Ashvani K. Singh

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway.

Novel Pseudomonas aeruginosa Quorum-Sensing Inhibitors Identified in an Ultra-High-Throughput Screen

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C4), and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC50) of 30 nM. The second compound, V-06-018, had an IC50 of 10 μM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

Inhibitory effect of silibinin on ligand binding to erbB1 and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostate carcinoma cells

We recently showed the inhibitory effect of a flavonoid antioxidant, silymarin, on erbB1‐Shc activation in prostate cancer (PCA) DU145 cells. In the present study, we performed more detailed mechanistic and molecular modeling studies with pure silibinin to assess and define its effect on membrane signaling related to erbB1 activation in human PCA LNCaP and DU145 cells. Studies also were performed to establish the biologic responses toward extracellular signal‐regulated protein kinase 1/2 (ERK1/2) activation, cell growth, and DNA synthesis. Treatment of serum‐starved cells with various doses of silibinin for 2 h followed by 125I‐epidermal growth factor (EGF) showed 30–75% inhibition in ligand binding and 55–95% inhibition in its internalization in LNCaP cells and 20–64% and 12–27% inhibition in these two events in DU145 cells. Time‐response studies showed similar effects. In further studies, treatment of serum‐starved cultures with silibinin followed by EGF showed strong inhibitory effects on membrane and cytoplasmic signaling molecules. In the case of erbB1 activation, silibinin showed a 58–75% decrease in LNCaP and a 40–100% decrease in DU145 cells at 50, 75, and 100‐μg/mL doses. Inhibitory effects of silibinin also were evident on ERK1/2 activation (20–80% inhibition) in both cell lines. Treatment of serum‐starved cultures with silibinin resulted in 20–40% and 30–55% inhibition of LNCaP and DU145 cell growth, respectively, at similar doses after 1–3 d of treatment, and 10–50% cell death in both cell lines. Under 10% serum conditions, identical silibinin treatments resulted in 20–65% inhibition of cell growth in LNCaP and DU145 cells but did not cause any cell death. Similar doses of silibinin treatments for 24 h also resulted in 25–60%, 35–40%, and 36–50% inhibition of DNA synthesis when cells were cultured in 10% serum, totally serum starved, and serum starved plus stimulated with EGF, respectively. Molecular modeling of silibinin showed that it is a highly lipophilic compound, suggesting that it interacts with lipid‐rich plasma membrane, including binding with erbB1, thereby competing with the EGF‐erbB1 interaction. Because the ligand‐erbB1 autocrine‐loop is causally involved in advanced and androgen‐independent PCA, the observed effects of silibinin and its strong lipophilic nature could be useful in developing this agent for the prevention and therapy of PCA.

Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells

Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIalpha and RIIalpha. In addition, immunogold electron microscopy showed that RIIalpha labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIalpha was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Calpha was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (approximately 100 microA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.Chloride exit across the apical membranes of secretory epithelial cells is acutely regulated by the cAMP-mediated second messenger cascade. To better understand the regulation of transepithelial chloride secretion, we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms present in the human colonic epithelial cell line T84. Our results show that both type I and type II PKA are present in T84 cells. Immunoprecipitation of 8-azido-[32P]cAMP-labeled cell lysates revealed that the major regulatory subunits of PKA were RIα and RIIα. In addition, immunogold electron microscopy showed that RIIα labeling was found on membranes of the trans Golgi network and on apical plasma membrane. In contrast, RIα was randomly distributed throughout the cytoplasm, with no discernible membrane association. Northern blot analysis of T84 RNA revealed that Cα was the predominantly expressed catalytic subunit. Short-circuit current measurements were performed in the presence of combinations of site-selective cAMP analog pairs to preferentially activate either PKA type I or PKA type II in intact T84 cell monolayers. Maximal levels of chloride secretion (∼100 μA/cm2) were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase chloride secretion. Thus activation of either type I or type II PKA is able to maximally stimulate chloride secretion in T84 colonic epithelial cells.

Therapeutic approaches to CFTR dysfunction: from discovery to drug development

Cystic fibrosis (CF) mutations have complex effects on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. They disrupt its processing to and stability at the plasma membrane and function as an ATP-gated Cl- channel. Here, we review therapeutic strategies to overcome defective CFTR processing and stability. Because CF mutations have multiple impacts on the assembly of CFTR protein, combination therapy with several pharmacological chaperones is likely to be required to rescue mutant CFTR expression at the plasma membrane. Alternatively, proteostasis regulators, proteins which regulate the synthesis, intracellular transport and membrane stability of CFTR might be targeted to enhance the plasma membrane expression of mutant CFTR. Finally, we consider an innovative approach to bypass CFTR dysfunction in CF, the delivery of artificial anion transporters to CF epithelia to shuttle Cl- across the apical membrane. The identification of therapies or combinations of therapies, which rescue all CF mutations, is now a priority.

A new treatment for neonatal scours

Scours account for significant losses to the US swine industry every year. A common treatment for scours is the administration of broad-spectrum antibiotics, a practice with increasing unpopularity in the eyes of consumers. Currently, no treatment is available to reduce or eliminate the fluid losses associated with scours that is both inexpensive and easy to use. In the present study, a variety of prospective drugs were used to determine if a single compound might inhibit the effects of bacterial toxins in a laboratory setting. The results indicate that a new class of drugs, which we call DASUs, likely will prove useful for the treatment of watery diarrhea. Additional studies are underway to validate this conclusion.