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The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCcf-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR. Short-circuit current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.

Gene transfer to exocrine glands, including the major salivary glands, presents an attractive method to deliver proteins for therapeutic applications. Previous efforts using nonviral gene delivery to these glands have resulted in limited success. In this report, zinc and other divalent transitions were coadministered with plasmid DNA in an effort to improve nonviral salivary gland transfection efficiency. The inclusion of zinc into plasmid DNA solutions resulted in a 20-fold enhancement in transgene expression without noticeable inflammation compared to a solution of plasmid DNA only. This observed enhancement in transgene expression was dependent upon the DNA dose and correlated with the accumulation of plasmid DNA by salivary gland tissues.

The oral delivery of functional DNA to the gastrointestinal system would constitute a desirable, noninvasive method for potentially treating a variety of diseases. The digestive process, however, remains a formidable barrier. This dilemma may be addressed by using targeted liposomes both to protect the polynucleotide and to deliver the therapeutic DNA with high tissue specificity. The present study represents the initial steps toward developing a novel gene delivery system designed to interact with the enterohepatic receptors of the small intestine. Two cholic acid esters were synthetically modified at position C(3) to incorporate a DNA-binding domain. These novel compounds were evaluated for their ability to protect DNA from the nucleases found in gastrointestinal segments. Additionally, the compounds were screened as a component of a gene delivery vector. Formulations containing the new bile salt derivatives protected DNA from degradation for more than 2 h and were capable of transfecting cultured NIH 3T3 cells.

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, non-viral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable enhanced non-viral gene transfer in salivary glands and other tissues. Our efforts to identify and transcend these barriers have resulted in the discovery of a group of compounds that enhance non-viral, salivary gland gene transfer by four orders of magnitude relative to unformulated DNA. These data as well as data directed towards understanding the mechanism-of-action responsible for the observed enhancement in non-viral gene transfer will be presented.