Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel gene. CF mutations like deltaF508 cause both a mistrafficking of the protein and a gating defect. Other mutations, like G551D, cause only a gating defect. Our aim was to find chemical compounds able to stimulate the activity of CFTR mutant proteins by screening a library containing approved drugs. Two thousand compounds were tested on Fischer rat thyroid cells coexpressing deltaF508-CFTR and a halide-sensitive yellow fluorescent protein (YFP) after correction of the trafficking defect by low-temperature incubation. The YFP-based screening allowed the identification of the antihypertensive 1,4-dihydropyridines (DHPs) nifedipine, nicardipine, nimodipine, isradipine, nitrendipine, felodipine, and niguldipine as compounds able to activate deltaF508-CFTR. This effect was not derived from the inhibition of voltage-dependent Ca2+ channels, the pharmacological target of antihypertensive DHPs. Indeed, methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine-5-carboxylate (BayK-8644), a DHP that is effective as an activator of such channels, also stimulated CFTR activity. DHPs were also effective on the G551D-CFTR mutant by inducing a 16- to 45-fold increase of the CFTR Cl- currents. DHP activity was confirmed in airway epithelial cells from patients with CF. DHPs may represent a novel class of therapeutic agents able to correct the defect caused by a set of CF mutations.

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Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

c-Myc is a transcription modulator proto-oncogene. When overexpressed, it becomes an important contributor to the multi-hit process of malignant transformation. In two earlier papers in this journal (see refs. 19 , 20) we reported that retro-inverso peptidomimetic molecules inspired by the Helix-1 of c-Myc motif could be sequence-specific antiproliferative agents active in the low micromolar range. We also found that our peptides were not opening the four-alpha-helix Myc:Max bundle. Their antiproliferative activity in cancer cell lines needs the presence of side chains projecting outside of the bundle in the corresponding native H1 motif. This observation suggested interference with an external partner. In this study we investigated the INI1:Myc interaction. INI1 is a subunit of the SWI/SNF complex (component of the enhanceosome surrounding Myc:Max heterodimer). The INI1:Myc interaction was confirmed via pull down, ELISA, and fluorescence anisotropy assays. According to the length of INI1 fragments used, we calculated Kds ranging between 1.3x10(-6) and 4.8x10(-7) M. The three different techniques applied showed that the INI1:Myc interaction was also the target of our retro-inverso peptidomimetic molecules, which seem to bind specifically at INI1. A Myc binding, 21aa INI1 fragment (minimum interacting sequence), could inspire the synthesis of a new class of more selective c-Myc inhibitors.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl(-) channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508-CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508-CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508-CFTR-expressing construct. Moreover, Matrine induced ΔF508-CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508-CFTR complexes with the co-chaperone BAG3 that in addition appeared to co-localize with the mutated protein on the cell surface. These results shed new light on ΔF508-CFTR interactions with proteins of the chaperones/co-chaperones system and could be useful in strategies for future medical treatments for CF.