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Multiple sclerosis (MS) is an inflammatory demyelinating disease often characterized by remission and relapse periods occurring at irregular intervals after an initial attack (clinically isolated syndrome) and followed by a gradual progression of disability. Clinical symptoms, magnetic resonance imaging and abnormalities in cerebrospinal fluid (CSF) immunoglobulin profile allow diagnosis with a good sensitivity. However, current biomarkers lack specificity or have poor individual prognostic value. To identify novel candidate biomarkers of MS, we analysed 1) the CSF proteome from symptomatic controls and patients with clinically isolated syndrome or remitting-relapsing multiple sclerosis (n=40), and 2) changes in oligodendrocyte secretome upon proinflammatory or pro-apoptotic treatment. Proteins exhibiting differences in abundance in both studies were combined with previously described MS biomarkers to build a list of 87 proteins that were quantified by parallel reaction monitoring (PRM) in CSF samples from a new cohort comprising symptomatic controls and MS patients at different disease stages (n=60). The eleven proteins that passed this qualification step were subjected to a new PRM assay from a larger cohort (n=158) comprising patients with MS at different disease stages or with other inflammatory or non-inflammatory neurological disorders. Collectively, these studies identified a biomarker signature of MS that might improve MS diagnosis and prognosis. These include the oligodendrocyte precursor cell proteoglycan Syndecan-1, which was more efficient than previously described biomarkers to discriminate MS from other inflammatory and non-inflammatory neurological disorders.

In metazoan cells, replication of genomic DNA initiates from thousands of discrete chromosomal loci known as origins. Proteins such as the Origin Recognition Complex (ORCs) associate with origins, but this does not show clear sequence specificity for DNA binding. Genome-wide origin mapping studies have shown that the region surrounding the replication initiation site contains motifs such as the Origin G-rich Repeated Element (OGRE), proximal to the majority of origins. Here, using an approach coupling DNA affinity purification to quantitative proteomics, we identified proteins that interact specifically with an OGRE. Three of the top-scoring interactors, Dhx36, Pura and Tial1, were selected for further study. We show that Dhx36 and Tial1 localise to the nucleus and their knockdown decreased cells in S-phase resulting in their accumulation in the G1phase of the cell cycle. Altogether these results indicate that these OGRE-binding factors may play roles in DNA synthesis in mammalian cells.

The K+-Cl–cotransporter KCC2, encoded by theSlc12a5gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAAreceptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENTFast synaptic inhibition in the brain is mediated by chloride-permeable GABAAreceptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

International audience; The serotonin 5-HT6 receptor (5-HT6R) is a promising target to improve cognitive symptoms of psychiatric diseases of neurodevelopmental origin, such as autism spectrum disorders and schizophrenia. However, its expression and localization at different stages of brain development remain largely unknown, due to the lack of specific antibodies to detect endogenous 5-HT6R. Here, we used transgenic mice expressing a GFP-tagged 5-HT6R under the control of its endogenous promoter (Knock-in) as well as embryonic stem cells expressing the GFP-tagged receptor to extensively characterize its expression at cellular and subcellular levels during development. We show that the receptor is already expressed at E13.5 in the cortex, the striatum, the ventricular zone, and to a lesser extent the subventricular zone. In adulthood, it is preferentially found in projection neurons of the hippocampus and cerebral cortex, in striatal medium-sized spiny neurons, as well as in a large proportion of astrocytes, while it is expressed in a minor population of interneurons. Whereas the receptor is almost exclusively detected in the primary cilia of neurons at embryonic and adult stages and in differentiated stem cells, it is located in the somatodendritic compartment of neurons from some brain regions at the neonatal stage and in the soma of undifferentiated stem cells. Finally, knocking-out the receptor induces a shortening of the primary cilium, suggesting that it plays a role in its function. This study provides the first global picture of 5-HT6R expression pattern in the mouse brain at different developmental stages. It reveals dynamic changes in receptor localization in neurons at the neonatal stage, which might underlie its key role in neuronal differentiation and psychiatric disorders of neurodevelopmental origin.

International audience; In line with recent clinical trials demonstrating that ondansetron, a 5-HT3 receptor (5-HT3R) antagonist, ameliorates cognitive deficits of schizophrenia and the known procognitive effects of 5-HT6 receptor (5-HT6R) antagonists, we applied the hybridization strategy to design dual-acting 5-HT3/5-HT6R antagonists. We identified the first-in-class compound FPPQ, which behaves as a 5-HT3R antagonist and a neutral antagonist 5-HT6R of the Gs pathway. FPPQ shows selectivity over 87 targets and decent brain penetration. Likewise, FPPQ inhibits phencyclidine (PCP)-induced hyperactivity and displays procognitive properties in the novel object recognition task. In contrast to FPPQ, neither 5-HT6R inverse agonist SB399885 nor neutral 5-HT6R antagonist CPPQ reversed (PCP)-induced hyperactivity. Thus, combination of 5-HT3R antagonism and 5-HT6R antagonism, exemplified by FPPQ, contributes to alleviating the positive-like symptoms. Present findings reveal critical structural features useful in a rational polypharmacological approach to target 5-HT3/5-HT6 receptors and encourage further studies on dual-acting 5-HT3/5-HT6R antagonists for the treatment of psychiatric disorders.

International audience; Psychedelic-assisted psychotherapy gained considerable interest as a novel treatment strategy for fear-related mental disorders but the underlying mechanism remains poorly understood. The serotonin 2A (5-HT2A) receptor is a key target underlying the effects of psychedelics on emotional arousal but its role in fear processing remains controversial. Using the psychedelic 5-HT2A/5-HT2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) and 5-HT2A receptor knockout (KO) mice we investigated the effect of 5-HT2A receptor activation on emotional processing. We show that DOI administration did not impair performance in a spontaneous alternation task but reduced anxiety-like avoidance behavior in the elevated plus maze and elevated zero maze tasks. Moreover, we found that DOI did not block memory recall but diminished fear expression in a passive avoidance task. Likewise, DOI administration reduced fear expression in an auditory fear conditioning paradigm, while it did not affect retention of fear extinction when administered prior to extinction learning. The effect of DOI on fear expression was abolished in 5-HT2A receptor KO mice. Administration of DOI induced a significant increase of c-Fos expression in specific amygdalar nuclei. Moreover, local infusion of the 5-HT2A receptor antagonist M100907 into the amygdala reversed the effect of systemic administration of DOI on fear expression while local administration of DOI into the amygdala was sufficient to suppress fear expression. Our data demonstrate that activation of 5-HT2A receptors in the amygdala suppresses fear expression but provide no evidence for an effect on retention of fear extinction.

Cancer stem cells alone can initiate and maintain tumors, but the function of non-cancer stem cells that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan Fibromodulin (FMOD) in the conditioned medium of non-cancer stem cells (DGCs; differentiated glioma cells) of glioma compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating Integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

G-protein coupled receptors (GPCRs) exist in an equilibrium of multiple conformational states, including different active states, which depend on the nature of the bound ligand. In consequence, different conformational states can initiate specific signal transduction pathways. The study identified compound 7e, which acts as a potent 5-hydroxytryptamine type 6 receptor (5-HT6R) neutral antagonist at Gs and does not impact neurite growth (process controlled by Cdk5). MD simulations highlighted receptor conformational changes for 7e and inverse agonist PZ-1444. In cell-based assays, neutral antagonists of the 5-HT6R (7e and CPPQ), but not inverse agonists (SB-258585, intepirdine, PZ-1444), displayed glioprotective properties against 6-hydroxydopamine-induced and doxorubicin-induced cytotoxicity. These suggest that targeting the activated conformational state of the 5-HT6R with neutral antagonists implicates the protecting properties of astrocytes. Additionally, 7e prevented scopolamine-induced learning deficits in the novel object recognition test in rats. We propose 7e as a probe for further understanding of the functional outcomes of different states of the 5-HT6R.

The emergence of additive manufacturing (AM) technologies, such as 3D printing and laser cutting, has created opportunities for new design practices covering a wide range of fields and a diversity of learning and teaching settings. The potential health impact of particulate matter and volatile organic compounds (VOCs) emitted from AM technologies is, therefore, a growing concern for makers. The research behind this paper addresses this issue by applying an indoor air quality assessment protocol in an educational fabrication laboratory. The paper presents the evaluation of the particle emission rate of different AM technologies. Real-time monitoring of multiple three-dimensional Polylactic Acid (PLA), Acrylonitrile Butadiene Styrene (ABS) and Thermoplastic Elastomers (TPE) printers and Polymethyl methacrylate (PMMA) laser cutters was performed in different usage scenarios. Non-contact electrical detectors and off-line gas chromatography–mass spectrometry (GC-MS) were used to detect VOCs. The results show that the emitted particle surface area concentrations vary between 294 and 406.2 μm2/cm3 for three-dimensional printers, and between 55.06 and 92.3 μm2/cm3 for laser cutters. The experiments demonstrate that the emission concentrations were highly dependent on the filtration systems in place. The highest quantities of VOCs emitted included Cyclohexene and Benzyl Alcohol for PLA, ABS and TPE 3D printers, and formic acid and Xylene for PMMA laser cutters. The experiment concludes that signature emissions are detectable for a given material type and an AM technology pair. A suitable mitigation strategy can be specified for each signature detected. Finally, this paper outlines some guidelines for improving indoor air quality in such specific environments. The data provided, as well as the proposed indoor air quality protocol, can be used as a baseline for future studies, and thus help to determine whether the proposed strategies can enhance operator and bystander safety.

International audience; In eukaryotes, there is now compelling evidence that in addition to the conventional endoplasmic reticulum-Golgi secretory pathway, there are additional routes for the export of cytoplasmic proteins with a critical role in numerous physio-pathological conditions. These alternative secretory pathways or unconventional protein secretion (UPS) start now to be molecularly dissected, and while UPS landscape appears to be governed by a striking diversity and heterogeneity of mechanisms, common principles are emerging. We review here the role of key molecular determinants as well as the role of central hubs for UPS, highlighting the plasticity and dynamic properties of membrane-bound compartments. We also describe recent findings that position UPS as an integral component of adaptive responses to cope with particular cellular needs and stresses.

This paper presents an off-line programming approach for the automatic generation of trajectories and the printing program for a 6-axis robot in order to print accurate conductive paths on 3D objects using a silver microparticle solvent-based ink. The aim of this study was to develop a semi-predictive model in order to - Adapt the printing parameters of a 6-axis robot arm and a piezo jetting print head to the printing speed and substrate type. - Print 3D electronic circuits matching the targeted geometry and conductivity. For the jetting printing process, 2D patterns printed on substrates with different roughness and wettability were analysed as a function of the print head translation speed and jetting frequency which were used as base variables to develop correlations in order to predict line width, thickness and conductivity. The model was based on the assumption that the behaviour of a single drop impacting the printing substrate is close to that of a train of drops (i.e. lines). Thus, the diameter of individual drops on a specific substrate was used to account for support properties and jetting conditions. The study also proposes a methodology to tune the circuit morphology by adapting the jetting parameters as a function of the trajectory and the speed of the 6-axis robot. As a representative case study, a 3D circuit was printed on a disposable paper cup obtaining an excellent agreement between measured and predicted conductivity values.

Background and ObjectivesTo evaluate the predictive value of serum neurofilament light chain (sNfL) and CSF NfL (cNfL) in patients with radiologically isolated syndrome (RIS) for evidence of disease activity (EDA) and clinical conversion (CC).MethodssNfL and cNfL were measured at RIS diagnosis by single-molecule array (Simoa). The risk of EDA and CC according to sNfL and cNfL was evaluated using the Kaplan-Meier analysis and multivariate Cox regression models including age, spinal cord (SC) or infratentorial lesions, oligoclonal bands, CSF chitinase 3–like protein 1, and CSF white blood cells.ResultsSixty-one patients with RIS were included. At diagnosis, sNfL and cNfL were correlated (Spearman r = 0.78,p< 0.001). During follow-up, 47 patients with RIS showed EDA and 36 patients showed CC (median time 12.6 months, 1–86). When compared with low levels, medium and high cNfL (>260 pg/mL) and sNfL (>5.0 pg/mL) levels were predictive of EDA (log rank,p< 0.01 andp= 0.02, respectively). Medium-high cNfL levels were predictive of CC (log rank,p< 0.01). In Cox regression models, cNfL and sNfL were independent factors of EDA, while SC lesions, cNfL, and sNfL were independent factors of CC.DiscussioncNfL >260 pg/mL and sNfL >5.0 pg/mL at diagnosis are independent predictive factors of EDA and CC in RIS. Although cNfL predicts disease activity better, sNfL is more accessible than cNfL and can be considered when a lumbar puncture is not performed.Classification of EvidenceThis study provides Class II evidence that in people with radiologic isolated syndrome (RIS), initial serum and CSF NfL levels are associated with subsequent evidence of disease activity or clinical conversion.

International audience; Serotonin (5-HT) appeared billions of years before 5-HT receptors and synapses. It is thus not surprising that 5-HT can control biological processes independently of its receptors. One example is serotonylation, which consists of covalent binding of 5-HT to the primary amine of glutamine. Over the past 20 years, serotonylation has been involved in the regulation of many signaling mechanisms. One of the most striking examples is the recent evidence that serotonylation of histone H3 constitutes an epigenetic mark. However, the pathophysiological role of histone H3 serotonylation remains to be discovered. All but one of the 5-HT receptors are G-protein-coupled receptors (GPCRs). The signaling pathways they control are finely tuned, and new, unexpected regulatory mechanisms are being uncovered continuously. Some 5-HT receptors (5-HT 2C , 5-HT 4 , 5-HT 6 , and 5-HT 7) signal through mechanisms that require neither G-proteins nor β-arrestins, the two classical and almost universal GPCR signal transducers. 5-HT 6 receptors are constitutively activated via their association with intracellular GPCR-interacting proteins (GIPs), including neurofibromin 1, cyclin-dependent kinase 5 (Cdk5), and G-protein-regulated inducer of neurite outgrowth 1 (GPRIN1). Interactions of 5-HT 6 receptor with Cdk5 and GPRIN1 are not concomitant but occur sequentially and play a key role in dendritic tree morphogenesis. Furthermore, 5-HT 6 receptor-mediated G-protein signaling in neurons is different in the cell body and primary cilium, where it is modulated by smoothened receptor activation. Finally, 5-HT 2A receptors form heteromers with mGlu 2 metabotropic glutamate receptors. This heteromerization results in a specific phosphorylation of mGlu 2 receptor on a serine residue (Ser 843) upon agonist stimulation of 5-HT 2A or mGlu 2 receptor. mGlu 2 receptor phosphorylation on Ser 843 is an essential step in engagement of G i/o signaling not only upon mGlu 2 receptor activation but also following 5-HT 2A receptor activation, and thus represents a key molecular event underlying functional crosstalk between both receptors.

The diverse signaling pathways engaged by serotonin type 6 receptor (5-HT

Open source design of hardware products is an emerging phenomenon that takes more and more importance today's in the society. However, open source (hardware) design implies a tremendous change in both design practices and philosophy because it is partly related to the movements of creative commons and the sharing economy. From this perspective one could think that participation is crucial in the success of open source design projects. In this paper, we analyse 9 case studies in the light of 3 hypotheses. If many studies highlight the potential of the crowd as a resource for design tasks, our study shows that for open source design communities the participation is not massive. In this study, we used an activity-based approach to build our model. As open source design processes are fairly unstructured and based on voluntary participation, it is impossible to adopt a classical task-based model. With the help of this model, we were able evaluate the overall size of the active community, the participation rate with regards to the activities. This study paves the way to deeper and extensive studies on how to support communities engaged in open source design of hardware products.

La fabricacion aditiva (AM) permite la produccion de geometrias complejas, que a veces son imposibles de obtener mediante los procesos convencionales. En los procesos de diseno tradicionales, el rendimiento obtenido en una pieza (como la resistencia mecanica) se evalua mediante un modelo numerico (calculo por Elementos Finitos) el cual es mas barato y rapido de obtener que un modelo fisico. Hoy en dia, al disenar un objeto dedicado a la AM, resulta mas facil fabricar una pieza fisica utilizando exactamente el mismo proceso metalico de AM que sera utilizado para la pieza final de serie. Por lo tanto, se dispone de dos opciones para evaluar el rendimiento de la pieza en cuestion, elaborar un modelo numerico y/o un modelo fisico. Posteriormente, se plantea como decidir entre estas dos opciones, labor que sigue causando confusion. El proposito del trabajo presentado aqui es proponer un metodo que ayude a los disenadores a realizar la mejor eleccion entre los modelos numerico y fisico a la hora de evaluar la validez de una pieza durante la fase de diseno. Para promover la comparacion entre estas opciones, el metodo presentado se basa en el modelo de la Metodologia de Investigacion de Diseno (DRM) y se utilizan varios ejemplos de estudio. El trabajo presenta dos casos que permiten proponer una metodologia y una lista de criterios que ayudan a los disenadores a comparar el Modelo Virtual y el Prototipo en una situacion dada, y finalmente a decidir que opcion se ajusta mejor a sus necesidades en este contexto. Palabras clave: modelo virtual, prototipo, fabricacion aditiva, proceso de diseno

Cancer stem-like cells (CSCs) and the differentiated bulk cells mainly contribute to cellular heterogeneity within a tumor. The dichotomous division pattern of the CSCs sitting atop the tumor hierarchy allow them to self-renew, and differentiate to initiate and repopulate aggressive tumors. These CSC-differentiation-derived bulk tumor cells reprogram to stem-like states, accounting for high plasticity within a tumor; a phenomenon governed by the distinct epigenetic profiles of the two cell populations. Reciprocal signaling between the tumor and its complex microenvironment is a vital driver of plasticity and heterogeneity in cancer. The indispensable role of CSCs in cellular cross-talk in the tumor niche as critical drivers of tumorigenesis is well-studied. However, the massive outnumbering of the CSCs by the differentiated cells raises several vital questions regarding the role of the latter in tumor growth; whether they contribute to the complex signaling within the tumor microenvironment (TME) to drive tumor growth remains unexplored. In this study, we aimed to mechanistically delineate the distinct roles of the cancer stem-like and differentiated cells by investigating their interactions with other stromal cells in the TME.In our model system, Glioblastoma (GBM), a highly aggressive brain cancer, we adopted a quantitative, label-free, high-throughput proteomics-based approach to identify the differentially abundant secreted proteins between the glioma stem-like cell (GSC) and differentiated glioma cell (DGC). This analysis showed a significantly higher abundance of the Fibromodulin (FMOD), a collagen-binding proteoglycan, in the conditioned medium of the DGCs compared with that of the GSCs. Our investigation revealed that an activated TGF-β signaling leads to p-SMAD2 occupancy on the FMOD promoter region, leading to increased FMOD secretion by the DGCs. DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth in a subcutaneous mouse model. We also observed that FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. Moreover, DGC-secreted FMOD activates integrin-dependent Notch signaling in endothelial cells, promoting angiogenesis. FMOD-silencing also inhibits the capability of the transdifferentiated endothelial cells (derived from the DGCs) to form angiogenic networks. Conditional silencing of FMOD in the de novo formed DGCs in an orthotopic intracranial C57BL/6 mouse model inhibited the growth of GSC-initiated tumors due to poorly developed vasculature, thereby increasing mouse survival. The same observation was recapitulated using patient-derived GSCs in an immunocompromised intracranial mouse model, thereby highlighting the human relevance of our findings. Collectively, our findings demonstrate how paracrine signaling between DGC-secreted FMOD and the stromal cells promotes tumor growth by enhancing angiogenesis, thereby highlighting a previously less studied supportive role of DGCs in tumor growth, albeit the GSCs retaining the tumor-initiating capacity. Thus our study identifies DGC-specific FMOD as a potential therapeutic target and emphasizes that targeting both cancer stem cells and differentiated cancer bulk cells is vital to achieving a durable response in GBM. Citation Format: Shreoshi Sengupta, Kumaravel Somasundaram, Dinorah Friedmann Morvinski, Philippe Marin, Prasasvi Reddy, Arani Mukherjee, Prerna Magod, Mainak Mondal, Serge Urbach. Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6383.

International audience; The serotonin 2B (5-HT2B) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT2B receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT2B receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.

Serotonin type 6 receptor (5-HT6R) has gained particular interest as a promising target for treating cognitive deficits, given the positive effects of its antagonists in a wide range of memory impairment paradigms. Herein, we report on degradation of the 1H-pyrrolo[3,2-c]quinoline scaffold to provide the 2-phenyl-1H-pyrrole-3-carboxamide, which is devoid of canonical indole-like skeleton and retains recognition of 5-HT6R. This modification has changed the compound’s activity at 5-HT6R-operated signaling pathways from neutral antagonism to inverse agonism. The study identified compound 27 that behaves as an inverse agonist of the 5-HT6R at the Gs and Cdk5 signaling pathways. Compound 27 showed high selectivity and metabolic stability and was brain penetrant. Finally, 27 reversed scopolamine-induced memory decline in the novel object recognition test and exhibited procognitive properties in the attentional set-shifting task in rats. In light of these findings, 27 might be considered for further evaluation as a new cognition-enhancing agent, while 2-phenyl-1H-pyrrole-3-carboxamide might be used as a template for designing 5-HT6R inverse agonists.

G-protein coupled receptors (GPCRs) exist in an equilibrium of multiple conformational states, including different active states, which depend on the nature of the bound ligand. In consequence, different conformational states can initiate specific signal transduction pathways. The study identified compound