Structural insights into constitutive activity of 5-HT6 receptor.

While most therapeutic research on G-protein-coupled receptors (GPCRs) focuses on receptor activation by (endogenous) agonists, significant therapeutic potential exists through agonist-independent intrinsic constitutive activity that can occur in various physiological and pathophysiological settings. For example, inhibiting the constitutive activity of 5-HT6R-a receptor that is found almost exclusively in the brain and mediates excitatory neurotransmission-has demonstrated a therapeutic effect on cognitive/memory impairment associated with several neuropsychiatric disorders. However, the structural basis of such constitutive activity remains unclear. Here, we present a cryo-EM structure of serotonin-bound human 5-HT6R-Gs heterotrimer at 3.0-Å resolution. Detailed analyses of the structure complemented by comprehensive interrogation of signaling illuminate key structural determinants essential for constitutive 5-HT6R activity. Additional structure-guided mutagenesis leads to a nanobody mimic Gαs for 5-HT6R that can reduce its constitutive activity. Given the importance of 5-HT6R for a large number of neuropsychiatric disorders, insights derived from these studies will accelerate the design of more effective medications, and shed light on the molecular basis of constitutive activity.

Impact of the Substitution Pattern at the Basic Center and Geometry of the Amine Fragment on 5-HT6 and D3R Affinity in the 1H-Pyrrolo[3,2-c]quinoline Series.

Salt bridge (SB, double-charge-assisted hydrogen bonds) formation is one of the strongest molecular non-covalent interactions in biological systems, including ligand-receptor complexes. In the case of G-protein-coupled receptors, such an interaction is formed by the conserved aspartic acid (D3.32) residue and the basic moiety of the aminergic ligand. This study aims to determine the influence of the substitution pattern at the basic nitrogen atom and the geometry of the amine moiety at position 4 of 1H-pyrrolo[3,2-c]quinoline on the quality of the salt bridge formed in the 5-HT6 receptor and D3 receptor. To reach this goal, we synthetized and biologically evaluated a new series of 1H-pyrrolo[3,2-c]quinoline derivatives modified with various amines. The selected compounds displayed a significantly higher 5-HT6R affinity and more potent 5-HT6R antagonist properties when compared with the previously identified compound PZ-1643, a dual-acting 5-HT6R/D3R antagonist; nevertheless, the proposed modifications did not improve the activity at D3R. As demonstrated by the in silico experiments, including molecular dynamics simulations, the applied structural modifications were highly beneficial for the formation and quality of the SB formation at the 5-HT6R binding site; however, they are unfavorable for such interactions at D3R.

New Triazine Derivatives as Serotonin 5-HT6 Receptor Ligands.

Since the number of people with Alzheimer's disease (AD) continues to rise, new and effective drugs are urgently needed to not only slow down the progression of the disease, but to stop or even prevent its development. Serotonin 5-HT6 receptor (5-HT6R) ligands are still a promising therapeutic target for the treatment of AD. 1,3,5-Triazine derivatives, as novel structures lacking an indole or a sulfone moiety, have proven to be potent ligands for this receptor. In present work, new derivatives of the compound MST4 (4-((2-isopropyl-5-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine), the potent 5-HT6R antagonist (Ki = 11 nM) with promising ADMET and in vivo properties, were designed. The synthesized compounds were tested for their affinity towards 5-HT6R and other receptor (off)targets (serotonin 5-HT2A, 5-HT7 and dopamine D2). Based on the new results, 4-(2-tert-butylphenoxy)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (3) was selected for extended in vitro studies as a potent and selective 5-HT6R ligand (Ki = 13 nM). Its ability to permeate the blood-brain barrier (BBB) and its hepatotoxicity were evaluated. In addition, X-ray crystallography and solubility studies were also performed. The results obtained confirm that 6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine derivatives, especially compound 3, are promising structures for further pharmacological studies as 5-HT6R ligands.

Dual 5-HT3 and 5-HT6 Receptor Antagonist FPPQ Normalizes Phencyclidine-Induced Disruption of Brain Oscillatory Activity in Rats.

Schizophrenia is a severe mental disorder featuring psychotic, depressive, and cognitive alterations. Current antipsychotic drugs preferentially target dopamine D2-R and/or serotonergic 5-HT2A/1A-R. They partly alleviate psychotic symptoms but fail to treat negative symptoms and cognitive deficits. Here we report on the putative antipsychotic activity of (1-[(3-fluorophenyl)sulfonyl]-4-(piperazin-1-yl)-1H-pyrrolo[3,2-c]quinoline dihydrochloride) (FPPQ), a dual serotonin 5-HT3-R/5-HT6-R antagonist endowed with pro-cognitive properties. FPPQ fully reversed phencyclidine-induced decrease of low-frequency oscillations in the medial prefrontal cortex of anaesthetized rats, a fingerprint of antipsychotic activity. This effect was mimicked by the combined administration of the 5-HT3-R and 5-HT6-R antagonists ondansetron and SB-399 885, respectively, but not by either drug alone. In freely moving rats, FPPQ countered phencyclidine-induced hyperlocomotion and augmentation of gamma and high-frequency oscillations in medial prefrontal cortex, dorsal hippocampus, and nucleus accumbens. Overall, this supports that simultaneous blockade of 5-HT3R and 5-HT6-R-like that induced by FPPQ-can be a new target in antipsychotic drug development.

N-Skatyltryptamines-Dual 5-HT6R/D2R Ligands with Antipsychotic and Procognitive Potential.

A series of N-skatyltryptamines was synthesized and their affinities for serotonin and dopamine receptors were determined. Compounds exhibited activity toward 5-HT1A, 5-HT2A, 5-HT6, and D2 receptors. Substitution patterns resulting in affinity/activity switches were identified and studied using homology modeling. Chosen hits were screened to determine their metabolism, permeability, hepatotoxicity, and CYP inhibition. Several D2 receptor antagonists with additional 5-HT6R antagonist and agonist properties were identified. The former combination resembled known antipsychotic agents, while the latter was particularly interesting due to the fact that it has not been studied before. Selective 5-HT6R antagonists have been shown previously to produce procognitive and promnesic effects in several rodent models. Administration of 5-HT6R agonists was more ambiguous-in naive animals, it did not alter memory or produce slight amnesic effects, while in rodent models of memory impairment, they ameliorated the condition just like antagonists. Using the identified hit compounds 15 and 18, we tried to sort out the difference between ligands exhibiting the D2R antagonist function combined with 5-HT6R agonism, and mixed D2/5-HT6R antagonists in murine models of psychosis.

Chemical update on the potential for serotonin 5-HT6 and 5-HT7 receptor agents in the treatment of Alzheimer's disease.

Despite the better understanding of the mechanisms underlying Alzheimer's Disease (AD) and launched clinical trials, no AD-modifying treatment based on a synthetic drug has been introduced for almost twenty years. The serotonin 5-HT6 and 5-HT7 receptors turned out to be promising biological targets for modulation of central nervous system dysfunctions including cognitive impairment. Within this paper, we evaluate the pharmacological potency of both, 5-HT6R and 5-HT7R, agents in search for novel AD treatment. An overview of chemical structures of the 5-HTRs ligands with simultaneous procognitive action which have undergone preclinical and clinical studies within the last 10 years has been performed.

The protective effect and potential mechanism of NRXN1 on learning and memory in ADHD rat models.

The learning and memory network is highly complex and remains unclear. The hippocampus is the location of learning and memory function. Impairment of synaptic morphology and synaptic plasticity (i.e., long-term potentiation) appears to cause learning and memory deficits. Several studies have indicated the role of NRXN1 in regulating the synaptic function, but little is known on its role in learning and memory dysfunction associated with attention deficit and hyperactivity disorder (ADHD). Our results showed that overexpression and interference of NRXN1 in vivo, respectively, affected learning and memory, as was assessed by Morris water maze tests, in spontaneously hypertensive rats (SHRs) and Sprague Dawley (SD) rats. We found that SD rats performed better after methylphenidate (MPH) treatment in salvage trials. Accordingly, the change of NRXN1 led to altered synapse-related gene (PSD95, SYN1, GAP43, NLGN1) expression, further providing evidence of its role in the maintenance of synaptic plasticity. We also verified that the expression of synapse-related genes synchronously changed with NRXN1expression in the behavioral assessment. The expression of NRXN1 was confirmed to affect the expression of synapse-related genes after its interference and overexpression in the primary hippocampal neurons in vitro. These results confirmed our hypothesis that NRXN1 might nucleate an overall trans-synaptic signaling network that controls synaptic plasticity and is responsible for impairments in learning and memory in ADHD. These findings suggest a possible protective role of NRXN1 in learning and memory in ADHD. Further RNA-seq sequencing revealed significant differences in the expression of 5-hydroxytryptamine receptor (5-HT6R), which was further verified at the cellular level, and the mechanism of NRXN1 affecting synaptic plasticity was preliminarily discussed.

5-HT6R null mutatrion induces synaptic and cognitive defects.

Serotonin 6 receptor (5-HT6R) is a promising target for a variety of human diseases, such as Alzheimer's disease (AD) and schizophrenia. However, the detailed mechanism underlying 5-HT6R activity in the central nervous system (CNS) is not fully understood. In the present study, 5-HT6R null mutant (5-HT6R-/- ) mice were found to exhibit cognitive deficiencies and abnormal anxiety levels. 5-HT6R is considered to be specifically localized on the primary cilia. We found that the loss of 5-HT6R affected the Sonic Hedgehog signaling pathway in the primary cilia. 5-HT6R-/- mice showed remarkable alterations in neuronal morphology, including dendrite complexity and axon initial segment morphology. Neurons lacking 5-HT6R exhibited increased neuronal excitability. Our findings highlight the complexity of 5-HT6R functions in the primary ciliary and neuronal physiology, supporting the theory that this receptor modulates neuronal morphology and transmission, and contributes to cognitive deficits in a variety of human diseases, such as AD, schizophrenia, and ciliopathies.

Development of Novel Potential Pleiotropic Compounds of Interest in Alzheimer's Disease Treatment through Rigidification Strategy.

The development of Multi-Target Directed Ligand is of clear interest for the treatment of multifactorial pathology such as Alzheimer's disease (AD). In this context, acetylcholinesterase (AChE) inhibitors have been modulated in order to generate novel pleiotropic compounds targeting a second protein of therapeutic interest in AD. Among them, donecopride was the first example of a dual acetylcholinesterase inhibitor and 5-HT4 receptor agonist. In order to explore the structural diversity around this preclinical candidate we have explored the preparation of novel constrained analogs through late-stage rigidification strategy. A series of phenylpyrazoles was prepared in a late-stage functionalization process and all compounds were evaluated in vitro towards AChE and 5-HTRs. A docking study was performed in order to better explain the observed SAR towards AChE, 5-HT4R and 5-HT6R and this study led to the description of novel ligand targeting both AChE and 5-HT6R.

Constitutive Activity of Serotonin Receptor 6 Regulates Human Cerebral Organoids Formation and Depression-like Behaviors.

Serotonin receptor 6 (5-HT6R), a typical G protein-coupled receptor (GPCR) mainly expressed in the neurogenic area with constitutive activity, is of particular interest as a promising target for emotional impairment. Here, we found that 5-HT6R was highly expressed in human NSCs and activation of the receptor promoted self-renewal of human NSCs, and thus induced the expansion and folding of human cerebral organoids; dysfunction of receptor or inhibition of its constitutive activity resulted in the premature differentiation of NSCs, which ultimately depleted the NSC pool. The following mechanistic study revealed that EPAC-CREB signaling was involved in 5-HT6R regulation. Furthermore, we showed that mice with genetic deletion of 5-HT6R or knockin A268R mutant presented depression-like behaviors and impaired hippocampal neurogenesis for progressive decrease of the NSC pool. Thus, this study indicates that the modulation of 5-HT6R and its constitutive activity may provide a therapeutic alternative to alleviate depression.

A dual-acting 5-HT6 receptor inverse agonist/MAO-B inhibitor displays glioprotective and pro-cognitive properties.

The complex etiology of Alzheimer's disease has initiated a quest for multi-target ligands to address the multifactorial causes of this neurodegenerative disorder. In this context, we designed dual-acting 5-HT6 receptor (5-HT6R) antagonists/MAO-B inhibitors using pharmacophore hybridization strategy. Our approach involved linking priviliged scaffolds of 5-HT6R with aryloxy fragments derived from reversible and irreversible MAO-B inhibitors. The study identified compound 48 that acts as an inverse agonist of 5-HT6R at Gs signaling and an irreversible MAO-B inhibitor. Compound 48 showed moderate metabolic stability in rat microsomal assay, artificial membrane permeability, no hepatotoxicity, and it was well distributed to the brain. Additionally, 48 showed glioprotective properties in a model of cultured astrocytes using 6-OHDA as the cytotoxic agent. Finally, compound 48 (MED = 1 mg/kg, p.o.) fully reversed memory deficits in the NOR task induced by scopolamine in rats. A better understanding of effects exerted by dual-acting 5-HT6R/MAO-B modulators may impact the future development of neurodegenerative-directed treatment strategies.

The Antagonism of 5-HT6 Receptor Attenuates Current-Induced Spikes and Improves Long-Term Potentiation via the Regulation of M-Currents in a Pilocarpine-Induced Epilepsy Model.

Recent studies have documented that reduced M-current promotes epileptogenesis and attenuates synaptic remodeling. Neurite growth is closely related to the level of 5-HT6 receptor (5-HT6R) in the central nervous system. However, little research is available regarding the relation between 5-HT6R and M-current and the role of 5-HT6R in M-current regulation. Herein, we found that the expression of 5-HT6R was notably increased and the expression of KNCQ2/3, the main components of the M channel, was decreased in a time-dependent manner in pilocarpine-induced chronic epileptic hippocampus. Interestingly, antagonism of 5-HT6R by SB271046 upregulated the expression of KCNQ2 but not KCNQ3. SB271046 greatly alleviated excitatory/inhibitory imbalance and improved the impaired LTP in the chronic epileptic hippocampus. Further mechanism exploration revealed that the above effects of SB271046 can be reversed by the M-channel inhibitor XE991, which also confirmed that SB271046 can indeed improve abnormal M current. These data indicate that the antagonism of 5-HT6R may decrease the excitability of hippocampal pyramidal neurons in chronic epileptic rats and improve the impaired long-term potentiation by upregulating the expression of KCNQ2 in the M-channel.

Are the Hydantoin-1,3,5-triazine 5-HT6R Ligands a Hope to a Find New Procognitive and Anti-Obesity Drug? Considerations Based on Primary In Vivo Assays and ADME-Tox Profile In Vitro.

Though the 5-HT6 serotonin receptor is an important target giving both agonists and antagonists similar therapeutic potency in the treatment of topic CNS-diseases, no 5-HT6R ligand has reached the pharmaceutical market yet due to the too narrow chemical space of the known 5-HT6R agents and insufficient "drugability." Recently, a new group of non-indole and non-sulfone hydantoin-triazine 5-HT6R ligands was found, where 3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-methyl-5-(naphthalen-2-yl)imidazolidine-2,4-dione (KMP-10) was the most active member. This study is focused on wider pharmacological and "druglikeness" characteristics for KMP-10. A computer-aided insight into molecular interactions with 5-HT6R has been performed. "Druglikeness" was examined using an eight-test panel in vitro, i.e., a parallel artificial membrane permeability assay (PAMPA), and Caco-2 permeability-, P-glycoprotein (Pgp) affinity-, plasma protein binding-, metabolic stability- and drug-drug interaction-assays, as well as mutagenicity- and HepG2-hepatotoxicity risk tests. Behavioral studies in vivo, i.e., elevated plus-maze (EPM) and novel object recognition (NOR) tests, were performed. Extended studies on the influence of KMP-10 on rats' metabolism, including biochemical tests, were conducted in vivo. Results indicated significant anxiolytic and precognitive properties, as well as some anti-obesity properties in vivo, and it was found to satisfy the "druglikeness" profile in vitro for KMP-10. The compound seems to be a good lead-structure and candidate for wider pharmacological studies in search for new CNS-drugs acting via 5-HT6R.

Synthesis and computer-aided SAR studies for derivatives of phenoxyalkyl-1,3,5-triazine as the new potent ligands for serotonin receptors 5-HT6.

This research has provided the most active 5-HT6R agents among 1,3,5-triazine derivatives investigated to date and has also identified the world's first selenium-containing 5-HT6R ligands. The studies are focused on design, synthesis, biological evaluation and docking-supported SAR analysis for novel 5-HT6R agents as derivatives of lead structure 4-(4-methylpiperazin-1-yl)-6-(phenoxymethyl)-1,3,5-triazin-2-amine (7). The lead modifications included an introduction of: (i) various small substituents at benzene ring, (ii) a branched ether linker or (iii) the ether oxygen replacement with other chalcogen (S, Se) or sulfonyl moiety. Hence, a series of new compounds (7-24) was synthesized and examined on their affinities for 5-HT6R and selectivity, in respect to the 5-HT1AR, 5-HT2AR, 5-HT7R and dopamine D2 receptor, in the radioligand binding assays. For representative most active compounds functional bioassays and toxicity profile in vitro and antidepressant-like activity in vivo were examined. The 2-isopropyl-5-methylphenyl derivative (10) was found as the most active triazine 5-HT6R antagonist (Ki = 11 nM). SAR analysis indicated, that an exchange of oxygen to selenium (7 vs. 22), and especially, to sulfur (7 vs. 19) was beneficial to increase both affinity and antagonistic action for 5-HT6R. Surprisingly, an introduction of SO2 caused a drastic decrease of the 5-HT6R affinity, which was explained at a molecular level based on docking studies. All in vivo tested compounds (10, 18 and 21) did not show any risk of toxicity in the safety studies in vitro.

Ligand-induced activation of ERK1/2 signaling by constitutively active Gs-coupled 5-HT receptors.

5-HT4R, 5-HT6R, and 5-HT7AR are three constitutively active Gs-coupled 5-HT receptors that have key roles in brain development, learning, memory, cognition, and other physiological processes in the central nervous system. In addition to Gs signaling cascade mediated by these three 5-HT receptors, the ERK1/2 signaling which is dependent on cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation downstream of Gs signaling has also been widely studied. In this study, we investigated these two signaling pathways originating from the three Gs-coupled 5-HT receptors in AD293 cells. We found that the phosphorylation and activation of ERK1/2 are ligand-induced, in contrast to the constitutively active Gs signaling. This indicates that Gs signaling alone is not sufficient for ERK1/2 activation in these three 5-HT receptors. In addition to Gs, we found that ß-arrestin and Fyn are essential for the activation of ERK1/2. Together, these results put forth a novel mechanism for ERK1/2 activation involving the cooperative action of Gs, ß-arrestin, and Fyn.

Dysregulation of 5-hydroxytryptamine 6 receptor accelerates maturation of bone-resorbing osteoclasts and induces bone loss.

Rationale: Characterizing the regulation of bone-resorbing osteoclasts is central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis and periodontitis. 5-hydroxytryptamine (5-HT) has drawn considerable attention for its role in bone; however, it remains unknown whether the intracellular signaling of 5-HT receptors (5-HTRs) is linked to any of the regulatory mechanisms in osteoclasts. Herein, we report 5-HT6R to be a key regulatory receptor for osteoclastogenesis. Methods: In order to explore the critical role of 5-HT6R in bone-resorbing osteoclasts, in vitro experiments were performed using mouse whole bone marrow cells isolated from femora and tibiae and In vivo animal experiments were performed using 5-HT6R-deficient (5-HT6RKO-/-) mice, bone resorption mice model, and osteoporosis mice model. Results: Compared to other 5HTRs, activation of 5-HT6R relatively increased TRAP (tartrate-resistant acid phosphatase) activity during osteoclastogenesis. 5-HT6RKO(-/-) mice and 5-HT6RKO(-/-) osteoclast lineages presented with an abnormal phenotype and impaired osteoclastogenesis and impaired osteoclastogenesis. Activation of 5-HT6R increased the number of TRAP-positive multinuclear osteoclasts, actin ring formation, and expression of early osteoclast markers with osteoclast lineage commitment. Intracellular 5-HT6R signaling was found to be linked to RhoA GTPase activation and was involved in the maturation of osteoclasts. This signaling pathway also showed enhanced bone destruction after lipopolysaccharide (LPS) administration in mice. Furthermore, inhibition of 5-HT6R-mediated RhoA GTPase signaling protected against ovariectomy(OVX)-induced bone loss in mice. Conclusion: Taken together, our findings place the 5-HT6R system in a new context of osteoclast lineages in both an in vitro and in vivo system, and also it may offer a novel molecular target for the treatment of bone diseases.

Synthesis of carbon-11-labeled 5-HT6R antagonists as new candidate PET radioligands for imaging of Alzheimer's disease.

Carbon-11-labeled serotonin (5-hydroxytryptamine) 6 receptor (5-HT6R) antagonists, 1-[(2-bromophenyl)sulfonyl]-5-[11C]methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (O-[11C]2a) and 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-[11C]methyl-1-piperazinyl)methyl]-1H-indole (N-[11C]2a), 5-[11C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (O-[11C]2b) and 5-methoxy-3-((4-[11C]methylpiperazin-1-yl)methyl)-1-(phenylsulfonyl)-1H-indole (N-[11C]2b), 1-((4-isopropylphenyl)sulfonyl)-5-[11C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[11C]2c) and 1-((4-isopropylphenyl)sulfonyl)-5-methoxy-3-((4-[11C]methylpiperazin-1-yl)methyl)-1H-indole (N-[11C]2c), 1-((4-fluorophenyl)sulfonyl)-5-[11C]methoxy-3-((4-methylpiperazin-1-yl)methyl)-1H-indole (O-[11C]2d) and 1-((4-fluorophenyl)sulfonyl)-5-methoxy-3-((4-[11C]methylpiperazin-1-yl)methyl)-1H-indole (N-[11C]2d), were prepared from their O- or N-desmethylated precursors with [11C]CH3OTf through O- or N-[11C]methylation and isolated by HPLC combined with SPE in 40-50% radiochemical yield, based on [11C]CO2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370-740 GBq/µmol with a total synthesis time of ∼40-min from EOB.

Tale of the Good and the Bad Cdk5: Remodeling of the Actin Cytoskeleton in the Brain.

Cdk5 kinase, a cyclin-dependent kinase family member, is a key regulator of cytoskeletal remodeling in the brain. Cdk5 is essential for brain development during embryogenesis. After birth, it is essential for numerous neuronal processes such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity is deregulated in various brain disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and ischemic stroke, resulting in profound remodeling of the neuronal cytoskeleton, loss of synapses, and ultimately neurodegeneration. This review focuses on the "good and bad" Cdk5 in the brain and its pleiotropic contribution in regulating neuronal actin cytoskeletal remodeling. A vast majority of physiological and pathological Cdk5 substrates are associated with the actin cytoskeleton. Thus, our special emphasis is on the numerous Cdk5 substrates identified in the past two decades such as ephexin1, p27, Mst3, CaMKv, kalirin-7, RasGRF2, Pak1, WAVE1, neurabin-1, TrkB, 5-HT6R, talin, drebrin, synapsin I, synapsin III, CRMP1, GKAP, SPAR, PSD-95, and LRRK2. These substrates have unraveled the molecular mechanisms by which Cdk5 plays divergent roles in regulating neuronal actin cytoskeletal dynamics both in healthy and diseased states.

Spiro[pyrrolidine-3,3'-oxindoles] and Their Indoline Analogues as New 5-HT6 Receptor Chemotypes.

Synthetic derivatives of spiro[pyrrolidinyl-3,3'-oxindole] alkaloids (coerulescine analogues) were investigated as new ligands for aminergic G-protein coupled receptors (GPCRs). The chemical starting point 2'-phenylspiro[indoline-3,3'-pyrrolidin]-2-one scaffold was identified by virtual fragment screening utilizing ligand- and structure based methods. As a part of the hit-to-lead optimization a structure-activity relationship analysis was performed to explore the differently substituted 2'-phenyl-derivatives, introducing the phenylsulphonyl pharmacophore and examining the corresponding reduced spiro[pyrrolidine-3,3'-indoline] scaffold. The optimization process led to ligands with submicromolar affinities towards the 5-HT6 receptor that might serve as viable leads for further optimization.

5-Hydroxytryptamine 6 Receptor (5-HT6R)-Mediated Morphological Changes via RhoA-Dependent Pathways.

The 5-HT6R has been considered as an attractive therapeutic target in the brain due to its exclusive expression in the brain. However, the mechanistic linkage between 5-HT6Rs and brain functions remains poorly understood. Here, we examined the effects of 5-HT6R-mediated cell morphological changes using immunocytochemistry, Western blot, and live-cell imaging assays. Our results showed that the activation of 5-HT6Rs caused morphological changes and increased cell surface area in HEK293 cells expressing 5-HT6Rs. Treatment with 5-HT specifically increased RhoA-GTP activity without affecting other Rho family proteins, such as Rac1 and Cdc42. Furthermore, live-cell imaging in hippocampal neurons revealed that activation of 5-HT6Rs using a selective agonist, ST1936, increased the density and size of dendritic protrusions along with the activation of RhoA-GTP activity and that both effects were blocked by pretreatment with a selective 5-HT6R antagonist, SB258585. Taken together, our results show that 5-HT6R plays an important role in the regulation of cell morphology via a RhoA-dependent pathway in mammalian cell lines and primary neurons.