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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).

C. elegans aex-3mutations cause pleiotropic behavioral defects that are suggestive of reduced synaptic transmission. aex-3mutations also show strong genetic interactions with mutations in unc-31and unc-64, two other genes implicated in synaptic transmission. Physiological and pharmacological studies indicate that aex-3defects are presynaptic. In aex-3mutants, the synaptic vesicle–associated RAB-3 protein aberrantly accumulates in neuronal cell bodies and is reduced in synapse-rich axons. This localization defect is specific to RAB-3, since other synaptic proteins are localized normally in aex-3mutants. aex-3encodes a 1409 amino acid protein with strong homology to DENN, a human protein of unknown function. In C. elegans, aex-3is expressed in all or nearly all neurons. These results suggest that AEX-3 is a novel regulator of presynaptic activity that interacts with RAB-3 to regulate synaptic vesicle release.

The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.

Prevailing drug discovery approaches focus on compounds with molecular selectivity, inhibiting disease-relevant targets over others in vitro. However in vivo, many such agents are not therapeutically selective, either because of undesirable activity at effective doses or because the biological system responds to compensate. In theory, drug combinations should permit increased control of such complex biology, but there is a common concern that therapeutic synergy will generally be mirrored by synergistic side-effects. Here we provide evidence, from 94,110 multi-dose combination experiments representing diverse disease areas and large scale flux balance simulations of inhibited bacterial metabolism, that multi-target synergies are more specific than single agent activities to particular cellular contexts. Using an anti-inflammatory combination, we show how multi-target synergy can achieve therapeutic selectivity in animals through differential target expression. Synergistic combinations can increase the number of selective therapies using the current pharmacopeia, and offer opportunities for more precise control of biological systems.

Rabphilin, a putative rab effector, interacts specifically with the GTP-bound form of the synaptic vesicle-associated protein rab3a. In this study, we define in vivo functions for rabphilin through the characterization of mutants that disrupt the Caenorhabditis elegans rabphilin homolog. The mutants do not display the general synaptic defects associated with rab3 lesions, as assayed at the pharmacological, physiological, and ultrastructural level. However, rabphilin mutants exhibit severe lethargy in the absence of mechanical stimulation. Furthermore, rabphilin mutations display strong synergistic interactions with hypomorphic lesions in the syntaxin, synaptosomal-associated protein of 25 kDa, and synaptobrevin soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) genes; double mutants were nonresponsive to mechanical stimulation. These synergistic interactions were independent of rab3 function and were not observed in rab3-SNARE double mutants. Our data reveal rab3-independent functions for rabphilin in the potentiation of SNARE function.

Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterizedCaenorhabditis elegans rab-3mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore,rab-3animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed inrab-3mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.