Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). A large number of nearly 2000 reported mutations, including the premature termination codon (PTC) mutations, urgently require new and personalized medicines. We have developed cell-based assays for readthrough modulators of CFTR PTC mutations (or nonsense mutation suppressors), based on the trafficking and surface expression of CFTR. Approximately 85,000 compounds have been screened for two PTC mutations (Y122X and W1282X). The hit rates at the threshold of 50% greater than vehicle response are 2% and 1.4% for CFTR Y122X and CFTR W1282X, respectively. The overlap of the two hit sets at this stringent hit threshold is relatively small. Only ~28% of the hits from the W1282X screen were also hits in the Y122X screen. The overlap increases to ~50% if compounds are included that in the second screen achieve only a less stringent hit criterion, that is, horseradish peroxidase (HRP) activity greater than three standard deviations above the mean of the vehicle. Our data suggest that personalization may not need to address individual genotypes, but that patients with different CFTR PTC mutations could benefit from the same medicines.

The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B 4 , a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3؉ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123-155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468 -872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation.The ability of cells to attain differentiated stages in development and to alter their functional phenotype in inflammatory settings depends on lineage-specific and cytokine-dependent transcription factors that either activate or repress target genes. Distinct patterns of transcription factors are associated with the myeloid and lymphoid lineages in hematopoiesis (1) and with the induction of pro-inflammatory genes in host defense (2). Transcription factor activity is context-dependent; a repressor of one gene can activate a different gene associated with an opposing function or alternative cell lineage (3). This has implications for the coordinated induction and repression of sets of genes that are functionally related. ZEB-1 (␦EF1, Zfhx1a) and ZEB-2 (SIP1, Zfhx1b) are recently characterized two-handed zinc finger transcription factors (4, 5) that may function as both gene silencers and activators. ZEBs repress transcription of genes that include IL-2, CD4, and GATA-3 in lymphocytes (6 -8), whereas their role in myeloid cells has not been determined. The C-terminal binding protein (CtBP) 1 is a corepressor for ZEBs and other factors but does not bind DNA directly (9). Recent evidence suggests that ZEBs can activate the vitamin D 3 receptor gene in colon carcinoma cells (10).Cytochrome P450 4F3 (CYP4F3) functions to control i...