Cystic fibrosis (CF) is an inherited, life-threatening disease caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter-class protein and ion channel that transports ions across epithelial cell membranes. The most common mutation leads to the deletion of a single phenylalanine, and the resulting protein, F508del-CFTR, shows reduced trafficking to the membrane and defective channel gating. The ideal therapeutic approach would address both of these defects and restore channel function at the same time. We describe here the application of a combination high-throughput screening to search for synergistic modulators of F508del-CFTR. With the adapted Fischer rat thyroid-yellow fluorescent protein halide flux assay to the combination high-throughput screening platform, we identified many interesting single agents as CFTR modulators from a library of approved drugs and mechanistic probe compounds, and combinations that synergistically modulate F508del-CFTR channel function in Fischer rat thyroid cells, demonstrating the potential for combination therapeutics to address the defects that cause CF.

Cystic fibrosis is an inherited, life-threatening disease associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, F508del CFTR, is found in 90% of CF patients. The loss of a single amino acid (phenylalanine at position 508) results in malformed CFTR with defective trafficking to the plasma membrane and impaired channel function. A functional assay with cells expressing F508del CFTR has been previously described by others using genetically engineered halide-sensitive yellow fluorescent protein to screen for CFTR modulators. We adapted this yellow fluorescent protein assay to 384-well plate format with a high-throughput screening plate reader, and optimized the assay in terms of data quality, resolution, and throughput, with target-specific protocols. The optimized assay was validated with reference compounds from cystic fibrosis foundation therapeutics. On the basis of the Z-factor range (≥0.5) and the potential productivity, this assay is well suited for high-throughput screening. It was successfully used to screen for active single agent and synergistic combinations of single agent modulators of F508del CFTR from a library collection of current active pharmaceutical ingredients (supported by Cystic Fibrosis Foundation Therapeutics).

Protein homeostasis is established and preserved by a collection of pathways and cellular processes known as the Proteostasis Network (PN). Protein misfolding disorders can arise through a reduced capacity of the PN to appropriately correct the folding, trafficking and degradation of disease‐associated proteins. We target PN pathways using small molecule Proteostasis Regulators (PRs) to identify modulators that confer functional rescue of misfolded proteins. Specifically, we have applied our PN approach to the correction of mutant Cystic Fibrosis Transductance Regulator (CFTR), the causative defect in Cystic Fibrosis (CF). A single mutation, deletion of phenylalanine 508 (F508del), is present in ~87% of all CF patients. The F508del mutation causes defective folding and trafficking, as well as enhanced degradation of the resultant CFTR protein. We carried out a high‐throughput screen using a multiplex gene expression platform to identify PRs that modulate proprietary PN pathway reporters. PRs found to correct mutant CFTR F508del exert effects on one or more pathways within the PN, suggesting they may target different mechanisms important in the maturation of CFTR. We present a novel approach for identifying therapeutics whose mechanism of action involves modulation of the PN. Funded in part by the Cystic Fibrosis Foundation Therapeutics, Inc.

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The in vitro dissection of the life cycle of dendritic cells and of the dynamics of T cell activation has been instrumental to identify some of the critical steps that control the generation of effector and memory T cells. I will first discuss how recruitment of dendritic cells into inflamed tissues, their activation by pathogens and inflammatory cytokines and their migration to secondary lymphoid organs provide the optimal conditions for T cell priming and polarization.In particular I will show that different T cell responses (type 1, type 2 or unpolarized T cells) can be generated by modulating the state of activation of dendritic cells and the duration of the dendritic cell-T cell interaction. I will also discuss the differential roles played by 'central memory' and 'effector memory' T cells in secondary immune responses. References: Lanzavecchia, A., G. Iezzi, and A. Viola. 1999. From TCR engagement to T cell activation: a kinetic view of T cell behavior. Cell Lanzavecchia. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J. Exp. Med. 189821-829. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712. Sallusto, F., C.R. Mackay, and A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector and memory immune responses. Annu. Rev. Immunol. (2000) 18,593-620 287 From genome sequences to protein functions Box 951570, UCLA, Lor AEgefes CA 90095-1570 USANew computational methods have been developed for assigning protein functions and interactions to genome sequences. These methods make use of information from all fully sequenced genomes and are distinct from standard homology methods. They have been applied to assign functions for roughly half of the previously uncharacterized proteins of Saccharomyces cervisiae and Mycobacterium tuberculosis.The first method is called the phylogenetic profile method; it examines the correlated inheritance of proteins in different species. It is based on the assumption that proteins that evolve in a correlated fashion are likely to function together in a pathway or XNCturd complex. During evolution, such functionally linked proteins tend to be either all preserved or eliminated in a new species. This property of c o r r e k d evohnion is described by characterizing each protein by irr phylogenetic profile, a string that encodes the presence or absence of a protein in every fully squenced genome.Studies show that proteins having matching or similar phylogenetic profiles strongly tend to be functionally linked. Conversely, proteins having similar function tend to have similar phylogenetic profiles. This correlation permits us to assign functions to uncharacterized proteins that have phylogenetic profiles similar to proteins of known function.The second method examines correlated domains in proteins and its termed the Rosetta Stone method. It is based on the observation that some pairs of interac...