Lars Brandén

Lars Brandén

Innovationsrådgivare
Avdelningen för externa relationer
Hus Culmen, Kalmar
Spara kontaktuppgifter Ladda ner bild

I work as innovation advisor at the Linné university, external relations department. By using my experience from research and contacts in the life sciences industry I help any researcher or employee at the university to bring their ideas and innovations into society for the betterment of life for everyone.

 

I have built my expertice on a combination of experience from both academic work and industry positions over the last 25 years. My background also includes an art eductation and several exhibitions that I think broadens my skillset and allows me to be a constructive sounding board for humanistic researchers that would like to bring an indea or a concept into society.

- Successfully built and operated two cutting edge High ThroughputCell Biology centers from ground and up.

- Successful in establishing multi-centre collaborations with highprofile global stakeholders.

-Successful in establishing pharma:academic collaborations

-Experienced in tech-transfer and company aquisition due dilligence

- Innovative approach to developing new technologies and newapplications.

- Strong background in design, implementation, and applicationsdevelopment for new technology.

-Art education at the Ölands folkhögskola and several exhibitions allows me to also hopefully better understand and work with artists and researchers in the humanistic disciplines in a collaborative and inspired manner.

I was recruited to three sequential senior director positions at Columbia University and Yale university and also as a post-doctoral research fellow at the Memorial Sloan-Kettering Cancer Center, MSKCC. I have experience in developing over 180 high content and low content cell based assays for HTS screening as well as experience in assembling and managing multi-disciplinary research teams to successfully meet goals and dead-lines. My work cover over a decade of hands-on management for multiple parallel external and internal projects with high impact stakeholders and with sharp attention to KPI's, finance and outreach. I generate success with a combination of high level analytical ability, broad knowledge base and an active and personal approach to teamwork and leadership.

Besides my academic work I have also gained a lot of experience from industry where I have worked as an external consultant with companies from the UK, USA, Israel, Sweden and Switzerland. I have primarily worked on senior director level and general senior scientific advisory positions. My work has stretched from building bioinformatics departments in industry from the bottom and up to evaluating and advising on basic scientific questions when translating bench-top work into industry-level applications.

Main areas of expertise in the life sciences/informatics:

-Virology.

-Immunology.

-In vitro diagnostics

-Cell signalling.

-Gene Therapy.

-Bioinformatics.

-AI

-Systems Biology.

-Drug discovery process.

-Laboratory automation.

Besides my life/informatics skill-set I also have a good grasp of the techniques and processes in the visual arts: Graphics, sculpting, drawing and painting. The process of creating art in a team is quiet different from how a life sciences team-projects are managed and my experience and education from Ölands folkhögskola was an experience that I think has broadened my understanding of how important it is to have a constructive as well as mutually respectful dialogue with anyone I work together with. The humanistic sciences of cource have a rigorous way of establishing results and evaluate concepts and ideas and is in no way less stringent than other scientiffic disciplines in the life sciences and other areas. The difference is more in the language used to descibe the processes, concepts and ideas and having a personal experience helps in bridging the gap of understanding that can exist between the humanistic disciplines and life sciences, physics, chemistry, computer sciences and more.

 

Publications:

    Potent inhibitors of Huntingtin protein aggregation in a cell-based assayPotent inhibitors of Huntingtin protein aggregation in a cell-based assay

Bioorg Med Chem Lett · Mar 1, 2009Bioorg Med Chem Lett · Mar 1, 2009

    Discovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocationDiscovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocation

Bioorg Med Chem Lett · Feb 1, 2009Bioorg Med Chem Lett · Feb 1, 2009

 

    Physiological and Pathological Role of Alpha-synuclein in Parkinson's Disease Through Iron Mediated Oxidative Stress; The Role of a Putative Iron-responsive ElementPhysiological and Pathological Role of Alpha-synuclein in Parkinson's Disease Through Iron Mediated Oxidative Stress; The Role of a Putative Iron-responsive Element

International Journal of Molecular Sciences · Jan 1, 2009International Journal of Molecular Sciences · Jan 1, 2009

 

    Discovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine phosphataseDiscovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine phosphatase

Bioorg Med Chem Lett · May 1, 2008Bioorg Med Chem Lett · May 1, 2008

 

    A convenient preparation of N-8-quinolinyl benzenesultams as novel NF-kB inhibitorsA convenient preparation of N-8-quinolinyl benzenesultams as novel NF-kB inhibitors

Tetrahedron Letters · Mar 1, 2008Tetrahedron Letters · Mar 1, 2008

 

    Identification of N-(quinoline-8-yl)benzenesulphonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NfkappaB activationIdentification of N-(quinoline-8-yl)benzenesulphonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NfkappaB activation

Bioorg Med Chem · Jan 1, 2008Bioorg Med Chem · Jan 1, 2008

 

    High-Throughput Identification of Combinatorial Ligands for DNA Delivery in Cell CultureHigh-Throughput Identification of Combinatorial Ligands for DNA Delivery in Cell Culture

AIP Conf. Pros · Jan 1, 2008AIP Conf. Pros · Jan 1, 2008

 

    Cell-based assays using primary endothelial cells to study multiple steps in inflammationCell-based assays using primary endothelial cells to study multiple steps in inflammation

Methods Enzymol. · Jan 1, 2006Methods Enzymol. · Jan 1, 2006

 

    Protease-induced release of functional peptides from bioplexesProtease-induced release of functional peptides from bioplexes

J Control Release · Jul 1, 2004J Control Release · Jul 1, 2004

 

    Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimerasCooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Biomol. Eng. · Apr 1, 2004Biomol. Eng. · Apr 1, 2004

 

    Human cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chainHuman cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chain

BLOOD · Jun 1, 2003BLOOD · Jun 1, 2003

 

    ER retention may play a role in sorting of the nuclear pore membrane protein POM121ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Exp.Cell Res. · Apr 1, 2003Exp.Cell Res. · Apr 1, 2003

 

    The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteinsThe human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins

J Virol. · Apr 1, 2003J Virol. · Apr 1, 2003

 

    Expression profiling in transformed human B cells: Influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arraysExpression profiling in transformed human B cells: Influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays

Eur. J. Immunol. · Apr 1, 2002Eur. J. Immunol. · Apr 1, 2002

 

    BIOPLEX TECHNOLOGY. A novel, synthtic Gene Delivery System Based on Peptides Anchored to Nucleic AcidsBIOPLEX TECHNOLOGY. A novel, synthtic Gene Delivery System Based on Peptides Anchored to Nucleic Acids

Methods Enzymol. · Jan 1, 2002Methods Enzymol. · Jan 1, 2002

 

    Bioplex technology: Novel synthetic gene delivery system based on peptides anchored to nucleic acidsBioplex technology: Novel synthetic gene delivery system based on peptides anchored to nucleic acids

Gene Therapy Methods · Jan 1, 2002Gene Therapy Methods · Jan 1, 2002

 

    In vivo delivery of nucleic acids via bi-functional peptide nucleic acid peptidesIn vivo delivery of nucleic acids via bi-functional peptide nucleic acid peptides

Gene Therapy · Jan 1, 2001Gene Therapy · Jan 1, 2001

 

    BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clonesBTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones

Gene Medicine · May 1, 2000J. Gene Medicine · May 1, 2000

 

    The role of Bruton's Tyrosine Kinase (btk) in Phosphoinositide-dependent SignalingThe role of Bruton's Tyrosine Kinase (btk) in Phosphoinositide-dependent Signaling

Allergy & Clinical Immunology International · May 1, 2000Allergy & Clinical Immunology International · May 1, 2000

 

    Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho family GTPasesRedistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho family GTPases

Eur. J. Immunol. · Jan 1, 2000Eur. J. Immunol. · Jan 1, 2000

 

    A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNAA peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA

Nature Biotechnol. · Aug 1, 1999Nature Biotechnol. · Aug 1, 1999

 

    XLA-Studies on the Btk tyrosine kinaseXLA-Studies on the Btk tyrosine kinase

Molecular Immunology · Aug 1, 1998Molecular Immunology · Aug 1, 1998

 

    X-linked agammaglobulinemia: lack of mature B lineage cells caused by mutations in the Btk kinaseX-linked agammaglobulinemia: lack of mature B lineage cells caused by mutations in the Btk kinase

Springer Semin Immunopathol. · Apr 19, 1998Springer Semin Immunopathol. · Apr 19, 1998

 

    BTKbase, mutation database for X-linked agammaglobulinemia (XLA)BTKbase, mutation database for X-linked agammaglobulinemia (XLA)

Nucleic Acids Res. · Jan 1, 1998Nucleic Acids Res. · Jan 1, 1998

 

    Reverse flow-through transduction: A new method for gene deliveryReverse flow-through transduction: A new method for gene delivery

Blood · Nov 15, 1997Blood · Nov 15, 1997

 

    Retroviral gene-transfer to XID bone-marrow cells- an experimental-model for gene-therapy in X-linked agammaglobulinemiaRetroviral gene-transfer to XID bone-marrow cells- an experimental-model for gene-therapy in X-linked agammaglobulinemia

Journal of cellular biochemistry · Mar 10, 1995Journal of cellular biochemistry · Mar 10, 1995

 

    Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci.Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci.

Immunol. · Dec 15, 1994J. Immunol. · Dec 15, 1994

 

    X-linked agammaglobulinemia (xla), a candidate disease for gene-therapyX-linked agammaglobulinemia (xla), a candidate disease for gene-therapy

journal of cellular biochemistry · Jan 4, 1994journal of cellular biochemistry · Jan 4, 1994

 

    Aggregation and Clearance of Mutant Huntingtin Protein [Primary Screening]Aggregation and Clearance of Mutant Huntingtin Protein [Primary Screening]

PubChemPubChem

        The mutation underlying Huntington's disease is an expansion of a polyglutamine tract in the N-terminus of the protein huntingtin (htt). Under nonpathogenic conditions, this stretch of glutamines range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the disease. In an inducible mouse model of Huntington's disease, we found that abolishing mutant htt expression led to complete recovery of symptomatic mice (Yamamoto et al. 2000). Tightly linked to the symptomatic reversal was a clearance of both the soluble and aggregated forms of the protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell based assay was developed to visualize and analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). This was accomplished by creating a stable cell line that inducibly expressed the N-terminus of huntingtin protein with 103Q repeats fused to monomeric CFP. This cell line permits high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells (Yamamoto et al. 2006) and therefore allows the screen of libraries of small molecules.The mutation underlying Huntington's disease is an expansion of a polyglutamine tract in the N-terminus of the protein huntingtin (htt). Under nonpathogenic conditions, this stretch of glutamines range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the disease. In an inducible mouse model of Huntington's disease, we found that abolishing mutant htt expression led to complete recovery of symptomatic mice (Yamamoto et al. 2000). Tightly linked to the symptomatic reversal was a clearance of both the soluble and aggregated forms of the protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell based assay was developed to visualize and analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). This was accomplished by creating a stable cell line that inducibly expressed the N-terminus of huntingtin protein with 103Q repeats fused to monomeric CFP. This cell line permits high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells (Yamamoto et al. 2006) and therefore allows the screen of libraries of small molecules.

 

    Cellular assay for TNF alpha induced NFkappaB translocation [Primary Screening]Cellular assay for TNF alpha induced NFkappaB translocation [Primary Screening]

PubChemPubChem

 

        Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB. Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response. Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events...Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB. Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response. Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events...

 

    Clearance of Mutant Huntingtin Protein - Confirmatory screen [Confirmatory]Clearance of Mutant Huntingtin Protein - Confirmatory screen [Confirmatory]

PubChemPubChem

 

        The mutation underlying Huntington's disease is an expansion of a polyglutamine tract in the N-terminus of the protein huntingtin (htt). Under nonpathogenic conditions, this stretch of glutamines range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the disease. In an inducible mouse model of Huntington's disease, we found that abolishing mutant htt expression led to complete recovery of symptomatic mice (Yamamoto et al. 2000). Tightly linked to the symptomatic reversal was a clearance of both the soluble and aggregated forms of the protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell based assay was developed to visualize and analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). This was accomplished by creating a stable cell line that inducibly expressed the N-terminus of huntingtin protein with 103Q repeats fused to monomeric CFP. This cell line permits high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells (Yamamoto et al. 2006) and therefore allows the screen of libraries of small molecules. Aggregation and Clearance of Mutant Huntingtin Protein HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076348-01; Aggregation and Clearance of Mutant Huntingtin Protein, Assay Provider Drs. A Yamamoto and JE Rothman, Department of Physiology and Cellular Biophysics at Columbia University. References: Yamamoto, A., J. J. Lucas and R. Hen (2000). "Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease." Cell 101(1): 57-66. Yamamoto, A., M. L. Cremona and J. E. Rothman (2006). "Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway." J Cell Biol 172(5): 719-31.The mutation underlying Huntington's disease is an expansion of a polyglutamine tract in the N-terminus of the protein huntingtin (htt). Under nonpathogenic conditions, this stretch of glutamines range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the disease. In an inducible mouse model of Huntington's disease, we found that abolishing mutant htt expression led to complete recovery of symptomatic mice (Yamamoto et al. 2000). Tightly linked to the symptomatic reversal was a clearance of both the soluble and aggregated forms of the protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell based assay was developed to visualize and analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). This was accomplished by creating a stable cell line that inducibly expressed the N-terminus of huntingtin protein with 103Q repeats fused to monomeric CFP. This cell line permits high throughput confocal microscopy to examine the state of the expressed mutant protein in live cells (Yamamoto et al. 2006) and therefore allows the screen of libraries of small molecules. Aggregation and Clearance of Mutant Huntingtin Protein HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076348-01; Aggregation and Clearance of Mutant Huntingtin Protein, Assay Provider Drs. A Yamamoto and JE Rothman, Department of Physiology and Cellular Biophysics at Columbia University. References: Yamamoto, A., J. J. Lucas and R. Hen (2000). "Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease." Cell 101(1): 57-66. Yamamoto, A., M. L. Cremona and J. E. Rothman (2006). "Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway." J Cell Biol 172(5): 719-31.

 

    Confirmatory Screen for chemical inhibitors of TNF alpha stimulated VCAM1 expression [Confirmatory]Confirmatory Screen for chemical inhibitors of TNF alpha stimulated VCAM1 expression [Confirmatory]

PubChemPubChem

 

        ...Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID = 802. Among the 508 compounds (active and inconclusive) identified during the primary screening, 493 were assessed in triplicate at 2uM to identify potentially false positive. 161 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10 uM......Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID = 802. Among the 508 compounds (active and inconclusive) identified during the primary screening, 493 were assessed in triplicate at 2uM to identify potentially false positive. 161 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10 uM...

 

 

    Confirmatory Screen for chemical modifiers of cytoskeleton assembly [Confirmatory]Confirmatory Screen for chemical modifiers of cytoskeleton assembly [Confirmatory]

PubChemPubChem

 

        ...During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported two high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808). The concomitant staining of F-actin filaments has allowed us to multiplex the output identifying compounds that perturb the cytoskeleton network and therefore potentially prevent VCAM1 attachment and function even though it is expressed properly. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical modifiers of cytoskeleton assembly" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=836. Among the 245 compounds (active and inconclusive) identified during the primary screening, 239 were assessed in triplicate at 2uM to identify potentially false positive. We also included 55 compounds showing a cytoskeleton modification phenotype from the triplicate 2uM screening of the confirmatory screen for VCAM1 expression inhibitors (AID 1013). 78 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10uM......During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported two high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808). The concomitant staining of F-actin filaments has allowed us to multiplex the output identifying compounds that perturb the cytoskeleton network and therefore potentially prevent VCAM1 attachment and function even though it is expressed properly. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical modifiers of cytoskeleton assembly" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=836. Among the 245 compounds (active and inconclusive) identified during the primary screening, 239 were assessed in triplicate at 2uM to identify potentially false positive. We also included 55 compounds showing a cytoskeleton modification phenotype from the triplicate 2uM screening of the confirmatory screen for VCAM1 expression inhibitors (AID 1013). 78 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10uM...

 

    Confirmatory Screen for chemical potentiatiors of TNF alpha stimulated VCAM1 expressionConfirmatory Screen for chemical potentiatiors of TNF alpha stimulated VCAM1 expression

PubChemPubChem

 

        Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical potentiators of TNF alpha stimulated VCAM1 expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=808. Among the 232 compounds (active and inconclusive) identified during the primary screening, 227 were assessed in triplicate at 2uM to identify potentially false positive. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16.Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical potentiators of TNF alpha stimulated VCAM1 expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID=808. Among the 232 compounds (active and inconclusive) identified during the primary screening, 227 were assessed in triplicate at 2uM to identify potentially false positive. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16.

 

 

    Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expression [Confirmatory]Confirmatory Screen: Chemical Inhibitors of TNF alpha stimulated E Selectin expression [Confirmatory]

PubChemPubChem

 

        Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. This assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor (1). This response induces the transcription of pro-inflammatory genes (2), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of TNF alpha stimulated E Selectin expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID = 1246. ...Among the 843 active compounds identified during the primary screening, 773 were assessed in triplicate at 2uM to identify potentially false positive. 80 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10 uM...Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. This assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor (1). This response induces the transcription of pro-inflammatory genes (2), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). Compounds identified from a previously described set of experiments entitled "Primary HTS assay for chemical inhibitors of TNF alpha stimulated E Selectin expression" were selected for testing in this assay. Further information on the primary screening can be found by searching on this website for PubChem AID = 1246. ...Among the 843 active compounds identified during the primary screening, 773 were assessed in triplicate at 2uM to identify potentially false positive. 80 compounds were selected and assessed in dose-response experiments in 8 data point, 1:2 dilution starting at a nominal test concentration of 10 uM...

 

    Confirmatory screen for Cellular assay for TNF alpha induced NFkappaB translocation [Confirmatory]Confirmatory screen for Cellular assay for TNF alpha induced NFkappaB translocation [Confirmatory]

PubChemPubChem

 

        ..Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events. Although the earliest signaling events initiated by each cytokine are unique, the pathways soon converge. A key event that is common to all pathways in the early inflammatory process is translocation of the transcription factor NFkappaB from the cytoplasm to the nucleus of the endothelial cell, an early event (20 min) after receptor stimulation. NFkappaB then activates the transcription of an array of pro-inflammatory proteins. These include proteins that regulate the attachment of lymphocytes to the wall of the blood vessel, mediated in part by proteins of the selectin family (Karman et al 1996). One of these, E-selectin, is clearly detectable on the endothelial cell surface at 4 hours post-stimulation. At later times, (24 h post-stimulation) lymphocyte recruitment is also mediated by VCAM-1, a cell adhesion molecule, on the endothelial cell surface (Gorczynski et al. 1996). VCAM-1 expression is dependent on activation of MAP kinase in addition to NFkappaB. Disruption of the mechanisms that regulate and limit the inflammatory response results in the development of chronic disease. Blocking the endothelial response at any point in the inflammatory pathway described above would assist in controlling the localized inflammatory response. In this proposal, we describe an assay to detect nuclear translocation of NFkappaB in primary human umbilical vein endothelial cells (HUVEC) after stimulation with cytokine.....Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events. Although the earliest signaling events initiated by each cytokine are unique, the pathways soon converge. A key event that is common to all pathways in the early inflammatory process is translocation of the transcription factor NFkappaB from the cytoplasm to the nucleus of the endothelial cell, an early event (20 min) after receptor stimulation. NFkappaB then activates the transcription of an array of pro-inflammatory proteins. These include proteins that regulate the attachment of lymphocytes to the wall of the blood vessel, mediated in part by proteins of the selectin family (Karman et al 1996). One of these, E-selectin, is clearly detectable on the endothelial cell surface at 4 hours post-stimulation. At later times, (24 h post-stimulation) lymphocyte recruitment is also mediated by VCAM-1, a cell adhesion molecule, on the endothelial cell surface (Gorczynski et al. 1996). VCAM-1 expression is dependent on activation of MAP kinase in addition to NFkappaB. Disruption of the mechanisms that regulate and limit the inflammatory response results in the development of chronic disease. Blocking the endothelial response at any point in the inflammatory pathway described above would assist in controlling the localized inflammatory response. In this proposal, we describe an assay to detect nuclear translocation of NFkappaB in primary human umbilical vein endothelial cells (HUVEC) after stimulation with cytokine...

 

    HTS for LYP Inhibitors-an Autoimmunity Target - Primary screen [Confirmatory]HTS for LYP Inhibitors-an Autoimmunity Target - Primary screen [Confirmatory]

PubChemPubChem

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Grave#s disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Grave#s disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560

 

    IL-1B Induced NFkB Translocation - Primary Screen [Primary Screening]IL-1B Induced NFkB Translocation - Primary Screen [Primary Screening]

PubChemPubChem

 

        Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage (Pober, 2002). Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB (Senftleben, et al., 2002). Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state (Aggarwal, 2003). A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response (Pober, 2002; Aggarwal, 2003). Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of...Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage (Pober, 2002). Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB (Senftleben, et al., 2002). Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state (Aggarwal, 2003). A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response (Pober, 2002; Aggarwal, 2003). Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of...

 

    Identification of Molecular Probes for Cellular assay for TNF alpha induced NFkappaB translocation [Confirmatory]Identification of Molecular Probes for Cellular assay for TNF alpha induced NFkappaB translocation [Confirmatory]

PubChemPubChem

 

 

        Eight compounds were confirmed as hits from the original TNFalpha induced NFkappaB translocation assay (primary AID: 438, confirmatory AID: 462). One of these hits (SID: 857745) was selected for analog follow-up and SAR studies. In this publication, we report six analogs of the original compound that show inhibitory activity when run against the original assay. Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB. Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes...Eight compounds were confirmed as hits from the original TNFalpha induced NFkappaB translocation assay (primary AID: 438, confirmatory AID: 462). One of these hits (SID: 857745) was selected for analog follow-up and SAR studies. In this publication, we report six analogs of the original compound that show inhibitory activity when run against the original assay. Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB. Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. The more common inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and psoriasis. Although each disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state. A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes...

 

    LYP Activators-an Autoimmunity Target - Primary screen [Primary Screening]LYP Activators-an Autoimmunity Target - Primary screen [Primary Screening]

PubChemPubChem

 

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant, suggesting that an animal model could be created with a small molecule activator. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm....LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant, suggesting that an animal model could be created with a small molecule activator. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm....

 

    LYP Activators-an Autoimmunity Target - Single Concentration Confirmatory ScreenLYP Activators-an Autoimmunity Target - Single Concentration Confirmatory Screen

PubChemPubChem

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant, suggesting that an animal model could be created with a small molecule activator. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant, suggesting that an animal model could be created with a small molecule activator. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560

 

    LYP Inhibitors - Confirmatory Screen [Confirmatory]LYP Inhibitors - Confirmatory Screen [Confirmatory]

PubChemPubChem

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at Columbia University as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01-MH077604-01; Assay: HTS for LYP Inhibitors-an Autoimmunity Target, Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA. Bottini N., Musumeci, L., Alonso, A., Rahmouni, S., Nika, K., Rostamkhani, M., MacMurray, J., Pellecchia, M., Eisenbarth, G.S., Comings, D., and Mustelin, T.(2004). A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genetics 36 337-38. PMID: 15004560

 

 

    LYP Inhibitors-an Autoimmunity Target - Primary screen [Primary Screening]LYP Inhibitors-an Autoimmunity Target - Primary screen [Primary Screening]

PubChemPubChem

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm...LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle. Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm...

 

    LYP_Inh_Infinite200_384_catalase_06132008 [Primary Screening]LYP_Inh_Infinite200_384_catalase_06132008 [Primary Screening]

PubChemPubChem

 

        LYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at the Columbia University Molecular Library Screening Center Network (MLSCN) Grant number: X01-MH077604-01 Assay: HTS for LYP Inhibitors-an Autoimmunity Target Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CALYP, a lymphocyte specific protein tyrosine phosphatase that plays a critical regulatory role in T cell receptor signaling, is encoded by the PTPN22 gene. A single-nucleotide polymorphism in PTPN22 is associated with a number of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and Graves disease. The autoimmunity-predisposing allele is a gain-of-function mutant suggesting that its effect could be eliminated by a specific small-molecule inhibitor. Assay Principle Full enzymatic activity is present in a 62 kDa N-terminal catalytic domain of the phosphatase that can be expressed in and purified from bacteria. Incubation of this recombinant protein with the appropriate substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) results in the conversion of substrate to a fluorescent derivative. The endpoint of the reaction is assessed by measuring fluorescence with excitation at 360 nm and emission at 465 nm. LYP screening was performed at the Columbia University Molecular Library Screening Center Network (MLSCN) Grant number: X01-MH077604-01 Assay: HTS for LYP Inhibitors-an Autoimmunity Target Assay provider: Dr. Tomas Mustelin, Burnham Institute for Medical Research, La Jolla, CA

 

    Novel sEH activators for the therapeutic treatment of hypertension and inflammation [Primary Screening]Novel sEH activators for the therapeutic treatment of hypertension and inflammation [Primary Screening]

PubChemPubChem

 

        Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. The importance of the biochemical pathways linking sEH to vascular and renal inflammation suggest that small molecule activators of this enzyme would be scientifically useful. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. The importance of the biochemical pathways linking sEH to vascular and renal inflammation suggest that small molecule activators of this enzyme would be scientifically useful. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.

 

    Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation [Primary Screening]Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation [Primary Screening]

PubChemPubChem

 

        Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. While these inhibitors are scientifically useful, the low solubility and relatively fast metabolism of the first generation inhibitors makes them less than therapeutically efficient, underlying the need for novel inhibitor structures. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. While these inhibitors are scientifically useful, the low solubility and relatively fast metabolism of the first generation inhibitors makes them less than therapeutically efficient, underlying the need for novel inhibitor structures. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.

 

    Primary HTS assay during TNFalpha stimulated VCAM1 expression to assess cytotoxicity.Primary HTS assay during TNFalpha stimulated VCAM1 expression to assess cytotoxicity.

PubChemPubChem

 

        During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported three high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808) as well as compounds that disturb the cytoskeleton integrity (AID 836). Because of the high content nature of the screening campaign, we were also able to assess the number of remaining cells after treatment with the compound library. These data give us an interesting preliminary insight of the level of toxic compounds in the library in the assay conditions used (24h incubation with the compounds). In this publication, we report the primary hits resulting of the MLSMR 100K library screen design to identify potential toxic compounds.During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported three high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808) as well as compounds that disturb the cytoskeleton integrity (AID 836). Because of the high content nature of the screening campaign, we were also able to assess the number of remaining cells after treatment with the compound library. These data give us an interesting preliminary insight of the level of toxic compounds in the library in the assay conditions used (24h incubation with the compounds). In this publication, we report the primary hits resulting of the MLSMR 100K library screen design to identify potential toxic compounds.

 

    Primary HTS assay for chemical inhibitors of TNF alpha stimulated E-Selectin expression [Primary Screening]Primary HTS assay for chemical inhibitors of TNF alpha stimulated E-Selectin expression [Primary Screening]

PubChemPubChem

 

        TNFalpha induced E-Selectin HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. This assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor (1). This response induces the transcription of pro-inflammatory genes (2), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). TNFalpha Induced E-Selectin Expression HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076509-01; Assay: TNFalpha Induced E-Selectin, Assay Provider Dr. Thomas Mayer, Department of Physiology and Cellular Biophysics at Columbia University.TNFalpha induced E-Selectin HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. This assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor (1). This response induces the transcription of pro-inflammatory genes (2), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). TNFalpha Induced E-Selectin Expression HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076509-01; Assay: TNFalpha Induced E-Selectin, Assay Provider Dr. Thomas Mayer, Department of Physiology and Cellular Biophysics at Columbia University.

 

    Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expression [Primary Screening]Primary HTS assay for chemical inhibitors of TNF alpha stimulated VCAM1 expression [Primary Screening]

PubChemPubChem

 

        Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16. Keywords: Inflammation, endothelial cells, Tumor necrosis factor alpha, VCAM-1, VCAM1, cell adhesion, vascular cell adhesion molecule, cytokine, lymphocyte, vascular endothelium bioassay, biological signal transduction, high content, microscopy, Columbia UniversityCytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16. Keywords: Inflammation, endothelial cells, Tumor necrosis factor alpha, VCAM-1, VCAM1, cell adhesion, vascular cell adhesion molecule, cytokine, lymphocyte, vascular endothelium bioassay, biological signal transduction, high content, microscopy, Columbia University

 

    Primary HTS assay for chemical modifiers of cytoskeleton assembly [Primary Screening]Primary HTS assay for chemical modifiers of cytoskeleton assembly [Primary Screening]

PubChemPubChem

 

        During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported two high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808). The concomitant staining of F-actin filaments has allowed us to multiplex the output identifying compounds that perturb the cytoskeleton network and therefore potentially prevent VCAM1 attachment and function even though it is expressed properly. In this publication, we report the primary hits resulting of the MLSMR 100K library screen designed to identify modifiers of cytoskeleton integrity. (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pozo M, de Nicolas R, Egido J, Gonzalez-Cabrero J. "Simvastatin inhibits the migration and adhesion of monocytic cells and disorganizes the cytoskeleton of activated endothelial cells". Eur J Pharmacol. 2006 Oct 24;548(1-3):53-63. (4) Hordijk PL. "Endothelial signalling events during leukocyte transmigration". FEBS J. 2006 Oct;273(19):4408-15 Key words: endothelial cells; Tumor necrosis factor alpha; cytoskeleton; F-actin filaments; Phalloidin; VCAM-1; cell adhesion; high content; microscopy, Columbia UniversityDuring inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression (1). Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 (2) anchored to the cytoskeleton using its cytoplasmic domain (3). Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion (4). We have previously reported two high-content, cell based assays that have enable us to identify inhibitors and potentiators of VCAM-1 expression and trafficking to the cell surface (AID 802 and 808). The concomitant staining of F-actin filaments has allowed us to multiplex the output identifying compounds that perturb the cytoskeleton network and therefore potentially prevent VCAM1 attachment and function even though it is expressed properly. In this publication, we report the primary hits resulting of the MLSMR 100K library screen designed to identify modifiers of cytoskeleton integrity. (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pozo M, de Nicolas R, Egido J, Gonzalez-Cabrero J. "Simvastatin inhibits the migration and adhesion of monocytic cells and disorganizes the cytoskeleton of activated endothelial cells". Eur J Pharmacol. 2006 Oct 24;548(1-3):53-63. (4) Hordijk PL. "Endothelial signalling events during leukocyte transmigration". FEBS J. 2006 Oct;273(19):4408-15 Key words: endothelial cells; Tumor necrosis factor alpha; cytoskeleton; F-actin filaments; Phalloidin; VCAM-1; cell adhesion; high content; microscopy, Columbia University

 

 

    Primary HTS assay for chemical potentiators of IL-1B stimulated NFkB nuclear translocation [Primary Screening]Primary HTS assay for chemical potentiators of IL-1B stimulated NFkB nuclear translocation [Primary Screening]

PubChemPubChem

 

        Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage (Pober, 2002). This assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB (Senftleben, et al., 2002). Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. Although each inflammatory disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state (Aggarwal, 2003). A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response (Pober, 2002; Aggarwal, 2003). Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events (Li et al., 2002)...Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage (Pober, 2002). This assay detects an early event in this process, the nuclear translocation of the transcription factor, NFkappaB (Senftleben, et al., 2002). Chronic inflammatory disease is believed to pose a tremendous medical burden in the developed world, not only in terms of patient suffering but also in the cost of treatment and loss of worker productivity. Although each inflammatory disease has unique aspects regarding the affected tissues and the clinical symptoms, they all share some common biological mechanisms for the establishment and maintenance of the disease state (Aggarwal, 2003). A major control point in the initiation of the inflammatory process is at the level of the endothelial cells lining the blood vessels in the affected tissue. Endothelial cells are of critical importance in detecting the earliest signals broadcast from the damaged tissue and amplifying this signal by attracting and transiently immobilizing lymphocytes. This ultimately allows the responding lymphocytes to transmigrate the vessel wall and invade the damaged tissue to produce a fully fledged inflammatory response (Pober, 2002; Aggarwal, 2003). Some of the major signaling molecules that act on the endothelial cell include cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin 1 (IL-1) and CD40L. Bacterial lipopolysaccharide (LPS) is also a known activator of the inflammatory response. In each case, the signaling molecule binding to an independent receptor on the surface of the endothelial cell activates a cascade of internal signaling events (Li et al., 2002)...

 

    Primary HTS assay for chemical potentiators of TNFalpha stimulated VCAM1 expression [Primary Screening]Primary HTS assay for chemical potentiators of TNFalpha stimulated VCAM1 expression [Primary Screening]

PubChemPubChem

 

        Cytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16. Keywords: Inflammation, endothelial cells, Tumor necrosis factor alpha, VCAM-1, VCAM1, cell adhesion, vascular cell adhesion molecule, cytokine, lymphocyte, vascular endothelium bioassay, biological signal transduction, high content, microscopy, Columbia UniversityCytokines such as TNF alpha and IL-1B activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkB transcription factor (1). This response induces the transcription of pro-inflammatory genes including, at late times, the cell adhesion molecule, VCAM-1 (Vascular Cell Adhesion Molecule - 1) (2). The resulting cell surface expression of VCAM-1 upon stimulation with cytokines in primary human umbilical vein cells (HUVEC cells) provides a read-out to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli (3). VCAM-1 expression was evaluated in a cell-based high-content primary screen; compounds affecting VCAM-1 expression were identified by measuring the relative number of cytoplasmic fluorescent grains resulting from VCAM-1 immunostaining. VCAM-1 HCS was developed and screened at the Columbia University Molecular Screening Center as part of the Molecular Screening Center Network (MLSCN). Grant Number: 1R03MH076343-01; Assay Provider: Thomas Mayer (1) Li, X. and G. R. Stark (2002). "NFkappaB-dependent signaling pathways." Exp Hematol 30(4): 285-96 (2) Zhou, Z., M. C. Connell, et al. (2007). "TNFR1-induced NF-kappaB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells." Cell Signal 19(6): 1238-48 (3) Pober, J. S. (2002). "Endothelial activation: intracellular signaling pathways." Arthritis Res 4 Suppl 3: S109-16. Keywords: Inflammation, endothelial cells, Tumor necrosis factor alpha, VCAM-1, VCAM1, cell adhesion, vascular cell adhesion molecule, cytokine, lymphocyte, vascular endothelium bioassay, biological signal transduction, high content, microscopy, Columbia University

 

 

    Primary HTS assay to asses cytotoxicity for IL-1B stimulated NFkB expression.Primary HTS assay to asses cytotoxicity for IL-1B stimulated NFkB expression.

PubChemPubChem

 

         This primary HTS screen has been developed to identify chemical compounds that potentiate NFkappaB translocation in primary human umbilical vein endothelial cells (HUVEC) after cytokine stimulation. The identification of molecules that would either potentiate cytokine stimulation or activate the nuclear translocation of NFKappaB will help with the understanding of the underlying cellular processes and signaling pathways implicated in endothelial cell stimulation in inflammatory disease. We have previously reported three high-content, cell based assays that have enable us to identify inhibitors and potentiators of IL-1B stimulated NFkB expression (AID 796 and 819). Because of the high content nature of the screening campaign, we were also able to assess the number of remaining cells after treatment with the compound library. These data give us an interesting preliminary insight of the level of toxic compounds in the library. In this publication, we report the primary hits resulting of the MLSMR 100K library screen design to identify potential toxic compounds.  This primary HTS screen has been developed to identify chemical compounds that potentiate NFkappaB translocation in primary human umbilical vein endothelial cells (HUVEC) after cytokine stimulation. The identification of molecules that would either potentiate cytokine stimulation or activate the nuclear translocation of NFKappaB will help with the understanding of the underlying cellular processes and signaling pathways implicated in endothelial cell stimulation in inflammatory disease. We have previously reported three high-content, cell based assays that have enable us to identify inhibitors and potentiators of IL-1B stimulated NFkB expression (AID 796 and 819). Because of the high content nature of the screening campaign, we were also able to assess the number of remaining cells after treatment with the compound library. These data give us an interesting preliminary insight of the level of toxic compounds in the library. In this publication, we report the primary hits resulting of the MLSMR 100K library screen design to identify potential toxic compounds.

 

 

 

    Primary HTS for Clearance of Mutant Huntintin Protein. [Primary Screening]Primary HTS for Clearance of Mutant Huntintin Protein. [Primary Screening]

PubChemPubChem

 

        The neurodegenerative disorder Huntington's Disease is caused by a polyglutamine expansion mutation in the HD gene that leads to a complex neurological disorder involving deterioration of psychiatric, cognitive and motoric function. Under nonpathogenic conditions, the N-terminus of the protein huntingtin (htt) presents stretch of glutamines(Q) that range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the pathogenic outcome. Abolishing mutant htt expression in an inducible mouse model of Huntington's led to complete recovery of symptomatic mice (Yamamoto et al. 2000), and this reversal was tightly dependent on clearance of the mutant protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell-based assay was developed to analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). The basis of this assay is a stable cell line that expresses in an inducible manner the N-terminus of huntingtin protein with 103 glutamines fused to monomeric CFP at the C-terminus. The end point of this assay measures the presence/absence of fluorescent intracellular inclusions. This readout allows for the use of high throughput confocal microscopy to examine the state of the expressed mutant protein in live or fixed cells (Yamamoto et al. 2006), and therefore screening of small molecules libraries can be performed...The neurodegenerative disorder Huntington's Disease is caused by a polyglutamine expansion mutation in the HD gene that leads to a complex neurological disorder involving deterioration of psychiatric, cognitive and motoric function. Under nonpathogenic conditions, the N-terminus of the protein huntingtin (htt) presents stretch of glutamines(Q) that range from 2 to 34 repeats, while greater than 37 repeats invariably leads to the pathogenic outcome. Abolishing mutant htt expression in an inducible mouse model of Huntington's led to complete recovery of symptomatic mice (Yamamoto et al. 2000), and this reversal was tightly dependent on clearance of the mutant protein. Can the elimination of the accumulated proteins be a means to bring about recovery in HD? A fluorescent cell-based assay was developed to analyze aggregation and clearance of mutant huntingtin protein (Yamamoto et al. 2006). The basis of this assay is a stable cell line that expresses in an inducible manner the N-terminus of huntingtin protein with 103 glutamines fused to monomeric CFP at the C-terminus. The end point of this assay measures the presence/absence of fluorescent intracellular inclusions. This readout allows for the use of high throughput confocal microscopy to examine the state of the expressed mutant protein in live or fixed cells (Yamamoto et al. 2006), and therefore screening of small molecules libraries can be performed...

 

    Primary screen for compounds that activate Alzheimer's amyloid precursor [Primary Screening]Primary screen for compounds that activate Alzheimer's amyloid precursor [Primary Screening]

PubChemPubChem

 

        Novel reagents that inhibit the Amyloid Precursor Protein (APP) translation and subsequently the Amyloid beta protein production, that is elevated in Alzheimer's disease, could lead to the discovery of therapeutic drugs for the disease. MLSMR 160K library was screened for small molecules that suppress the Amyloid Precursor Protein (APP) translation by binding to the 5'Untranslated Region of the APP mRNA. It utilizes a stable neuroblastoma SH-SY5Y transfectants that express Luciferase under the translational control of 146 nucleotides from the 5'UTR of APP mRNA. It had been shown that this region forms a stem loop which is, in turn, a significant regulator of the APP levels in the brain (1, 2). The effects of compounds addition on APP levels is measured by quantification of luciferase expression levels with a bioluminescence assay system. The assay was previously utilized by Jack Rogers for HTS screen of a 110,000 compounds library from the Laboratory for Drug Discovery on Neurodegeneration (LDDN)(3). (1) Werstuck G, Green MR. Science. 1998 Oct 9;282(5387):296-8. Controlling gene expression in living cells through small molecule-RNA interactions. (2)Lahiri DK, Ge YW, Maloney B. FASEB J. 2005 Apr;19(6):653-5. Epub 2005 Feb 9. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. (3) Bandyopadhyay S, Ni J, Ruggiero A, Walshe K, Rogers MS, Chattopadhyay N, Glicksman MA, Rogers JT. J Biomol Screen, 2006 Aug;11(5):469-80. Epub 2006 Apr 28. A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA. Keywords: Alzheimer's disease, APP mRNA, HTS, luciferase, bioluminescence, RNA binding small molecules, Columbia UniversityNovel reagents that inhibit the Amyloid Precursor Protein (APP) translation and subsequently the Amyloid beta protein production, that is elevated in Alzheimer's disease, could lead to the discovery of therapeutic drugs for the disease. MLSMR 160K library was screened for small molecules that suppress the Amyloid Precursor Protein (APP) translation by binding to the 5'Untranslated Region of the APP mRNA. It utilizes a stable neuroblastoma SH-SY5Y transfectants that express Luciferase under the translational control of 146 nucleotides from the 5'UTR of APP mRNA. It had been shown that this region forms a stem loop which is, in turn, a significant regulator of the APP levels in the brain (1, 2). The effects of compounds addition on APP levels is measured by quantification of luciferase expression levels with a bioluminescence assay system. The assay was previously utilized by Jack Rogers for HTS screen of a 110,000 compounds library from the Laboratory for Drug Discovery on Neurodegeneration (LDDN)(3). (1) Werstuck G, Green MR. Science. 1998 Oct 9;282(5387):296-8. Controlling gene expression in living cells through small molecule-RNA interactions. (2)Lahiri DK, Ge YW, Maloney B. FASEB J. 2005 Apr;19(6):653-5. Epub 2005 Feb 9. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. (3) Bandyopadhyay S, Ni J, Ruggiero A, Walshe K, Rogers MS, Chattopadhyay N, Glicksman MA, Rogers JT. J Biomol Screen, 2006 Aug;11(5):469-80. Epub 2006 Apr 28. A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA. Keywords: Alzheimer's disease, APP mRNA, HTS, luciferase, bioluminescence, RNA binding small molecules, Columbia University

 

    Primary screen for compounds that activate Insulin promoter activity in TRM-6 cells [Primary Screening]Primary screen for compounds that activate Insulin promoter activity in TRM-6 cells [Primary Screening]

PubChemPubChem

 

        The assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity (3, 4); thus, the cells express substantial levels of endogenous insulin mRNA although less than produced by a healthy pancreatic Beta-cell. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature Beta-cells. (1) Ohneda K, Ee H, German M. Semin Cell Dev Biol. 2000 Aug;11(4):227-33. Regulation of insulin gene transcription. (2) Wang S, Beattie GM, Mally MI, Lopez AD, Hayek A, Levine F. Transplant Proc. 1997 Jun;29(4):2219. Analysis of a human fetal pancreatic islet cell line. (3)Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F. Dev Dyn. 2005 Jul;233(3):946-53. NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor. (4) Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F. PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol. 2000 Jun;14(6):814-22. Keywords: Insulin, Diabetes, reporter construct, GFP, inducible expression, high content, microscopy, Columbia UniversityThe assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity (3, 4); thus, the cells express substantial levels of endogenous insulin mRNA although less than produced by a healthy pancreatic Beta-cell. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature Beta-cells. (1) Ohneda K, Ee H, German M. Semin Cell Dev Biol. 2000 Aug;11(4):227-33. Regulation of insulin gene transcription. (2) Wang S, Beattie GM, Mally MI, Lopez AD, Hayek A, Levine F. Transplant Proc. 1997 Jun;29(4):2219. Analysis of a human fetal pancreatic islet cell line. (3)Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F. Dev Dyn. 2005 Jul;233(3):946-53. NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor. (4) Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F. PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol. 2000 Jun;14(6):814-22. Keywords: Insulin, Diabetes, reporter construct, GFP, inducible expression, high content, microscopy, Columbia University

 

    Primary screen for compounds that inhibit Alzheimer's amyloid precursor protein (APP) translation [Primary Screening]Primary screen for compounds that inhibit Alzheimer's amyloid precursor protein (APP) translation [Primary Screening]

PubChemPubChem

 

        Novel reagents that inhibit the Amyloid Precursor Protein (APP) translation and subsequently the Amyloid beta protein production, that is elevated in Alzheimer's disease, could lead to the discovery of therapeutic drugs for the disease. MLSMR 160K library was screened for small molecules that suppress the Amyloid Precursor Protein (APP) translation by binding to the 5'Untranslated Region of the APP mRNA. It utilizes a stable neuroblastoma SH-SY5Y transfectants that express Luciferase under the translational control of 146 nucleotides from the 5'UTR of APP mRNA. It had been shown that this region forms a stem loop which is, in turn, a significant regulator of the APP levels in the brain (1, 2). The effects of compounds addition on APP levels is measured by quantification of luciferase expression levels with a bioluminescence assay system. The assay was previously utilized by Jack Rogers for HTS screen of a 110,000 compounds library from the Laboratory for Drug Discovery on Neurodegeneration (LDDN)(3). (1) Werstuck G, Green MR. Science. 1998 Oct 9;282(5387):296-8. Controlling gene expression in living cells through small molecule-RNA interactions. (2)Lahiri DK, Ge YW, Maloney B. FASEB J. 2005 Apr;19(6):653-5. Epub 2005 Feb 9. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. (3) Bandyopadhyay S, Ni J, Ruggiero A, Walshe K, Rogers MS, Chattopadhyay N, Glicksman MA, Rogers JT. J Biomol Screen, 2006 Aug;11(5):469-80. Epub 2006 Apr 28. A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA. Keywords: Alzheimer's disease, APP mRNA, HTS, luciferase, bioluminescence, RNA binding small molecules, Columbia UniversityNovel reagents that inhibit the Amyloid Precursor Protein (APP) translation and subsequently the Amyloid beta protein production, that is elevated in Alzheimer's disease, could lead to the discovery of therapeutic drugs for the disease. MLSMR 160K library was screened for small molecules that suppress the Amyloid Precursor Protein (APP) translation by binding to the 5'Untranslated Region of the APP mRNA. It utilizes a stable neuroblastoma SH-SY5Y transfectants that express Luciferase under the translational control of 146 nucleotides from the 5'UTR of APP mRNA. It had been shown that this region forms a stem loop which is, in turn, a significant regulator of the APP levels in the brain (1, 2). The effects of compounds addition on APP levels is measured by quantification of luciferase expression levels with a bioluminescence assay system. The assay was previously utilized by Jack Rogers for HTS screen of a 110,000 compounds library from the Laboratory for Drug Discovery on Neurodegeneration (LDDN)(3). (1) Werstuck G, Green MR. Science. 1998 Oct 9;282(5387):296-8. Controlling gene expression in living cells through small molecule-RNA interactions. (2)Lahiri DK, Ge YW, Maloney B. FASEB J. 2005 Apr;19(6):653-5. Epub 2005 Feb 9. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. (3) Bandyopadhyay S, Ni J, Ruggiero A, Walshe K, Rogers MS, Chattopadhyay N, Glicksman MA, Rogers JT. J Biomol Screen, 2006 Aug;11(5):469-80. Epub 2006 Apr 28. A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA. Keywords: Alzheimer's disease, APP mRNA, HTS, luciferase, bioluminescence, RNA binding small molecules, Columbia University

 

    Primary screen for compounds that inhibit Insulin promoter activity in TRM-6 cells [Primary Screening]Primary screen for compounds that inhibit Insulin promoter activity in TRM-6 cells [Primary Screening]

PubChemPubChem

 

        The assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity (3, 4); thus, the cells express substantial levels of endogenous insulin mRNA although less than produced by a healthy pancreatic Beta-cell. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature Beta-cells. (1) Ohneda K, Ee H, German M. Semin Cell Dev Biol. 2000 Aug;11(4):227-33. Regulation of insulin gene transcription. (2) Wang S, Beattie GM, Mally MI, Lopez AD, Hayek A, Levine F. Transplant Proc. 1997 Jun;29(4):2219. Analysis of a human fetal pancreatic islet cell line. (3)Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F. Dev Dyn. 2005 Jul;233(3):946-53. NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor. (4) Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F. PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol. 2000 Jun;14(6):814-22. Keywords: Insulin, Diabetes, reporter construct, GFP, inducible expression, high content, microscopy, Columbia UniversityThe assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity (3, 4); thus, the cells express substantial levels of endogenous insulin mRNA although less than produced by a healthy pancreatic Beta-cell. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature Beta-cells. (1) Ohneda K, Ee H, German M. Semin Cell Dev Biol. 2000 Aug;11(4):227-33. Regulation of insulin gene transcription. (2) Wang S, Beattie GM, Mally MI, Lopez AD, Hayek A, Levine F. Transplant Proc. 1997 Jun;29(4):2219. Analysis of a human fetal pancreatic islet cell line. (3)Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F. Dev Dyn. 2005 Jul;233(3):946-53. NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor. (4) Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F. PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol. 2000 Jun;14(6):814-22. Keywords: Insulin, Diabetes, reporter construct, GFP, inducible expression, high content, microscopy, Columbia University

 

    TNFalpha Induced E-Selectin Expression - Confirmatory screen [Confirmatory]TNFalpha Induced E-Selectin Expression - Confirmatory screen [Confirmatory]

PubChemPubChem

 

        ...Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor. This response induces the transcription of pro-inflammatory genes (Li and Stark, 2002; Pober, 2002), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli. In this publication we report the confirmatory screening results of AID 487, TNFalpha Induced E-Selectin Expression - Primary screen......Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor. This response induces the transcription of pro-inflammatory genes (Li and Stark, 2002; Pober, 2002), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli. In this publication we report the confirmatory screening results of AID 487, TNFalpha Induced E-Selectin Expression - Primary screen...

 

 

    TNFalpha Induced E-Selectin Expression - Primary screen [Primary Screening]TNFalpha Induced E-Selectin Expression - Primary screen [Primary Screening]

PubChemPubChem

 

        Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor. This response induces the transcription of pro-inflammatory genes (Li and Stark, 2002; Pober, 2002), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli. TNFalpha Induced E-Selectin Expression. HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076509-01; Assay: TNFalpha Induced E-Selectin, Assay Provider Dr. Thomas Mayer, Department of Physiology and Cellular Biophysics at Columbia University. Li, X. and Stark, G.R. (2002). NFkappaB-dependent signaling pathways. Exp Hematol 30 285-96. Pober, J.S. (2002). Endothelial activation: intracellular signaling pathways. Arthritis Res 4 S109-16Inflammatory disease requires that endothelial cells detect and amplify a pro-inflammatory signal. This results in the adherence, activation, and ultimately transmigration of lymphocytes at the site of damage. Drugs that block this process would significantly alleviate the symptoms of inflammation. Our assay detects an early event in this process, the expression of E-selectin on the surface of endothelial cells, which is essential for lymphocyte adherence. Assay Principle. Cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1) activate a pro-inflammatory response in endothelial cells by nuclear translocation of the NFkappaB transcription factor. This response induces the transcription of pro-inflammatory genes (Li and Stark, 2002; Pober, 2002), including E-selectin. Cell surface expression of E-selectin upon stimulation with cytokines in primary human umbilical vein cells (HUVEC) provides a readout to study the effects of bioactive compounds on the responsiveness of endothelium to pro-inflammatory stimuli. TNFalpha Induced E-Selectin Expression. HCS/HTS was developed and run at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: 1R03MH076509-01; Assay: TNFalpha Induced E-Selectin, Assay Provider Dr. Thomas Mayer, Department of Physiology and Cellular Biophysics at Columbia University. Li, X. and Stark, G.R. (2002). NFkappaB-dependent signaling pathways. Exp Hematol 30 285-96. Pober, J.S. (2002). Endothelial activation: intracellular signaling pathways. Arthritis Res 4 S109-16

 

 

    sEH_DR_Inh_Infinite200_Fluorescence_01072008 [Confirmatory]sEH_DR_Inh_Infinite200_Fluorescence_01072008 [Confirmatory]

PubChemPubChem

 

        Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. While these inhibitors are scientifically useful, the low solubility and relatively fast metabolism of the first generation inhibitors makes them less than therapeutically efficient, underlying the need for novel inhibitor structures. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.Hypertension and vascular inflammation are associated with cardiovascular diseases, the primary cause of death in our society. Because a large proportion of patients are not responding to current therapies, the next generation of drugs will not only need to reduce blood pressure but also treat vascular and renal inflammation as well as reduce smooth muscle cell proliferation, which in turn should also reduce hypertension related organ damage. Using inhibitors developed in the Hammock laboratory, it was shown that the inhibition of soluble epoxide hydrolase (sEH) has therapeutic application in the treatment of hypertension and several inflammatory diseases. While these inhibitors are scientifically useful, the low solubility and relatively fast metabolism of the first generation inhibitors makes them less than therapeutically efficient, underlying the need for novel inhibitor structures. Assay Principle. A new fluorescent assay has been developed for sEH that is adequate for high throughput screening, using the novel substrate (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl) methyl ester (PHOME). The assay is based on the hydrolysis of the epoxide moiety of the substrate by sEH, followed by intramolecular cyclization, and resulting in the release of a cyanohydrin. Under basic conditions, the cyanohydrin rapidly decomposes into a cyanide ion as well as the highly fluorescent 6-methoxy-2-naphthaldehyde, which can be detected with a fluorescence plate reader with excitation at 320 nm and emission at 460 nm. sEH screening was performed at the Columbia University Molecular Screening Center as part of the Molecular Library Screening Center Network (MLSCN). Grant number: X01 MH078954-01; Assay: Novel sEH inhibitors for the therapeutic treatment of hypertension and inflammation, Assay provider: Dr. Bruce D. Hammock, UC, Davis, CA.