Illkirch-Graffenstaden, France
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Jung-Deyon L.,Prestwick Chemical | Giethlen B.,Prestwick Chemical | Mann A.,CNRS Laboratory for Therapeutic Innovation
European Journal of Organic Chemistry | Year: 2011

An expeditious route to (±)-desethyleburnamonine (1), a direct precursor of vindeburnol, is reported. Microwave irradiation of an indolo-tetrahydropyridine ethyl ester 2 in CH3CN in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave the pentacyclic (±)-desethyleburnamonine in a one-pot process. Experimental evidence support an allylamine-enamine isomerization, followed by a rare Pictet-Spengler condensation in basic media. From commercially available 3-(2-bromo-ethyl)indole and ethyl pyridin-3-yl acetate, (±)-desethyleburnamonine was obtained in 52 % yield over three steps. A one-pot synthesis of (±)- desethyleburnamonine is reported. With 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the base, the double-bond isomerization of allylamine 2 to an enamine took place, which then undergoes a final Pictet-Spengler condensation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zeder-Lutz G.,CNRS Biotechnology and Cell Signaling Laboratory | Choulier L.,CNRS Biotechnology and Cell Signaling Laboratory | Besse M.,CNRS Biotechnology and Cell Signaling Laboratory | Besse M.,French National Center for Scientific Research | And 6 more authors.
Analytical Biochemistry | Year: 2012

We investigated the suitability of surface plasmon resonance (SPR) for providing quantitative binding information from direct screening of a chemical library on protein tyrosine phosphatase 1b (PTP1B). The experimental design was established from simulations to detect binding with K D < 10 -4 M. The 1120 compounds (cpds) were injected sequentially at concentrations [C(cpd)] of 0.5 or 10 μM over various target surfaces. An optimized evaluation procedure was applied. More than 90% of cpds showed no detectable signal in four screens. The 30 highest responders at C(cpd) = 10 μM, of which 25 were selected in at least one of three screens at C(cpd) = 0.5 μM, contained 22 promiscuous binders and 8 potential PTP1B-specific binders with K D ∼ 10 -5 M. Inhibition of PTP1B activity was assayed and confirmed for 6 of these, including sanguinarine, a known PTP1B inhibitor. C(cpd) dependence studies fully confirmed screening conclusions. The quantitative consistency of SPR data led us to propose a structure-activity relationship (SAR) model for developing selective PTP1B inhibitors based on the ranking of 10 arylbutylpiperidine analogs. © 2011 Elsevier Inc. All rights reserved.

Deyon-Jung L.,Prestwick Chemical | Morice C.,Prestwick Chemical | Chery F.,Prestwick Chemical | Gay J.,Prestwick Chemical | And 5 more authors.
MedChemComm | Year: 2016

Through a process of fragmentation, functionalization, and recombination of market approved molecules for cosmetic usage, we customized an in-house virtual library comprising molecules ideally suited for virtual screening. Computational pharmacophore-based screening of this virtual library followed by a 3 month optimization phase led to the identification of an optimized lead with all its expected properties in hand to be developed as a candidate molecule for skin care in cosmetic applications. The success of this pilot project paves the way for other cosmetic targets of interest. © The Royal Society of Chemistry 2016.

Scior T.,Autonomous University of Puebla | Bender A.,University of Cambridge | Tresadern G.,Research Informatics and Integrative Genomics | Medina-Franco J.L.,Torrey Pines Institute for Molecular Studies | And 4 more authors.
Journal of Chemical Information and Modeling | Year: 2012

The aim of virtual screening (VS) is to identify bioactive compounds through computational means, by employing knowledge about the protein target (structure-based VS) or known bioactive ligands (ligand-based VS). In VS, a large number of molecules are ranked according to their likelihood to be bioactive compounds, with the aim to enrich the top fraction of the resulting list (which can be tested in bioassays afterward). At its core, VS attempts to improve the odds of identifying bioactive molecules by maximizing the true positive rate, that is, by ranking the truly active molecules as high as possible (and, correspondingly, the truly inactive ones as low as possible). In choosing the right approach, the researcher is faced with many questions: where does the optimal balance between efficiency and accuracy lie when evaluating a particular algorithm; do some methods perform better than others and in what particular situations; and what do retrospective results tell us about the prospective utility of a particular method? Given the multitude of settings, parameters, and data sets the practitioner can choose from, there are many pitfalls that lurk along the way which might render VS less efficient or downright useless. This review attempts to catalogue published and unpublished problems, shortcomings, failures, and technical traps of VS methods with the aim to avoid pitfalls by making the user aware of them in the first place. © 2012 American Chemical Society.

Wermuth C.G.,Prestwick Chemical | Aldous D.,Boston | Raboisson P.,Janssen Infectious Diseases and Diagnostics BVBA | Rognan D.,CNRS Laboratory for Therapeutic Innovation
The Practice of Medicinal Chemistry: Fourth Edition | Year: 2015

The Practice of Medicinal Chemistry, Fourth Edition provides a practical and comprehensive overview of the daily issues facing pharmaceutical researchers and chemists. In addition to its thorough treatment of basic medicinal chemistry principles, this updated edition has been revised to provide new and expanded coverage of the latest technologies and approaches in drug discovery. With topics like high content screening, scoring, docking, binding free energy calculations, polypharmacology, QSAR, chemical collections and databases, and much more, this book is the go-to reference for all academic and pharmaceutical researchers who need a complete understanding of medicinal chemistry and its application to drug discovery and development. Includes updated and expanded material on systems biology, chemogenomics, computer-aided drug design, and other important recent advances in the field Incorporates extensive color figures, case studies, and practical examples to help users gain a further understanding of key concepts Provides high-quality content in a comprehensive manner, including contributions from international chapter authors to illustrate the global nature of medicinal chemistry and drug development research An image bank is available for instructors at © 2015 Elsevier Ltd. All rights reserved.

Choulier L.,University of Strasbourg | Nomine Y.,University of Strasbourg | Zeder-Lutz G.,University of Strasbourg | Charbonnier S.,University of Strasbourg | And 3 more authors.
Analytical Chemistry | Year: 2013

We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (K D = 0.8 μM) interaction between an E6-derived peptide (E6 peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with K D in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening. © 2013 American Chemical Society.

Human ES cell line H9 (WA-09) and derivatives (SOX10::GFP; SYN::ChR2-EYFP; SYN::EYFP;PHOX2B:GFP;EF1::RFP Ednrb−/−) as well as two independent human iPS cell lines (healthy and familial dysautonomia, Sendai-based, OMSK (Cytotune)) were maintained on mouse embryonic fibroblasts (Global Stem) in knockout serum replacement (KSR; Life Technologies, 10828-028) containing human ES cell medium as described previously7. Cells were subjected to mycoplasma testing at monthly intervals and short tandem repeats (STR) profiled to confirm cell identity at the initiation of the study. Human ES cells were plated on matrigel (BD Biosciences, 354234)-coated dishes (105 cells cm−2) in ES cell medium containing 10 nM FGF2 (R&D Systems, 233-FB-001MG/CF). Differentiation was initiated in KSR medium (knockout DMEM plus 15% KSR (Life Technologies, 10828-028), l-glutamine (Life Technologies, 25030-081), NEAA (Life Technologies, 11140-050)) containing LDN193189 (100 nM, Stemgent) and SB431542 (10 μM, Tocris). The KSR medium was gradually replaced with increasing amounts of N2 medium from day 4 to day 10 as described previously7. For CNC induction, cells were treated with 3 μM CHIR99021 (Tocris Bioscience, 4423) in addition to LDN193189 and SB431542 from day 2 to day 11. ENC differentiation involves additional treatment with retinoic acid (1 μM) from day 6 to day 11. For deriving MNCs, LDN193189 is replaced with BMP4 (10 nM, R&D, 314-BP) and EDN3 (10 nM, American Peptide company, 88-5-10B) from day 6 to day 11 (ref. 3). The differentiated cells are sorted for CD49D at day 11. CNS precursor control cells were generated by treatment with LDN193189 and SB431542 from day 0 to day 11 as previously described7. Throughout the manuscript, day 0 is the day the medium is switched from human ES cell medium to LDN193189 and SB431542 containing medium. Days of differentiation in text and figures refer to the number of days since the pluripotent stage (day 0). For immunofluorescence, the cells were fixed with 4% paraformaldehyde (Affymetrix-USB, 19943) for 20 min, then blocked and permeabilized using 1% bovine serum albumin (BSA) (Thermo Scientific, 23209) and 0.3% Triton X-100 (Sigma, T8787). The cells were then incubated in primary antibody solutions overnight at 4 °C and stained with fluorophore-conjugated secondary antibodies at room temperature for 1 h. The stained cells were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before imaging. For flow cytometry analysis, the cells are dissociated with Accutase (Innovative Cell Technologies, AT104) and fixed and permeabilized using BD Cytofix/Cytoperm (BD Bioscience, 554722) solution, then washed, blocked and permeabilized using BD Perm/Wash buffer (BD Bioscience, 554723) according to manufacturer’s instructions. The cells are then stained with primary (overnight at 4 °C) and secondary (30 min at room temperature) antibodies and analysed using a flow Cytometer (Flowjo software). A list of primary antibodies and working dilutions is provided in Supplementary Table 4. The PHOX2A antibody was provided by J.-F. Brunet (rabbit, 1:800 dilution). Fertilized eggs (from Charles River Farms) were incubated at 37 °C for 50 h before injections. A total of 2 × 105 CD49D-sorted, RFP-labelled NC cells were injected into the intersomitic space of the vagal region of the embryos targeting a region between somite 2 and 6 (HH 14 embryo, 20–25 somite stage). The embryos were collected 36 h later for whole-mount epifluorescence and histological analyses. For RNA sequencing, total RNA was extracted using RNeasy RNA purification kit (Qiagen, 74106). For qRT–PCR assay, total RNA samples were reverse transcribed to cDNA using Superscript II Reverse Transcriptase (Life Technologies, 18064-014). qRT–PCR reactions were set up using QuantiTect SYBR Green PCR mix (Qiagen, 204148). Each data point represents three independent biological replicates. ENC cells from the 11-day induction protocol were aggregated into 3D spheroids (5 million cells per well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF). After 4 days of suspension culture, the spheroids are plated on poly-ornithine/laminin/fibronectin (PO/LM/FN)-coated dishes (prepared as described previously26) in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1–2 weeks. The cells were fixed for immunostaining or collected for gene expression analysis at days 25, 40 and 60 of differentiation. Mesoderm specification is carried out in STEMPRO-34 (Gibco, 10639-011) medium. The ES cells are subjected to activin A treatment (100 ng ml−1, R&D, 338-AC-010) for 24 h followed by BMP4 treatment (10 ng ml−1, R&D, 314-BP) for 4 days9. The cells are then differentiated into SMC progenitors by treatment with PDGF-BB (5 ng ml−1, Peprotech, 100-14B), TGFb3 (5 ng ml−1, R&D systems, 243-B3-200) and 10% FBS. The SMC progenitors are expandable in DMEM supplemented with 10% FBS. The SMC progenitors were plated on PO/LM/FN-coated culture dishes (prepared as described previously26) 3 days before addition of ENC-derived neurons. The neurons were dissociated (using accutase, Innovative Cell Technologies, AT104) at day 30 of differentiation and plated onto the SMC monolayer cultures. The culture is maintained in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng ml−1, Peprotech, 450-10) and ascorbic acid (100 μM, Sigma, A8960-5G). Functional connectivity was assessed at 8–16 weeks of co-culture. SMC-only and SMC-ENC-derived neuron co-cultures were subjected to acetylcholine chloride (50 μM, Sigma, A6625), carbamoylcholine chloride (10 μM, Sigma,C4382) and KCl (55 mM, Fisher Scientific, BP366–500) treatment, 3 months after initiating the co-culture. Optogenetic stimulations were performed using a 450-nm pigtailed diode pumped solid state laser (OEM Laser, PSU-III LED, OEM Laser Systems, Inc.) achieving an illumination between 2 and 4 mW mm−2. The pulse width was 4 ms and stimulation frequencies ranged from 2 to 10 Hz. For the quantification of movement, images were assembled into a stack using Metamorph software and regions with high contrast were identified (labelled yellow in Supplementary Fig. 5). The movement of five representative high-contrast regions per field was automatically traced (Metamorph software). Data are presented in kinetograms as movement in pixels in x and y direction (distance) with respect to the previous frame. We used the previously described method for generation of tissue-engineered colon11. In brief, the donor colon tissue was collected and digested into organoid units using dispase (Life Technologies, 17105-041) and collagenase type 1 (Worthington, CLS-1). The organoid units were then mixed immediately (without any in vitro culture) with CD49D-purified human ES-cell-derived ENC precursors (day 15 of differentiation) and seeded onto biodegradable polyglycolic acid scaffolds (2-mm sheet thickness, 60 mg cm−3 bulk density; porosity >95%, Concordia Fibres) shaped into 2 mm long tubes with poly-l lactide (PLLA) (Durect Corporation). The seeded scaffolds were then placed onto and wrapped in the greater omentum of the adult (>2 months old) NSG mice. Just before the implantation, these mice were irradiated with 350 cGy. The seeded scaffolds were differentiated into colon-like structures inside the omentum for 4 additional weeks before they were surgically removed for tissue analysis. All mouse procedures were performed following NIH guidelines, and were approved by the local Institutional Animal Care and Use Committee (IACUC), the Institutional Biosafety Committee (IBC) as well as the Embryonic Stem Cell Research Committee (ESCRO). We used 3–6-week-old male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice or 2–3-week-old Ednrbs-l/s-l (SSL/LeJ) mice27 (n = 12, 6 male, 6 female) for these studies. Animal numbers were based on availability of homozygous hosts and on sufficient statistical power to detect large effects between treatment versus control (Ednrbs-l/s-l) as well as for demonstrating robustness of migration behaviour (NSG). Animals were randomly selected for the various treatment models (NSG and Ednrbs-l/s-l) but assuring for equal distribution of male/female ratio in each group (Ednrbs-l/s-l). All in vivo experiments were performed in a blinded manner. Animals were anaesthetized with isoflurane (1%) throughout the procedure, a small abdominal incision was made, abdominal wall musculature lifted and the caecum is exposed and exteriorized. Warm saline is used to keep the caecum moist. Then 20 μl of cell suspension (2–4 million RFP+ CD49D-purified human ES-cell-derived ENC precursors) in 70% Matrigel (BD Biosciences, 354234) in PBS or 20 μl of 70% Matrigel in PBS only (control-grafted animals) were slowly injected into the caecum (targeting the muscle layer) using a 27-gauge needle. Use of 70% matrigel as carrier for cell injection assured that the cells stayed in place after the injection and prevented backflow into the peritoneum. After injection that needle was withdrawn, and a Q-tip was placed over the injection site for 30 s to prevent bleeding. The caecum was returned to the abdominal cavity and the abdominal wall was closed using 4-0 vicryl and a taper needle in an interrupted suture pattern and the skin was closed using sterile wound clips. After wound closure animals were put on paper on top of their bedding and attended until conscious and preferably eating and drinking. The tissue was collected at different time points (ranging from two weeks to four months) after transplantation for histological analysis. Ednrbs-l/s-l mice were immunosuppressed by daily injections of cyclosporine (10 mg kg−1 i.p, Sigma, 30024). The collected colon samples were fixed in 4% paraformaldehyde at 4 °C overnight before imaging. Imaging is performed using Maestro fluorescence imaging system (Cambridge Research and Instrumentation). The tissue samples were incubated in 30% sucrose (Fisher Scientific, BP220-1) solutions at 4 °C for 2 days, and then embedded in OCT (Fisher Scientific, NC9638938) and cryosectioned. The sections were then blocked with 1% BSA (Thermo Scientific, 23209) and permeabilized with 0.3% Triton X-100 (Sigma, T8787). The sections are then stained with primary antibody solution at 4 °C overnight and fluorophore-conjugated secondary antibody solutions at room temperature for 30 min. The stained sectioned were then incubated with DAPI (1 ng ml−1, Sigma, D9542-5MG) and washed several times before they were mounted with Vectashield Mounting Medium (vector, H1200) and imaged using fluorescent (Olympus IX70) or confocal microscopes (Zeiss SP5). Mice are gavaged with 0.3 ml of dye solution containing 6% carmine (Sigma, C1022-5G), 0.5% methylcellulose (Sigma, 274429-5G) and 0.9 NaCl, using a #24 round-tip feeding needle. The needle was held inside the mouse oesophagus for a few seconds after gavage to prevent regurgitation. After 1 h, the stool colour was monitored for gavaged mice every 10 min. For each mouse, total gastrointestinal transit time is between the time of gavage and the time when red stool is observed. The double nickase CRISPR/Cas9 system28 was used to target the EDNRB locus in EF1–RFP H9 human ES cells. Two guide RNAs were designed (using the CRISPR design tool; to target the coding sequence with PAM targets ~20 base pairs apart (qRNA #1 target specific sequence: 5′-AAGTCTGTGCGGACGCGCCCTGG-3′, RNA #2 target specific sequence: 5′-CCAGATCCGCGACAGGCCGCAGG-3′). The cells were transfected with guide RNA constructs and GFP-fused Cas9-D10A nickase. The GFP-expressing cells were FACS purified 24 h later and plated in low density (150 cells cm−2) on mouse embryonic fibroblasts. The colonies were picked 7 days later and passaged twice before genomic DNA isolation and screening. The targeted region of EDNRB gene was PCR amplified (forward primer: 5′-ACGCCTTCTGGAGCAGGTAG-3′, reverse primer: 5′-GTCAGGCGGGAAGCCTCTCT-3′) and cloned into Zero Blunt TOPO vector (Invitrogen, 450245). To ensure that both alleles (from each ES cell colony) are represented and sequenced, we picked 10 bacterial clones (for each ES cell clone) for plasmid purification and subsequent sequencing. The clones with bi-allelic nonsense mutations were expanded and differentiated for follow-up assays. The ENC cells are plated on PO/LM/FN coated (prepared as described previously26) 96-well or 48-well culture plates (30,000 cm−2). After 24 h, the culture lawn is scratched manually using a pipette tip. The cells are given an additional 24–48 h to migrate into the scratch area and fixed for imaging and quantification. The quantification is based on the percentage of the nuclei that are located in the scratch area after the migration period. The scratch area is defined using a reference well that was fixed immediately after scratching. Migration of cells was quantified using the open source data analysis software KNIME29 ( with the ‘quantification in ROI’ plug-in as described in detail elsewhere30. To quantify proliferation, FACS-purified ENC cells were assayed using CyQUANT NF cell proliferation Assay Kit (Life Technologies, C35006) according to manufacturer’s instructions. In brief, to generate a standard, cells were plated at various densities and stained using the fluorescent DNA binding dye reagent. Total fluorescence intensity was then measured using a plate reader (excitation at 485 nm and emission detection at 530 nm). After determining the linear range, the CD49D+ wild-type and Ednrb−/− ENC precursors were plated (6,000 cell cm−2) and assayed at 0, 24, 48 and 72 h. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To monitor the viability of wild-type and Ednrb−/− ENC precursors, cells were assayed for lactate dehydrogenase (LDH) activity using CytoTox 96 cytotoxicity assay kit (Promega, G1780). In brief, the cells are plated in 96-well plates at 30,000 cm−2. The supernatant and the cell lysate is collected 24 h later and assayed for LDH activity using a plate reader (490 nm absorbance). Viability is calculated by dividing the LDH signal of the lysate by total LDH signal (from lysate plus supernatant). The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. The chemical compound screening was performed using the Prestwick Chemical Library. The ENC cells were plated in 96-well plates (30,000 cm−2) and scratched manually 24 h before addition of the compounds. The cells were treated with two concentrations of the compounds (10 μM and 1 μM). The plates were fixed 24 h later for total plate imaging. The compounds were scored based on their ability to promote filling of the scratch in 24 h. The compounds that showed toxic effects (based on marked reduction in cell numbers assessed by DAPI staining) were scored 0, compounds with no effects were scored 1, compounds with moderate effects were scored 2, and compounds with strong effects (that resulted in complete filling of the scratch area) were scored 3 and identified as hit compounds. The hits were further validated to ensure reproducibility. The cells were treated with various concentrations of the selected hit compound (pepstatin A) for dose response analysis. The optimal dose (10 μM based on optimal response and viability) was used for follow-up experiments. For the pre-treatment experiments, cells were CD49D purified at day 11 and treated with pepstatin A from day 12 to day 15 followed by transplantation into the colon wall of NSG mice. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. To inhibit BACE2, the ENC precursors were treated with 1 μM β-secretase inhibitor IV (CAS 797035-11-1; Calbiochem). To knockdown BACE2, cells were dissociated using accutase (Innovative Cell Technologies, AT104) and reverse-transfected (using Lipofectamine RNAiMAX-Life Technologies, 13778-150) with an siRNA pool (SMARTpool: ON-TARGETplus BACE2 siRNA, Dharmacon, L-003802-00-0005) or four different individual siRNAs (Dharmacon, LQ-003802-00-0002, 2 nmol). The knockdown was confirmed by qRT–PCR measurement of BACE2 mRNA levels in cells transfected with the BACE2 siRNAs versus the control siRNA pool (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05). The transfected cells were scratched 24 h after plating and fixed 48 h later for migration quantification. The cells were cultured in neurobasal (NB) medium supplemented with l-glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3 μM, Tocris Bioscience, 4423) and FGF2 (10 nM, R&D Systems, 233-FB-001MG/CF) during the assay. Data are presented as mean ± s.e.m. and were derived from at least three independent experiments. Data on replicates (n) is given in figure legends. Statistical analysis was performed using the Student’s t-test (comparing two groups) or ANOVA with Dunnett test (comparing multiple groups against control). Distribution of the raw data approximated normal distribution (Kolmogorov–Smirnov normality test) for data with sufficient number of replicates to test for normality. Survival analysis was performed using a log-rank (Mantel–Cox) test. Z-scores for primary hits were calculated as Z = (x − μ)/σ, in which x is the migration score value and is 3 for all hit compounds; μ is the mean migration score value, and σ is the standard deviation for all compounds and DMSO controls (n = 224).

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