Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells.

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

MetaMiner (CF): a disease-oriented bioinformatics analysis environment.

MetaMiner (CF) is a data analysis platform for a broad range of CF researchers including wet lab biologists, bioinformaticians, clinicians, and chemists. To understand disease mechanisms and gain insight into complex biological actions, analysis of even simple gene interactions often requires integration of a variety of separate data resources such as literature, 3D molecular models, metabolic pathways, ontologies, small molecules, and drugs. Large-scale data sets from high-throughput screening assays, microarrays, and other data intensive procedures present an even greater challenge in data handling and analysis which now requires interdisciplinary teams of scientists with strikingly diverse backgrounds including computer scientists, statisticians, biologists, and clinicians. To address the issues raised by the complexity of analysis and resource limitations of many research laboratories, MetaMiner (CF) was developed by GeneGo under direction and funding of Cystic Fibrosis Foundation Therapeutics. The platform was designed to provide the CF community with a single tool for analyzing experimental data in a disease-centered environment. To that end, the most important biological and chemical experimental data available today in cystic fibrosis research have been assembled and integrated with data analysis and visualization tools to highlight the key pathways leading to and perturbed by the disease. GeneGo developers assembled and edited CF-specific content and designed the disease-specific interface under the guidance and review of a team of leading cystic fibrosis experts. Updates and revisions will be processed quarterly under the direction of the CF Foundation Therapeutics.

A pipeline of therapies for cystic fibrosis.

Therapeutics development for cystic fibrosis (CF) involves a coordinated effort among many groups, including individuals with CF and their caregivers, clinical research teams, and those in academia and industry who have discovered and developed the therapeutic strategies. In the United States, the Cystic Fibrosis Foundation (CFF) has devoted over $875 million to facilitate and coordinate this process since 1986, resulting in the clinical development and/or assessment of ~50 drug candidates during that time. The more than 30 compounds currently in the pipeline of Foundation-funded therapeutics are used as a platform to discuss why and how therapeutic strategies are brought into clinical development. Consideration is also given to the funding, management, and infrastructure necessary and practical to support the progression of drug candidates and the availability of therapeutics for use by individuals with CF. The importance of the clinical trial process and relevant outcome measures to assess the efficacy of drug candidates is also discussed. Finally, the potential impact of the pipeline for individuals with CF is summarized.

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A ß loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.

Serum metabolomics indicate altered cellular energy metabolism in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a multi-system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis. AIM: We hypothesized that metabolomics using serum from children would differentiate CF from non-CF lung disease subjects and would provide insight into metabolism in CF. METHODS: Serum collected from children with CF (n = 31) and 31 age and gender matched children with other lung diseases was used for metabolomic profiling by gas- and liquid-chromatography. Relative concentration of metabolites was compared between the groups using partial least square discriminant analyses (PLS-DA) and linear modeling. RESULTS: A clear separation of the two groups was seen in PLS-DA. Linear model found that among the 459 detected metabolites 92 differed between CF and non-CF. These included known biochemicals in lipid metabolism, oxidants, and markers consistent with abnormalities in bile acid processing. Bacterial metabolites were identified and differed between the groups indicating intestinal dysbiosis in CF. As a novel finding several pathways were markedly different in CF, which jointly point towards decreased activity in the ß-oxidation of fatty acids. These pathways include low ketone bodies, low medium chain carnitines, elevated di-carboxylic acids and decreased 2-hydroxybutyrate from amino acid metabolism in CF compared to non-CF. CONCLUSION: Serum metabolomics discriminated CF from non-CF and show altered cellular energy metabolism in CF potentially reflecting mitochondrial dysfunction. Future studies are indicated to examine their relation to the underlying CF defect and their use as biomarkers for disease severity or for cystic fibrosis transmembrane regulator (CFTR) function in an era of CFTR modifying drugs.

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis.

The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.