Synthesis, in vitro and in vivo biological evaluation of novel dual compounds targeting both acetylcholinesterase and serotonergic 5-HT receptors with potential interest in the treatment of Alzheimer's disease.

In this work, we exemplified the "copride" family of drug candidates able to both inhibit acetylcholinesterase and to activate 5-HT receptors, with anti-amnesiant and promnesiant activities in mice. Twenty-one analogs of donecopride, the first-in class representative of the series, were synthesized exploring the influence on the biological activities of the substituents (methoxy, amine and chlorine) carried by its phenyl ring. This work was the support of an intensive structure-activity relationship study and allowed to obtain some interesting derivatives of donecopride.

Donecopride, a Swiss army knife with potential against Alzheimer's disease.

Background And Purpose: We recently identified donecopride as a pleiotropic compound able to inhibit AChE and to activate 5-HT receptors. Here, we have assessed the potential therapeutic effects of donecopride in treating Alzheimer's disease (AD).

Experimental Approach: We used two in vivo animal models of AD, transgenic 5XFAD mice and mice exposed to soluble amyloid-β peptides and, in vitro, primary cultures of rat hippocampal neurons.

Chronic treatments with a 5-HT receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia and a major health issue worldwide. The complexity of the pathology continues to challenge its comprehension and the implementation of effective treatments. In the last decade, a number of possible targets of intervention have been pointed out, among which the stimulation of 5-HT receptors (5-HTRs) seems very promising.

Novel multitarget-directed ligands (MTDLs) with acetylcholinesterase (AChE) inhibitory and serotonergic subtype 4 receptor (5-HT4R) agonist activities as potential agents against Alzheimer's disease: the design of donecopride.

In this work, we describe the synthesis and in vitro evaluation of a novel series of multitarget-directed ligands (MTDL) displaying both nanomolar dual-binding site (DBS) acetylcholinesterase inhibitory effects and partial 5-HT4R agonist activity, among which donecopride was selected for further in vivo evaluations in mice. The latter displayed procognitive and antiamnesic effects and enhanced sAPPα release, accounting for a potential symptomatic and disease-modifying therapeutic benefit in the treatment of Alzheimer's disease.

Primary culture of mouse dopaminergic neurons.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson's disease (PD).

Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment.

RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-β peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer's disease (AD).

Early administration of RS 67333, a specific 5-HT4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer's disease.

Amyloid β (Aβ) accumulation is considered the main culprit in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that decreasing Aβ production at very early stages of AD could be a promising strategy to slow down disease progression. Serotonin 5-HT4 receptor activation stimulates α-cleavage of the amyloid precursor protein (APP), leading to the release of the soluble and neurotrophic sAPPα fragment and thus precluding Aβ formation.

Serotonin type 4 receptor dimers.

Numerous class A G protein-coupled receptors and especially biogenic amine receptors have been reported to form homodimers. Indeed, the dimerization process might occur for all the metabotropic serotonergic receptors. Moreover, dimerization appears to be essential for the function of serotonin type 2C (5-HT2C) and type 4 (5-HT4) receptors and required to obtain full receptor activity.

5-HT4 receptors constitutively promote the non-amyloidogenic pathway of APP cleavage and interact with ADAM10.

In addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-β. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT(4)Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons.

Pharmacological profile of engineered 5-HT₄ receptors and identification of 5-HT₄ receptor-biased ligands.

G protein-coupled receptors (GPCRs) can activate simultaneously multiple signaling pathways upon agonist binding. The combined use of engineered GPCRs, such as the receptors activated solely by synthetic ligands (RASSLs), and of biased ligands that activate only one pathway at a time might help deciphering the physiological role of each G protein signaling. In order to find serotonin type 4 receptor (5-HT₄R) biased ligands, we analyzed the ability of several compounds to activate the Gs and G(q/11) pathways in COS-7 cells that transiently express wild type 5-HT₄R, the 5-HT₄R-D(100)A mutant (known also as 5-HT₄-RASSL, or Rs1) or the 5-HT₄R-T(104)A mutant, which modifies agonist-induced 5-HT₄R activation.

5-HT(6) receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia.

Cognitive deficits in schizophrenia severely compromise quality of life and are poorly controlled by current antipsychotics. While 5-HT(6) receptor blockade holds special promise, molecular substrates underlying their control of cognition remain unclear. Using a proteomic strategy, we show that 5-HT(6) receptors physically interact with several proteins of the mammalian target of rapamycin (mTOR) pathway, including mTOR.

Gα(olf) mutation allows parsing the role of cAMP-dependent and extracellular signal-regulated kinase-dependent signaling in L-3,4-dihydroxyphenylalanine-induced dyskinesia.

Although L-3,4-dihydroxyphenylalanine (L-DOPA) remains the reference treatment of Parkinson's disease, its long-term beneficial effects are hindered by L-DOPA-induced dyskinesia (LID). In the dopamine (DA)-denervated striatum, L-DOPA activates DA D₁ receptor(D₁R) signaling, including cAMP-dependent protein kinase A (PKA) and extracellular signal-regulated kinase (ERK), two responses associated with LID. However, the cause of PKA and ERK activation, their respective contribution to LID, and their relationship are not known.

G protein activation by serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient.

The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and β(2)-adrenergic receptors, activate G proteins.

Beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling.

G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT(4) receptor (5-HT(4)R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the G(s) pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons.

Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors.

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R(*). However, different structural active R(*) complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R(*) states of 5-hydroxytryptamine-4 (5-HT(4)) receptors involve different side-chain conformational toggle switches.

Synthesis and biological evaluation of 1-amino-2-phosphonomethylcyclopropanecarboxylic acids, new group III metabotropic glutamate receptor agonists.

Stereoisomers of 1-amino-2-phosphonomethylcyclopropanecarboxylic acid (APCPr), conformationally restricted analogues of L-AP4 (2-amino-4-phosphonobutyric acid), have been prepared and evaluated at recombinant group III metabotropic glutamate receptors. They activate these receptors over a broad range of potencies. The most potent isomer (1S,2R)-APCPr displays a similar pharmacological profile as that of L-AP4 (EC50 0.

5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling.

The 5-hydroxytryptamine(4) (5-HT(4)) receptors have recently emerged as key modulators of learning, memory, and cognitive processes. In neurons, 5-hydroxytryptamine(4) receptors (5-HT(4)Rs) activate cAMP production and protein kinase A (PKA); however, nothing is known about their ability to activate another key signaling pathway involved in learning and memory: the extracellular signal-regulated kinase (ERK) pathway. Here, we show that 5-HT(4)R stimulation, in primary neurons, produced a potent but transient activation of the ERK pathway.

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements.

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively.

Palmitoylation of the 5-hydroxytryptamine4a receptor regulates receptor phosphorylation, desensitization, and beta-arrestin-mediated endocytosis.

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase.

Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors.

Although agonists bind directly in the heptahelical domain (HD) of most class-I rhodopsin-like G protein coupled receptors (GPCRs), class-III agonists bind in the extracellular domain of their receptors. Indeed, the latter possess a large extracellular domain composed of a cysteine-rich domain and a Venus flytrap module. Both the low sequence homology and the structural organization of class-III GPCRs raised the question of whether or not the HD of these receptors functions the same way as rhodopsin-like GPCRs.

Confinement but not microgravity alters NMDA NR1 receptor expression in rat inner ear ganglia.

Space flight produces changes in neuronal activity in the vestibular system. We studied the protein expression of the NMDA receptor subunit NR1 in the vestibular ganglia of rats exposed to microgravity for 17 days, beginning on postnatal day 8, as part of the NASA Neurolab mission. As a control, we studied the cochlear ganglia in the same way.

Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists.

Ca2+, pheromones, sweet taste compounds, and the main neurotransmitters glutamate and gamma-aminobutyric acid activate G protein-coupled receptors (GPCRs) that constitute the GPCR family 3. These receptors are dimers, and each subunit has a large extracellular domain called a Venus flytrap module (VFTM), where agonists bind. This module is connected to a heptahelical domain that activates G proteins.