Targeting the NFAT:AP-1 transcriptional complex on DNA with a small-molecule inhibitor III
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE125445 | GEO | 2019/01/23
REPOSITORIES: GEO
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Project description:The transcription factor NFAT has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of AP-1, and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ~202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite ARRE-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other CsA-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.
2019-01-23 | GSE125443 | GEO
Project description:The transcription factor NFAT has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of AP-1, and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ~202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite ARRE-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other CsA-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.
2019-01-23 | GSE125444 | GEO
Project description:Fungal infections are a serious health problem in the clinic especially in the immunocompromised patient. Disease ranges from widespread superficial vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antimycotica are available, including azoles, polyenes, echinocandines and amphothericin B. Due to emerging resistance to standard therapy and significant side effects for some antimycotica there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we screened compound libraries, including combinatorial libraries as well as more than 30 000 pure compounds derived from organic synthesis for antimycotic activity. In total more than 100 000 compounds were screened using an innovative AS (activity-selectivity) assay analyzing both the antifungal activity and the compatability with human cells at the same time. One promising hit, a Benzimidazol-2-yl-alkylamine derivative, was developed in a series of lead compounds showing potent antifungal activity. ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12) showed the highest antifungal activity and best compatability with human cells in several cell culture models and against a number of different yeasts and clinical isolates. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol-pathway. In total, three biological replicates were performed. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed. Hybridization experiments included an untreated reference sample and a sample of cells treated with ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12). The array included one technical replicate of each probe.
2011-07-14 | E-GEOD-21622 | biostudies-arrayexpress
Project description:Fungal infections are a serious health problem in the clinic especially in the immunocompromised patient. Disease ranges from widespread superficial vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antimycotica are available, including azoles, polyenes, echinocandines and amphothericin B. Due to emerging resistance to standard therapy and significant side effects for some antimycotica there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we screened compound libraries, including combinatorial libraries as well as more than 30 000 pure compounds derived from organic synthesis for antimycotic activity. In total more than 100 000 compounds were screened using an innovative AS (activity-selectivity) assay analyzing both the antifungal activity and the compatability with human cells at the same time. One promising hit, a Benzimidazol-2-yl-alkylamine derivative, was developed in a series of lead compounds showing potent antifungal activity. ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12) showed the highest antifungal activity and best compatability with human cells in several cell culture models and against a number of different yeasts and clinical isolates. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol-pathway.
2011-07-15 | GSE21622 | GEO
Project description:Fungal infections are a serious health problem in clinics especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes and echinocandines. Due to emerging resistance to standard therapy and significant side effects and high costs for several antifungals.,there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we previously screened a compound library, using a new type of activity-selectivity (AS) assay analysing both the antifungal activity and the compatibility with human cells at the same time. One compound, ((S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole (EMC120B12)), showed high antifungal activity against several species of pathogenic yeasts including C. glabrata and C. krusei, species which are highly refractory to antifungals, especially to the commonly used azoles. Here we could show by transcriptional profiling and sterol analysis that the target of this new antifungal compound is the ergosterol pathway. The effects of EMC120B12 on sterol biosynthesis mimic those of fluconazole, strongly indicating that EMC120B12 also targets ERG11 like the azols. But not only the marker sterol 14 methylergosta 8,24(28) dien 3β,6α diol accumulated in C. krusei under EMC120B12 treatment, but also hitherto unknown related sterols. The novel sterols have a 3β,6α diol structure. Furthermore, this is the first time that a benzimidazole structure has been shown to result in a block of the sterol pathway by accumulating marker sterols connected to ERG11 inactivation. In total, three biological replicates were performed. All experiments were performed as dye swaps. Thus, in total 18 arrays have been hybridzed. Hybridization experiments included an untreated reference sample and a sample of cells treated with either ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12), Fluconazole or Nocodazole. The array included one technical replicate of each probe.
2015-06-30 | E-GEOD-64976 | biostudies-arrayexpress
Project description:Calcineurin/NFAT signaling pathway has been shown to play important roles in various tissues such as the immune system, cardiac muscle and neuron. Although recent studies have shown that the pathway is involved in the regulation of hair growth, the precise mechanism is still unclear. In this study, we examine the molecular mechanism which is regulated by calcineurin/NFAT pathway, using two specific inhibitors for the pathway. Transcriptional profiling of human keratinocyte cell line, PHK cells, comparing control untreated PHK cells with cyclosporin A (CsA)-treated or 11R-VIVIT-treated PHK cells. Goal was to identify the effects of NFAT inhibitors on PHK cell proliferation. Cyclosporine A is a chemical compound, which strongly inhibits calcineurin phosphatase activity cooperating with cyclophilin, resulting in inhibition of NFAT signaling pathway. 11R-VIVIT is a cell-permeable peptide, which specifically interacts with NFAT and inhibits its binding to calcineurin, resulting in competitive inhibition of NFAT signaling pathway. Three-condition experiment; untreated PHK cells, CsA-treated PHK cells and 11R-VIVIT-treated PHK cells. Supplementary files: Significantly up-regulated genes across 4 different comparisons.
2010-11-08 | E-GEOD-25175 | biostudies-arrayexpress
Project description:Calcineurin/NFAT signaling pathway has been shown to play important roles in various tissues such as the immune system, cardiac muscle and neuron. Although recent studies have shown that the pathway is involved in the regulation of hair growth, the precise mechanism is still unclear. In this study, we examine the molecular mechanism which is regulated by calcineurin/NFAT pathway, using two specific inhibitors for the pathway. Transcriptional profiling of human keratinocyte cell line, PHK cells, comparing control untreated PHK cells with cyclosporin A (CsA)-treated or 11R-VIVIT-treated PHK cells. Goal was to identify the effects of NFAT inhibitors on PHK cell proliferation. Cyclosporine A is a chemical compound, which strongly inhibits calcineurin phosphatase activity cooperating with cyclophilin, resulting in inhibition of NFAT signaling pathway. 11R-VIVIT is a cell-permeable peptide, which specifically interacts with NFAT and inhibits its binding to calcineurin, resulting in competitive inhibition of NFAT signaling pathway.
2010-11-08 | GSE25175 | GEO
Project description:Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (Mixed Lymphocyte Reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC inhibitor that potently inhibits human T cell functions. PBMC from healthy donors (n=4) were stimulated with anti-CD3/CD28 in the presence or absence of CRAC inhibitor, CsA or FK506 for 24 hrs
2013-01-31 | E-GEOD-43210 | biostudies-arrayexpress
Project description:We have used computational and experimental biology approaches to identify candidate mechanisms of action of a traditional Chinese medicine; Compound Kushen Injection (CKI), in a breast cancer cell line in which CKI has been shown to cause apoptosis. Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis in MDA-MB-231 cells. Our initial fractionation separated major from minor compounds, and showed that the major compounds accounted for little of the activity of CKI. By systematically perturbing the major compounds in CKI we found that removal of no single major compound could alter the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical compounds with respect to CKI activity. We then used RNA sequencing and transcriptome analysis to correlate compound removal with gene expression and phenotype data. We determined that many compounds in CKI are required for its effectiveness in triggering apoptosis but that significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.
2019-05-29 | GSE125743 | GEO
Project description:Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (Mixed Lymphocyte Reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC inhibitor that potently inhibits human T cell functions.
2013-01-31 | GSE43210 | GEO