Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

Alkaloids Purified from Aristotelia chilensis Inhibit the Human α3ß4 Nicotinic Acetylcholine Receptor with Higher Potencies Compared with the Human α4ß2 and α7 Subtypes.

The alkaloids aristoteline (1), aristoquinoline (2), and aristone (3) were purified from the leaves of the Maqui tree Aristotelia chilensis and chemically characterized by NMR spectroscopy. The pharmacological activity of these natural compounds was evaluated on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results suggest that these alkaloids do not have agonistic, but inhibitory, activity on each receptor subtype. The obtained IC50 values indicate the following receptor selectivity: hα3ß4 > hα4ß2 ≫ hα7. In the particular case of hα3ß4 AChRs, 1 (0.40 ± 0.20 µM) and 2 (0.96 ± 0.38 µM) show higher potencies compared with 3 (167 ± 3 µM). Molecular docking and structure-activity relationship results indicate that ligand lipophilicity is important for the interaction with the luminal site located close to the cytoplasmic side of the hα3ß4 ion channel between positions -2' and -4'. Compound 1 could be used as a molecular scaffold for the development of more potent noncompetitive inhibitors with higher selectivity for the hα3ß4 AChR that could serve for novel addiction and depression therapies.

Drimane Sesquiterpenoids Noncompetitively Inhibit Human α4ß2 Nicotinic Acetylcholine Receptors with Higher Potency Compared to Human α3ß4 and α7 Subtypes.

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree ( Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4ß2, hα3ß4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4ß2 > hα3ß4 > hα7. In the case of hα4ß2 AChRs, the following potency rank order was determined (IC50's in µM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) ≫ dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4ß2 AChRs, additional structure-activity relationship and molecular docking experiments were performed. The Ca2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-ß2 intrasubunit) sites. The structure-activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4ß2 AChR by a cooperative mechanism, as shown experimentally ( nH > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4ß2 AChR.

Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer's disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4ß2 and α3ß4 subtypes.

To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca2+ influx results established that: (a) varenicline activates (µM range) nAChR subtypes with the following rank sequence: hα7>hα4ß4>hα4ß2>hα3ß4>>>hα1ß1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4ß2~hα4ß4>hα3ß4>hα7>>>Torpedo α1ß1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4ß4 (93%), and a partial agonist at hα4ß2 (20%) and hα3ß4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3ß4 and hα4ß2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4ß2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4ß2 nAChRs, hα4ß4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3ß4- and hα4ß2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.

A Placebo-Controlled Trial of AQW051 in Patients With Moderate to Severe Levodopa-Induced Dyskinesia.

BACKGROUND: This phase 2 randomized, double-blind, placebo-controlled study evaluated the efficacy and safety of the nicotinic acetylcholine receptor α7 agonist AQW051 in patients with Parkinson's disease and levodopa-induced dyskinesia. METHODS: Patients with idiopathic Parkinson's disease and moderate to severe levodopa-induced dyskinesia were randomized to AQW051 10 mg (n = 24), AQW051 50 mg (n = 24), or placebo (n = 23) once daily for 28 days. Coprimary end points were change in Modified Abnormal Involuntary Movement Scale and Unified Parkinson's Disease Rating Scale part III scores. Secondary outcomes included pharmacokinetics. RESULTS: In total, 67 patients completed the study. AQW051-treated patients experienced no significant improvements in Modified Abnormal Involuntary Movement Scale or Unified Parkinson's Disease Rating Scale part III scores by day 28. AQW051 was well tolerated; the most common adverse events were dyskinesia, fatigue, nausea, and falls. CONCLUSIONS: AQW051 did not significantly reduce dyskinesia or parkinsonian severity. © 2016 International Parkinson and Movement Disorder Society.

Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the Venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Differential pharmacological activity of JN403 between α7 and muscle nicotinic acetylcholine receptors.

The differential action of the novel agonist JN403 at neuronal α7 and muscle nicotinic receptors (AChRs) was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations ~1000-fold lower and ~20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking the ε subunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α-δ but not at the α-ε binding site. JN403 does not elicit detectable Ca²âº influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [³H]cytisine and [³H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs.

Functional and structural interaction of (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor.

The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4ß2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4ß2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4ß2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4ß2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4ß2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4ß2 nAChRs.

Sustained Trem2 stabilization accelerates microglia heterogeneity and Aß pathology in a mouse model of Alzheimer's disease.

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aß pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor.

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4ß2, α3ß4, α7, and α1ß1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4ß2 and hα3ß4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner ß-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states.

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.

Different interaction between the agonist JN403 and the competitive antagonist methyllycaconitine with the human alpha7 nicotinic acetylcholine receptor.

The interaction of the agonist JN403 with the human (h) alpha7 nicotinic acetylcholine receptor (AChR) was compared to that for the competitive antagonist methyllycaconitine (MLA). The receptor selectivity of JN403 was studied on the halpha7, halpha3beta4, and halpha4beta2 AChRs. The results established that the cationic center and the hydrophobic group found in JN430 and MLA are important for the interaction with the AChRs. MLA preincubation inhibits JN403-induced Ca(2+) influx in GH3-halpha7 cells with a potency 160-fold higher than that when MLA is co-injected with JN403. The most probable explanation, based on our dynamics results, is that MLA (more specifically the 3-methyl-2,5-dioxopyrrole ring and the B-D rings) stabilizes the resting conformational state. The order of receptor specificity for JN403 is as follows: halpha7 > halpha3beta4 ( approximately 40-fold) > halpha4beta2 ( approximately 500-fold). This specificity is based on a larger number of hydrogen bonds between the carbamate group (another pharmacophore) of JN403 and the halpha7 sites, the electrostatic repulsion between the positively charged residues around the halpha3beta4 sites and the cationic center of JN403, fewer hydrogen bonds for the interaction of JN403 with the halpha3beta4 AChR, and an unfavorable van der Waals interaction between JN403 and the alpha4-beta2 interface. The higher receptor specificity for JN403 could be important for the treatment of alpha7-related disorders, including dementias, pain-related ailments, depression, anxiety, and wound healing.

Interaction of selective serotonin reuptake inhibitors with neuronal nicotinic acetylcholine receptors.

We compared the interaction of fluoxetine and paroxetine, two selective serotonin reuptake inhibitors (SSRIs), with the human (h) alpha4beta2, alpha3beta4, and alpha7 nicotinic acetylcholine receptors (AChRs) in different conformational states, using Ca(2+) influx, radioligand binding, and molecular docking approaches. The results established that (1) fluoxetine was more potent than paroxetine in inhibiting agonist-activated Ca(2+) influx on halpha4beta2 and halpha7 AChRs, whereas the potency of both SSRIs was practically the same in the halpha3beta4 AChR. [corrected] (2) SSRIs bind to the [(3)H]imipramine locus with a [corrected] higher affinity when the AChRs are in the desensitized states compared to the resting states. (3) The different receptor specificity for fluoxetine determined by their inhibitory potencies or binding affinities suggests different modes of interaction when the AChR is in the closed or activated state. (4) Neutral and protonated fluoxetine interacts with a binding domain located in the middle of the AChR ion channel. In conclusion, SSRIs inhibit the most important neuronal AChRs with potencies and affinities that are clinically relevant by binding to a luminal site that is shared with tricyclic antidepressants.

Interaction of bupropion with muscle-type nicotinic acetylcholine receptors in different conformational states.

To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion (a) inhibits epibatidine-induced Ca(2+) influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with approximately 100-fold higher potency compared to that in the open state, (c) increases the desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits binding of [(3)H]TCP and [(3)H]imipramine to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/alpha-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively, our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process.

The selective nicotinic acetylcholine receptor alpha7 agonist JN403 is active in animal models of cognition, sensory gating, epilepsy and pain.

Several lines of evidence suggest that the nicotinic acetylcholine receptor alpha7 (nAChR alpha7) is involved in central nervous system disorders like schizophrenia and Alzheimer's disease as well as in inflammatory disorders like sepsis and pancreatitis. The present article describes the in vivo effects of JN403, a compound recently characterized to be a potent and selective partial nAChR alpha7 agonist. JN403 rapidly penetrates into the brain after i.v. and after p.o. administration in mice and rats. In the social recognition test in mice JN403 facilitates learning/memory performance over a broad dose range. JN403 shows anxiolytic-like properties in the social exploration model in rats and the effects are retained after a 6h pre-treatment period and after subchronic administration. The effect on sensory inhibition was investigated in DBA/2 mice, a strain with reduced sensory inhibition under standard experimental conditions. Systemic administration of JN403 restores sensory gating in DBA/2 mice, both in anaesthetized and awake animals. Furthermore, JN403 shows anticonvulsant potential in the audiogenic seizure paradigm in DBA/2 mice. In the two models of permanent pain tested, JN403 produces a significant reversal of mechanical hyperalgesia. The onset was fast and the duration lasted for about 6h. Altogether, the present set of data suggests that nAChR alpha7 agonists, like JN403 may be beneficial for improving learning/memory performance, restoring sensory gating deficits, and alleviating pain, epileptic seizures and conditions of anxiety.

Gamma-lactams--a novel scaffold for highly potent and selective alpha 7 nicotinic acetylcholine receptor agonists.

A novel class of alpha7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-throughput screening. The cis gamma-lactam scaffold has been optimized to reveal highly potent and selective alpha7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice.

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3ß4* and α9α10 subtypes.

The inhibitory activity of (±)-citalopram on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3ß4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in µM) hα3ß4 AChRs (5.1 ±â€¯1.3) with higher potency than that for hα7 (18.8 ±â€¯1.1) and hα4ß2 (19.1 ±â€¯4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3ß4 with >2-fold higher affinity than that for hα4ß2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3ß4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3ß4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3ß4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders.

Histamine H3 receptor (H3R) inverse agonists that have been in clinical trials for the treatment of excessive sleep disorders, have been plagued with insomnia as a mechanism-based side effect. We focused on the identification of compounds that achieve high receptor occupancy within a short time, followed by rapid disengagement from the receptor, a target profile that could provide therapeutic benefits without the undesired side effect of insomnia. This article describes the optimization work that led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18 b, LML134).

Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in µM) rank: amitriptyline (2.7 ±â€¯0.3) > doxepin (5.9 ±â€¯1.1) ∼ imipramine (6.6 ±â€¯1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ±â€¯8.5 µM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ±â€¯0.05 µM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in µM): imipramine (1.6 ±â€¯0.2) > amitriptyline (2.4 ±â€¯0.3) > doxepin (4.9 ±â€¯0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action.