A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate.

Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure. As more drugs are being sought, it will be critical to develop assays that accurately determine cGMP levels. Here, we present cGMP Lumit, a sensitive and specific bioluminescent assay to detect cGMP. We demonstrate the utility of the detection system in enzyme assays, cell-based assays, and high-throughput screening formats. It is anticipated that this assay will be of significant value to aid in further understanding the role of cGMP in physiology and support further drug discovery efforts toward the treatment of human disease.

Monitoring PROTAC interactions in biochemical assays using Lumit immunoassays.

The discovery of new PROTAC molecules is dependent on robust and high-throughput assays to measure PROTAC-protein interactions and ternary complex formation. Here we present the optimization and execution of Lumit Immunoassays to measure PROTAC binding and ternary complex formation in a biochemical format. We demonstrate how Lumit can be used to rank order affinities of small molecules and PROTACs to BRD4(BD1, BD2) and how to measure PROTAC-mediated ternary complex formation of BRD4(BD1, BD2) and E3 Ligase VHL. Results from both biochemical assays correlate with live and lytic cell assays, indicating that Lumit Immunoassays can be used as a high-throughput compatible screening methodology to test new small molecules.

Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.

Role of miR-214 in regulation of ß-catenin and the malignant phenotype of melanoma.

Wnt/ß-catenin signaling plays an important role in melanocyte biology, especially in the early stages of melanocyte transformation and melanomagenesis. ß-catenin, encoded by the gene CTNNB1, is an intracellular signal transducer of Wnt signaling and activates transcription of genes important for cell proliferation and survival. Wnt/ß-catenin signaling is frequently activated in melanoma through oncogenic mutations of ß-catenin and elevated ß-catenin levels are positively correlated with melanoma aggressiveness. Molecular mechanisms that regulate ß-catenin expression in melanoma are not fully understood. MicroRNA-214 is known to function as a tumor suppressor by targeting ß-catenin in several types of cancer cells. Here, we investigated the regulation of ß-catenin by miR-214 and its role in melanoma. We show that ß-catenin mRNA levels are negatively correlated with miR-214 in melanoma. However, overexpression of miR-214 paradoxically increased ß-catenin protein levels and promoted malignant properties of melanoma cells including resistance to mitogen-activated protein kinase inhibitors (MAPKi). RNA-seq analysis revealed that melanoma cells predominantly express a ß-catenin mRNA isoform lacking miR-214 target site. Using matched miRNA and mRNA-seq and bioinformatics analysis, we identified novel miR-214 targets, ankyrin repeat domain 6 (ANKRD6) and C-terminal binding protein 1 (CTBP1), that are involved in negative regulation of Wnt signaling. Overexpression of miR-214 or knockdown of the novel miR-214 targets, ANKRD6 or CTBP1, increased melanoma cell proliferation, migration, and decreased sensitivity to MAPKi. Our data suggest that in melanoma cells ß-catenin is not regulated by miR-214 and the functions of miR-214 in melanoma are mediated partly by regulating proteins involved in attenuation of Wnt/ß-catenin signaling.

Melanoma Progression Inhibits Pluripotency and Differentiation of Melanoma-Derived iPSCs Produces Cells with Neural-like Mixed Dysplastic Phenotype.

Melanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced pluripotent stem cell (iPSCs) to investigate the relationship between cellular plasticity and melanoma progression and mitogen-activated protein kinase (MAPK) inhibitor resistance. We found that melanocyte reprogramming is prevented by the expression of oncogenic BRAF, and in melanoma cells harboring oncogenic BRAF and sensitive to MAPK inhibitors, reprogramming can be restored by inhibition of the activated oncogenic pathway. Our data also suggest that melanoma tumor progression acts as a barrier to reprogramming. Under conditions that promote melanocytic differentiation of fibroblast- and melanocyte-derived iPSCs, melanoma-derived iPSCs exhibited neural cell-like dysplasia and increased MAPK inhibitor resistance. These data suggest that iPSC-like reprogramming and drug resistance of differentiated cells can serve as a model to understand melanoma cell plasticity-dependent mechanisms in recurrence of aggressive drug-resistant melanoma.

Notch signaling activation induces cell death in MAPKi-resistant melanoma cells.

The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, migration or the sensitivity of BRAFV600E-melanoma cells to MAPK inhibition (MAPKi). We found that NOTCH1 is downregulated in melanoma cell lines with intrinsic and acquired resistance to MAPKi. Forced expression of NICD1, the active form of Notch1, caused apoptosis of the NOTCHlo , MAPKi-resistant cells, but not the NOTCHhi , MAPKi-sensitive melanoma cell lines. Whole transcriptome-sequencing analyses of NICD1-transduced MAPKi-sensitive and MAPKi-resistant cells revealed differential regulation of endothelin 1 (EDN1) by NICD1, that is, downregulation in MAPKi-resistant cells and upregulation in MAPKi-sensitive cells. Knockdown of EDN1 partially mimicked the effect of NICD1 on the survival of MAPKi-resistant cells. We show that the opposite regulation of EDN1 by Notch signaling is mediated by the differential regulation of c-JUN by NICD1. Our data show that MAPKi-resistant melanoma cells acquire vulnerability to Notch signaling activation and suggest that Notch-c-JUN-EDN1 axis is a potential therapeutic target in MAPKi-resistant melanoma.

Role of the flagellar basal-body protein, FlgC, in the binding of Salmonella enterica serovar Enteritidis to host cells.

Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.

Role of StdA in adhesion of Salmonella enterica serovar Enteritidis phage type 8 to host intestinal epithelial cells.

BACKGROUND: Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry. METHODS AND RESULTS: To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. CONCLUSIONS: These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.

Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.