaptosomes with the opioid agonist, morphine, results in a reduction of depolarization-induced CGRP release. This study has demonstrated the use of a dorsal spinal cord homogenate assay for investigation of natively expressed TRPA1 channels and for modulation of depolarizing stimuli at the level of the dorsal spinal cord.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain.

BACKGROUND: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. RESULTS: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. CONCLUSIONS: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

Monocytes expressing CX3CR1 orchestrate the development of vincristine-induced pain.

A major dose-limiting side effect associated with cancer-treating antineoplastic drugs is the development of neuropathic pain, which is not readily relieved by available analgesics. A better understanding of the mechanisms that underlie pain generation has potential to provide targets for prophylactic management of chemotherapy pain. Here, we delineate a pathway for pain that is induced by the chemotherapeutic drug vincristine sulfate (VCR). In a murine model of chemotherapy-induced allodynia, VCR treatment induced upregulation of endothelial cell adhesion properties, resulting in the infiltration of circulating CX3CR1⁺ monocytes into the sciatic nerve. At the endothelial-nerve interface, CX3CR1⁺ monocytes were activated by the chemokine CX3CL1 (also known as fractalkine [FKN]), which promoted production of reactive oxygen species that in turn activated the receptor TRPA1 in sensory neurons and evoked the pain response. Furthermore, mice lacking CX3CR1 exhibited a delay in the development of allodynia following VCR administration. Together, our data suggest that CX3CR1 antagonists and inhibition of FKN proteolytic shedding, possibly by targeting ADAM10/17 and/or cathepsin S, have potential as peripheral approaches for the prophylactic treatment of chemotherapy-induced pain.

Pharmacological characterisation of ligand- and voltage-gated ion channels expressed in human iPSC-derived forebrain neurons.

INTRODUCTION: Genetic causes, or predisposition, are increasingly accepted to be part of the ethiopathogenesis of many neuropsychiatric diseases. While genes can be studied in any type of cells, their physiological function in human brain cells is difficult to evaluate, particularly in living subjects. METHODS: As a first step towards the characterisation of human inducible pluripotent stem cell (iPSC)-derived neurons from autism spectrum disorder (ASD) patients, we used gene expression and functional studies to define the regional identity of the typical forebrain differentiation, demonstrate expression patterns of genes of interest in ASD and understand the properties of 'control' iPSC-derived neurons (iCell-Neurons™), with a focus on receptors and ion channels that play a central role in synaptic physio-pathology. RESULTS AND DISCUSSION: The gene expression profile of the iCell-Neurons™ closely resembled that observed in neonatal prefrontal cortex tissues. Functional studies, performed mainly using calcium flux assays, demonstrated the presence of ionotropic glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) and gamma-aminobutyric acid type A receptors. Voltage-gated sodium and calcium channels were also identified using similar techniques. CONCLUSIONS: Overall, the results reported here suggest that iCell-Neurons™ are a good cellular model of a relatively immature forebrain human neuron population that can be used both as a control in comparison to patients cells, and as host cells in which mutations, insertions and deletions can be used in order to study the molecular mechanisms of ASD and other neurological disorders in an isogenic cellular background.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACH: Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTS: A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies.

The subtype-selective nicotinic acetylcholine receptor positive allosteric potentiator 2087101 differentially facilitates neurotransmission in the brain.

Positive allosteric modulators of centrally expressed nicotinic acetylcholine receptors have therapeutic potentials in areas of cognition, motor function and reward. Several chemical classes of allosteric modulators that are selective for alpha7 nicotinic receptors have been characterised, but potentiators for the most widely expressed alpha4beta2 nicotinic receptor subtype are few and less defined, owing probably to the difficulty to achieve selectivity over other heteromeric receptor subtypes. 2087101 (2-amino-5-keto)thiazole) is a potent potentiator of both alpha7 and alpha4beta2 receptors and it has selectivity against the alpha3beta4 subtype, which may be responsible for the undesirable peripheral side effects. To further characterise its ability to differentiate between native nicotinic receptors, we examined the effects of 2087101 on alpha7, alpha4beta2* and alpha3beta4* receptor-mediated responses in the rat brain in electrophysiological and neurochemical experiments. 2087101 significantly potentiated agonist-induced, alpha7 and non-alpha7 receptor-mediated, GABAergic postsynaptic currents in cultured hippocampal neurones, but not the nicotine-stimulated [(3)H]noradrenaline release from hippocampal slices, which was primarily mediated by alpha3beta4* receptors, confirming its selectivity for alpha7 and alpha4beta2* receptors in native systems. 2087101 also significantly enhanced nicotine-stimulated firing increase in dopamine neurones of the ventral tegmental area, an effect that was dihydro-beta-erythroidine-sensitive and thereby mediated by alpha4beta2* nicotinic receptors. 2087101 can therefore enhance native nicotinic activities mediated by alpha7 and alpha4beta2*, but not alpha3beta4* receptors, showing its unique ability to discriminate between native heteromeric nicotinic receptor subtypes and its therapeutic potential for treating brain disorders by concurrent modulation of both alpha7 and alpha4beta2* nicotinic receptors.

Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties.

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.

TRP channels as emerging targets for pain therapeutics.

BACKGROUND: The transient receptor potential (TRP) superfamily of ion channels are a large and diverse group that have received increased attention in recent years. The sub-family of thermo-TRPs which are regulated by temperature, among other physical and chemical stimuli, are of particular interest for the development of potential pain therapeutics. OBJECTIVE/METHODS: We review the advances in the field in recent years, focusing on a rationale for pain therapy and potential challenges associated with these targets. RESULTS/CONCLUSIONS: Vanilloid-type TRP 1 (TRPV1) is the most well studied and advanced member of the family, with selective agonists and antagonists already in clinical use or development, respectively. Among other thermo-TRPs (including TRPV2 - 4, Ankyrin type TRP 1 (TRPA1) and melastatin type TRP 8 (TRPM8)), TRPA1 and TRPM8 are emerging as promising novel pain targets.