Publications by authors named "Thomas Roux"

32 Publications

Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

Anal Chem 2021 Apr 7. Epub 2021 Apr 7.

Centre de Recherche en Cancérologie de Toulouse (CRCT), INSERM, Université de Toulouse, CNRS, UPS, Toulouse 31037, France.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 μg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.
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http://dx.doi.org/10.1021/acs.analchem.0c05137DOI Listing
April 2021

Interaction between Neurons and the Oligodendroglial Lineage in Multiple Sclerosis and Its Preclinical Models.

Life (Basel) 2021 Mar 11;11(3). Epub 2021 Mar 11.

Paris Brain Institute (ICM), Sorbonne Université, CNRS, Inserm, GH Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75013 Paris, France.

Multiple sclerosis (MS) is a complex central nervous system inflammatory disease leading to demyelination and associated functional deficits. Though endogenous remyelination exists, it is only partial and, with time, patients can enter a progressive phase of the disease, with neurodegeneration as a hallmark. Though major therapeutic advances have been made, with immunotherapies reducing relapse rate during the inflammatory phase of MS, there is presently no therapy available which significantly impacts disease progression. Remyelination has been shown to favor neuroprotection, and it is thus of major importance to better understand remyelination mechanisms in order to promote them and hence preserve neurons. A crucial point is how this process is regulated through the neuronal crosstalk with the oligodendroglial lineage. In this review, we present the current knowledge on neuron interaction with the oligodendroglial lineage, in physiological context as well as in MS and its experimental models. We further discuss the therapeutic possibilities resulting from this research field, which might allow to support remyelination and neuroprotection and thus limit MS progression.
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http://dx.doi.org/10.3390/life11030231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999210PMC
March 2021

COVID-19 infection in NMO/SD patients: a French survey.

J Neurol 2021 Apr 12;268(4):1188-1190. Epub 2020 Sep 12.

Department of Neurology, Pitié Salpêtrière Hospital, APHP, Sorbonne University, 75013, Paris, France.

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http://dx.doi.org/10.1007/s00415-020-10112-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486806PMC
April 2021

Beyond COVID-19: DO MS/NMO-SD patients treated with anti-CD20 therapies develop SARS-CoV2 antibodies?

Mult Scler Relat Disord 2020 Nov 3;46:102482. Epub 2020 Sep 3.

AP-HP, Pitié-Salpêtrière Hospital, Department of Neurology, Paris, France; CRC SEP Paris, France; Sorbonne University, Paris Brain Institute, APHP, Inserm, CNRS, CIC neuroscience, Pitié-Salpêtrière Hospital, Paris, France.

Since 2019, a new coronavirus infection (COVID-19) due to an agent called SARS-CoV-2 spread rapidly worldwide. Patients with multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMO-SD) are often treated with immunosuppressants. Beyond their effect on the risk of COVID-19 infection, the consequences on the long-term immune response against the coronavirus remain unknown. Among 13 MS or NMOSD patients with confirmed COVID-19 included, all 5 patients treated with anti-CD20 therapies had a negative SARS-CoV-2 serology. To date, maximal precautions to prevent coronavirus infection should be maintained in MS/NMOSD patients already exposed to COVID-19 during anti-CD20 therapy.
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http://dx.doi.org/10.1016/j.msard.2020.102482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468278PMC
November 2020

Clinical, neuropathological, and genetic characterization of STUB1 variants in cerebellar ataxias: a frequent cause of predominant cognitive impairment.

Genet Med 2020 11 27;22(11):1851-1862. Epub 2020 Jul 27.

Sorbonne Université, Institut du Cerveau-Paris Brain Institute (ICM), AP-HP, INSERM, CNRS, University Hospital Pitié-Salpêtrière, Paris, France.

Purpose: Pathogenic variants in STUB1 were initially described in autosomal recessive spinocerebellar ataxia type 16 and dominant cerebellar ataxia with cerebellar cognitive dysfunction (SCA48).

Methods: We analyzed a large series of 440 index cerebellar ataxia cases, mostly with dominant inheritance.

Results: STUB1 variants were detected in 50 patients. Age at onset and severity were remarkably variable. Cognitive impairment, predominantly frontal syndrome, was observed in 54% of STUB1 variant carriers, including five families with Huntington or frontotemporal dementia disease-like phenotypes associated with ataxia, while no STUB1 variant was found in 115 patients with frontotemporal dementia. We report neuropathological findings of a STUB1 heterozygous patient, showing massive loss of Purkinje cells in the vermis and major loss in the cerebellar hemispheres without atrophy of the pons, hippocampus, or cerebral cortex. This screening of STUB1 variants revealed new features: (1) the majority of patients were women (70%) and (2) "second hits" in AFG3L2, PRKCG, and TBP were detected in three families suggesting synergic effects.

Conclusion: Our results reveal an unexpectedly frequent (7%) implication of STUB1 among dominantly inherited cerebellar ataxias, and suggest that the penetrance of STUB1 variants could be modulated by other factors, including sex and variants in other ataxia-related genes.
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http://dx.doi.org/10.1038/s41436-020-0899-xDOI Listing
November 2020

Clinical Characteristics and Outcomes in Patients With Coronavirus Disease 2019 and Multiple Sclerosis.

JAMA Neurol 2020 09;77(9):1079-1088

Service de Neurologie, Clinical Investigation Center Institut National de la Santé et de la Recherche Médicale 1434, Centre Hospitalier Universitaire de Strasbourg, Strasbourg, France.

Importance: Risk factors associated with the severity of coronavirus disease 2019 (COVID-19) in patients with multiple sclerosis (MS) are unknown. Disease-modifying therapies (DMTs) may modify the risk of developing a severe COVID-19 infection, beside identified risk factors such as age and comorbidities.

Objective: To describe the clinical characteristics and outcomes in patients with MS and COVID-19 and identify factors associated with COVID-19 severity.

Design, Setting, And Participants: The Covisep registry is a multicenter, retrospective, observational cohort study conducted in MS expert centers and general hospitals and with neurologists collaborating with MS expert centers and members of the Société Francophone de la Sclérose en Plaques. The study included patients with MS presenting with a confirmed or highly suspected diagnosis of COVID-19 between March 1, 2020, and May 21, 2020.

Exposures: COVID-19 diagnosed with a polymerase chain reaction test on a nasopharyngeal swab, thoracic computed tomography, or typical symptoms.

Main Outcomes And Measures: The main outcome was COVID-19 severity assessed on a 7-point ordinal scale (ranging from 1 [not hospitalized with no limitations on activities] to 7 [death]) with a cutoff at 3 (hospitalized and not requiring supplemental oxygen). We collected demographics, neurological history, Expanded Disability Severity Scale score (EDSS; ranging from 0 to 10, with cutoffs at 3 and 6), comorbidities, COVID-19 characteristics, and outcomes. Univariate and multivariate logistic regression models were used to estimate the association of collected variables with COVID-19 outcomes.

Results: A total of 347 patients (mean [SD] age, 44.6 [12.8] years, 249 women; mean [SD] disease duration, 13.5 [10.0] years) were analyzed. Seventy-three patients (21.0%) had a COVID-19 severity score of 3 or more, and 12 patients (3.5%) died of COVID-19. The median EDSS was 2.0 (range, 0-9.5), and 284 patients (81.8%) were receiving DMT. There was a higher proportion of patients with a COVID-19 severity score of 3 or more among patients with no DMT relative to patients receiving DMTs (46.0% vs 15.5%; P < .001). Multivariate logistic regression models determined that age (odds ratio per 10 years: 1.9 [95% CI, 1.4-2.5]), EDSS (OR for EDSS ≥6, 6.3 [95% CI. 2.8-14.4]), and obesity (OR, 3.0 [95% CI, 1.0-8.7]) were independent risk factors for a COVID-19 severity score of 3 or more (indicating hospitalization or higher severity). The EDSS was associated with the highest variability of COVID-19 severe outcome (R2, 0.2), followed by age (R2, 0.06) and obesity (R2, 0.01).

Conclusions And Relevance: In this registry-based cohort study of patients with MS, age, EDSS, and obesity were independent risk factors for severe COVID-19; there was no association found between DMTs exposure and COVID-19 severity. The identification of these risk factors should provide the rationale for an individual strategy regarding clinical management of patients with MS during the COVID-19 pandemic.
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http://dx.doi.org/10.1001/jamaneurol.2020.2581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320356PMC
September 2020

Revisit ligand-receptor interaction at the human vasopressin V receptor: A kinetic perspective.

Eur J Pharmacol 2020 Aug 30;880:173157. Epub 2020 Apr 30.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, China. Electronic address:

The vasopressin V receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target for water balance disorders such as polycystic kidney disease. Traditionally, the discovery of novel agents for the vasopressin V receptor has been guided by evaluating their receptor affinity, largely ignoring the binding kinetics. However, the latter is receiving increasing attention in the drug research community and has been proved to be a more complete descriptor of the dynamic process of ligand-receptor interaction. Herein we aim to revisit the molecular basis of ligand-vasopressin V receptor interaction from the less-investigated kinetic perspective. A homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was set up and optimized, which enabled accurate kinetic profiling of unlabeled vasopressin V receptor ligands. Receptor occupancy profiles of two representative antagonists with distinct target residence time were simulated. Their functional effects were further explored in cAMP assays. Our results showed that the antagonist with longer receptor residence time (lixivaptan) displayed sustained target occupancy than the antagonist with shorter receptor residence time (mozavaptan). In accordance, lixivaptan displayed insurmountable antagonism and wash-resistant inhibitory effect on the cellular cAMP level, while not so for mozavaptan. Together, our data provide evidence that binding kinetics, next to their affinity, offers additional information for the dynamic process of ligand-receptor interaction. Hopefully, this study may lead to more kinetics-directed medicinal chemistry efforts and aid the design and discovery of different-in-class of vasopressin V receptor ligands for clinical applications.
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http://dx.doi.org/10.1016/j.ejphar.2020.173157DOI Listing
August 2020

Multiple cervical dissections after Rituximab.

Mult Scler Relat Disord 2020 Jul 17;42:102105. Epub 2020 Apr 17.

Neurology Department, Pitié-Salpêtrière hospital, Paris, France.

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http://dx.doi.org/10.1016/j.msard.2020.102105DOI Listing
July 2020

An alternative mechanism of early nodal clustering and myelination onset in GABAergic neurons of the central nervous system.

Glia 2020 09 2;68(9):1891-1909. Epub 2020 Mar 2.

Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle épinière, ICM-GH Pitié-Salpêtrière, Paris, France.

In vertebrates, fast saltatory conduction along myelinated axons relies on the node of Ranvier. How nodes assemble on CNS neurons is not yet fully understood. We previously described that node-like clusters can form prior to myelin deposition in hippocampal GABAergic neurons and are associated with increased conduction velocity. Here, we used a live imaging approach to characterize the intrinsic mechanisms underlying the assembly of these clusters prior to myelination. We first demonstrated that their components can partially preassemble prior to membrane targeting and determined the molecular motors involved in their trafficking. We then demonstrated the key role of the protein β2Na for node-like clustering initiation. We further assessed the fate of these clusters when myelination proceeds. Our results shed light on the intrinsic mechanisms involved in node-like clustering prior to myelination and unravel a potential role of these clusters in node of Ranvier formation and in guiding myelination onset.
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http://dx.doi.org/10.1002/glia.23812DOI Listing
September 2020

The pharmacologic characterization of allosteric molecules: Gq protein activation.

J Recept Signal Transduct Res 2019 Apr 19;39(2):106-113. Epub 2019 Jul 19.

b Department of Pharmacology, University of North Carolina School of Medicine , Chapel Hill , NC , USA.

Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts . This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τ), effects on affinity (α cooperativity), and efficacy (β cooperativity). It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these where a range of tissue sensitivities is encountered.
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http://dx.doi.org/10.1080/10799893.2019.1634101DOI Listing
April 2019

Healthcare accessibility in the rural plains (terai) of Nepal: physical factors and associated attitudes of the local population.

Int Health 2019 11;11(6):528-535

School of Public Health, Physiotherapy and Sports Science, College of Health and Agricultural Sciences, University College Dublin, Dublin, Ireland.

Background: While access to healthcare has been a focus of international development, populations around the world continue to lack proper access to care. Identifying at-risk demographic groups can help advance efforts both regionally and internationally. There are only a small number of studies that previously have assessed physical barriers and attitudes in Nepal.

Methods: This study assessed the factors and attitudes associated with healthcare accessibility in a rural population outside of Lumbini, Nepal. This descriptive cross-sectional study used a volunteer-sampling approach to collect 585 questionnaire responses from the area formerly known as the Madhuwani Village Development Committee.

Results: The study found that the population was more likely to access private care than public, and reported longer times to access a hospital than the national average. Across almost all findings, those with lower than a secondary education had significantly larger barriers, lower satisfaction and higher reported difficulty in accessing healthcare. Females were shown to have significantly larger transportation barriers in accessing care and lower satisfaction compared with males.

Conclusions: Results identify women and the less-educated as having larger barriers to accessing healthcare. Further research should focus on how inequities in access affect health outcomes among these identified vulnerable groups.
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http://dx.doi.org/10.1093/inthealth/ihz008DOI Listing
November 2019

HTRF Total and Phospho-YAP (Ser127) Cellular Assays.

Methods Mol Biol 2019 ;1893:153-166

IGF, Univ Montpellier, CNRS, INSERM, Montpellier, France.

The YAP protein is a co-transcription factor increasing the expression of genes involved in cell proliferation and repressing the expression of genes important for cell differentiation and apoptosis. It is regulated by several inputs, like the Hippo pathway, through the action of kinases that phosphorylate YAP on several residues. The level of phosphorylation of the residues serine 127 (S127) of YAP is generally assessed in cellular models, native tissues, and organs, as a marker of YAP activity and location, and is regulated by numerous partners. This phosphorylation event is classically detected using a western blot technical approach. Here, we describe a novel approach to detect both the relative amount of total YAP (T-YAP assay) and the phosphorylation of the residue S127 of YAP (S127-P-YAP assay) using a HTRF-based method. This easy-to-run method can easily be miniaturized and allows for a high-throughput analysis in 96/384-well plate format, requiring less cellular material and being more rapid than other approaches.
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http://dx.doi.org/10.1007/978-1-4939-8910-2_13DOI Listing
June 2019

Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay.

Biochem Pharmacol 2018 12 17;158:45-59. Epub 2018 Sep 17.

Institut Cochin, Inserm U1016, CNRS UMR 8104, University Paris Descartes, University Sorbonne Paris Cité, Paris, France. Electronic address:

Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.
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http://dx.doi.org/10.1016/j.bcp.2018.09.014DOI Listing
December 2018

Rituximab in chronic inflammatory demyelinating polyradiculoneuropathy with associated diseases.

J Peripher Nerv Syst 2018 12 7;23(4):235-240. Epub 2018 Oct 7.

Department of Clinical Neurophysiology, APHP, Pitié-Salpêtrière Hospital, Paris, France.

We aimed to analyse the response to rituximab in a cohort of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients with associated disorders. We conducted a clinical and electrophysiological retrospective monocentric study in 28 CIDP patients. Response to rituximab was defined as (a) a five-point increase in the Medical Research Council sum score or a one-point decrease in the Overall Neuropathy Limitations Scale score, compared to the score at the first rituximab infusion, or (b) the discontinuation of, or reduced need for, the last treatments before rituximab initiation. Twenty-one patients (75%) were responders to rituximab. The median time before response was 6 months (1-10 months). Only two patients needed to be treated again during a median follow-up of 2.0 years (0.75-9 years). Interestingly, the response rate was good in patients with associated autoimmune disease (5/8) and similar to the response rate observed in patients with a haematological disease (16/20) (P = 0.63). A shorter disease duration was associated with a better clinical response to rituximab (odds ratio 0.81, P = 0.025) and the response rate was better (P = 0.05) in common forms (83.3%) than in sensory forms (42.9%). No major adverse events were recorded. Rituximab is efficacious in CIDP patients with haematological or autoimmune disease. It improves clinical response and decreases dependence on first-line treatments.
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http://dx.doi.org/10.1111/jns.12287DOI Listing
December 2018

Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors.

Mol Pharmacol 2018 09 28;94(3):992-1006. Epub 2018 Jun 28.

Cisbio Bioassays, Codolet, France (S.B., J.V., N.P., E.T., T.R.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.)

This paper discusses the process of determining the activity of candidate molecules targeting Gq-protein activation through G-protein-coupled receptors for possible therapeutic application with two functional assays; calcium release and inositol phosphate metabolism [inositol monophosphate (IP1)]. While both are suitable for detecting ligand activity (screening), differences are seen when these assays are used to quantitatively measure ligand parameters for therapeutic activity. Specifically, responses for Gq-related pathways present different and dissimulating patterns depending on the functional assay used to assess them. To investigate the impact of functional assays on the accuracy of compound pharmacological profiles, five exemplar molecules [partial agonist, antagonist, inverse agonist, positive allosteric modulator (PAM) agonist, and positive -PAM] targeting either muscarinic M1 or ghrelin receptors were tested using two functional assays (calcium release and IP1) and the results were compared with theoretical pharmacological models. The IP1 assay is an equilibrium assay that is able to determine the correct (i.e., internally consistent) pharmacological profiles of all tested compounds. In contrast, the nonequilibrium nature of calcium assays yields misleading classification of most of the tested compounds. Our study suggests that the use of an equilibrium assay, such as IP1, is mandatory for the optimal use of pharmacological models that can both identify mechanisms of action and also convert descriptive-to-predictive data for therapeutic systems. Such assays allow the identification of consistent and simple scales of activity that can guide medicinal chemistry in lead optimization of candidate molecules for therapeutic use.
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http://dx.doi.org/10.1124/mol.118.112573DOI Listing
September 2018

From the Promiscuous Asenapine to Potent Fluorescent Ligands Acting at a Series of Aminergic G-Protein-Coupled Receptors.

J Med Chem 2018 01 22;61(1):174-188. Epub 2017 Dec 22.

Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR7200 CNRS, Université de Strasbourg , 74 Route du Rhin, 67412 Illkirch, France.

Monoamine neurotransmitters such as serotonin, dopamine, histamine, and noradrenaline have important and varied physiological functions and similar chemical structures. Representing important pharmaceutical drug targets, the corresponding G-protein-coupled receptors (termed aminergic GPCRs) belong to the class of cell membrane receptors and share many levels of similarity as well. Given their pharmacological and structural closeness, one could hypothesize the possibility to derivatize a ubiquitous ligand to afford rapidly fluorescent probes for a large set of GPCRs to be used for instance in FRET-based binding assays. Here we report fluorescent derivatives of the nonselective agent asenapine which were designed, synthesized, and evaluated as ligands of 34 serotonin, dopamine, histamine, melatonin, acetylcholine, and adrenergic receptors. It appears that this strategy led rapidly to the discovery and development of nanomolar affinity fluorescent probes for 14 aminergic GPCRs. Selected probes were tested in competition binding assays with unlabeled competitors in order to demonstrate their suitability for drug discovery purposes.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01220DOI Listing
January 2018

Efficacy and Safety of Fingolimod in Daily Practice: Experience of an Academic MS French Center.

Front Neurol 2017 5;8:183. Epub 2017 May 5.

AP-HP, Neurology Department, Pitié-Salpétrière Hospital, Paris, France.

Introduction: Fingolimod (Fg), a sphingosine 1-phosphate receptor modulator, decreases the annual relapse rate (ARR) in relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to assess the efficacy and safety of Fg in daily practice in patients with RRMS, previously treated with natalizumab (Nz) or not, and systematically followed during at least 1 year.

Methods: Data were collected from the patient files. Primary endpoint was the comparison between the ARR the year before Fg onset and after 1 and 2 years of Fg treatment. The secondary endpoints were the difference between Expanded Disability Status Scale (EDSS) at Fg onset and after 1 and 2 years of treatment, and safety.

Results: In the whole sample, we confirmed Fg efficacy on the ARR (0.895 before vs. 0.364 1 year after,  < 0.0001). Between our two groups (with or without Nz before Fg), the ARR was higher in the Nz group during the first year but similar during the second year. The EDSS was stable during the first year of Fg but significantly higher after 2 years (3.33 vs. 3.72,  = 0.02). Concerning safety, only three patients had to discontinue Fg because of tolerance issues.

Conclusion: Our study showed that Fg is safe in RRMS and can be used either after first-line treatments or after Nz. However we observed a mild disability progression after 2 years.
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http://dx.doi.org/10.3389/fneur.2017.00183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418224PMC
May 2017

A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.

ChemMedChem 2017 06 20;12(12):925-931. Epub 2017 Apr 20.

Université de Strasbourg, CNRS, Laboratoire d'Innovation Thérapeutique (LIT), UMR7200, Labex MEDALIS, 6700, Strasbourg, France.

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.
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http://dx.doi.org/10.1002/cmdc.201700106DOI Listing
June 2017

Astrocytic tumor with large cells and worrisome features in two patients with tuberous sclerosis: drastically different diagnoses and prognoses.

Clin Neuropathol 2017 May/Jun;36 (2017)(3):102-107

Introduction: Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic disease, characterized by the development of benign tumors in several organs. During infancy, 6 - 20% of patients develop brain tumors called subependymal giant cell astrocytoma (SEGA).

Case Reports: Here we present two patients with TSC, who displayed acute intracranial tumors requiring surgery. Although both tumors shared similar histological aspects with large astrocytic cells and worrisome features, immunohistochemical and genetic analysis successfully distinguished an opposite diagnosis for the two patients.
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http://dx.doi.org/10.5414/NP301008DOI Listing
January 2018

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Nat Chem Biol 2017 04 30;13(4):372-380. Epub 2017 Jan 30.

Institut de Génomique Fonctionnelle, (IGF), CNRS, INSERM, Université de Montpellier, Montpellier, France.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABA, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.
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http://dx.doi.org/10.1038/nchembio.2286DOI Listing
April 2017

Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery.

ACS Chem Biol 2016 08 3;11(8):2140-8. Epub 2016 Jun 3.

CNRS, INSERM, Aix-Marseille Université, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille , CS30059, 13273 Marseille Cedex 9, France.

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.
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http://dx.doi.org/10.1021/acschembio.6b00286DOI Listing
August 2016

Governing the toxics and the pollutants. France, Great Britain, 1750-1850.

Authors:
Thomas Le Roux

Endeavour 2016 Jun 16;40(2):70-81. Epub 2016 Apr 16.

Centre de Recherches Historiques (CNRS/EHESS), 190 avenue de France, 75013 Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.endeavour.2016.03.002DOI Listing
June 2016

Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins.

J Med Chem 2016 Feb 6;59(4):1634-41. Epub 2016 Jan 6.

Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), UMR 7258; INSERM U1068; Institut Paoli-Calmettes; Aix-Marseille Université, UM105 , 13273 Marseille, France.

A midthroughput screening follow-up program targeting the first bromodomain of the human BRD4 protein, BRD4(BD1), identified an acetylated-mimic xanthine derivative inhibitor. This compound binds with an affinity in the low micromolar range yet exerts suitable unexpected selectivity in vitro against the other members of the bromodomain and extra-terminal domain (BET) family. A structure-based program pinpointed a role of the ZA loop, paving the way for the development of potent and selective BET-BRDi probes.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01708DOI Listing
February 2016

Fecundity in women with multiple sclerosis: an observational mono-centric study.

J Neurol 2015 12;262(4):957-60. Epub 2015 Feb 12.

AP-HP, Neurology Department, Pitié-Salpêtrière Hospital, 43-87 Boulevard de l'Hôpital, 75013, Paris, France.

Multiple sclerosis (MS) is a neurological disease mostly affecting women of childbearing age. When counseling MS patients, many questions arise on the reciprocal influence of MS and pregnancy. However, little is known on the impact of MS and its treatments on the time to pregnancy. The objective was to evaluate fecundity (pregnancy and time to pregnancy) in a French cohort of MS women. One hundred and fifteen women with MS were included consecutively in this observational retrospective study. Pregnancy and time to pregnancy were collected using self-questionnaires. Among the 115 patients, 216 pregnancies (from 84 women) were reported. Mean time to pregnancy, which was available for 124 of these pregnancies, was 8.57 months when pregnancy occurred before MS onset, and 7.53 months after MS onset. Among the 95 patients who had a parental project, 2.27 spontaneous pregnancies per woman were recorded. The mean number of children per woman with MS was 1.37. Spontaneous pregnancies per woman and time to pregnancy were not different from the general French population. However, despite a normal fecundity, the mean number of children per woman with MS (1.37) was lower than in the general French population (1.99).
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http://dx.doi.org/10.1007/s00415-015-7663-1DOI Listing
January 2016

A single glycine in extracellular loop 1 is the critical determinant for pharmacological specificity of dopamine D2 and D3 receptors.

Mol Pharmacol 2013 Dec 23;84(6):854-64. Epub 2013 Sep 23.

Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York (M.M., L.S.); Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York (P.D., L.D., Y.H., J.A.J.); Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York (P.D., L.D., Y.H., J.A.J.); Schrödinger, Inc., New York, New York (T.B.); Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, Intramural Research Program of the National Institutes of Health National Institute on Drug Abuse, Baltimore, Maryland (A.B., A.H.N.); and Cisbio Bioassays, Codolet, France (T.R., E.T.).

Subtype-selective agents for the dopamine D3 receptor (D3R) have been considered as potential medications for drug addiction and other neuropsychiatric disorders. Medicinal chemistry efforts have led to the discovery of 4-phenylpiperazine derivatives that are >100-fold selective for the dopamine D3 receptor over dopamine D2 receptor (D2R), despite high sequence identity (78% in the transmembrane domain). Based on the recent crystal structure of D3R, we demonstrated that the 4-phenylpiperazine moiety in this class of D3R-selective compounds binds to the conserved orthosteric binding site, whereas the extended aryl amide moiety is oriented toward a divergent secondary binding pocket (SBP). In an effort to further characterize molecular determinants of the selectivity of these compounds, we modeled their binding modes in D3R and D2R by comparative ligand docking and molecular dynamics simulations. We found that the aryl amide moiety in the SBP differentially induces conformational changes in transmembrane segment 2 and extracellular loop 1 (EL1), which amplify the divergence of the SBP in D3R and D2R. Receptor chimera and site-directed mutagenesis studies were used to validate these binding modes and to identify a divergent glycine in EL1 as critical to D3R over D2R subtype selectivity. A better understanding of drug-dependent receptor conformations such as these is key to the rational design of compounds targeting a specific receptor among closely related homologs, and may also lead to discovery of novel chemotypes that exploit subtle differences in protein conformations.
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http://dx.doi.org/10.1124/mol.113.087833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834143PMC
December 2013

Time-resolved fluorescence ligand binding for G protein-coupled receptors.

Nat Protoc 2013 13;8(7):1307-20. Epub 2013 Jun 13.

Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.

G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.
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http://dx.doi.org/10.1038/nprot.2013.073DOI Listing
January 2014

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

Anal Biochem 2013 May 17;436(1):1-9. Epub 2013 Jan 17.

Institut National de la Santé et de la Recherche Médicale (INSERM), U1016, Institut Cochin, 75014 Paris, France.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.
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http://dx.doi.org/10.1016/j.ab.2012.12.013DOI Listing
May 2013

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

Proc Natl Acad Sci U S A 2013 Jan 7;110(4):1512-7. Epub 2013 Jan 7.

Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.
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http://dx.doi.org/10.1073/pnas.1212137110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557016PMC
January 2013

A fluorescent ligand-binding alternative using Tag-lite® technology.

J Biomol Screen 2010 Dec 25;15(10):1248-59. Epub 2010 Oct 25.

Cisbio Bioassays, Bagnols-sur-Cèze, France.

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(®). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.
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http://dx.doi.org/10.1177/1087057110384611DOI Listing
December 2010