The cystic fibrosis transmembrane conductance regulator (CFTR)1 is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common diseaseassociated mutant ΔF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na + /H + Exchanger-3 Regulatory Factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as ΔF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued ΔF508-CFTR in patients with cystic fibrosis.CFTR is a cAMP-activated, ATP-gated chloride channel. It plays a central role in maintaining fluid and ion homeostasis in epithelial tissues and is mutated in patients with cystic fibrosis (CF) (1). Although CFTR is subject to rapid endocytosis (2), this appears to be coupled with a highly efficient constitutive recycling mechanism (e.g refs. 3,4). As a result, mature CFTR exhibits a long functional half-life (5,6), requiring individual molecules to cycle through the endocytic pathway dozens or even hundreds of times. † This work was supported in part by grants from the Cystic Fibrosis Foundation (MADDEN06P0 and STANTO97R0) and the NIH (grants P20-RR018787 from the Institutional Development Award (IDeA) Program of the NCRR and R01-DK075309 from NIDDK). P.B. was supported by the Deutsche Forschungsgemeinschaft (DFG grant VO 885/3-1). 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PDZ, PSD-95, discs large, zonula occludens-1; CAL, CFTR-Associated Ligand; NHERF1, Na + /H + Exchanger-3 Regulatory Factor-1; NHERF2, Na + /H + Exchanger-3 Regulatory Factor-2; CF, cystic fibrosis; DTT, dithiothreitol; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; SPR, surface-plasmon resonance; ITC, isothermal titration calorimetry; FP, fluorescence polarizatio...

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators (“potentiators” and “correctors”), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called “stabilizers”) that rescue ΔF508-CFTR activity. To design the “stabilizers”, we extended our structural ensemble-based computational protein redesign algorithm to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

Cell-penetrating peptides (CPPs) are short peptides able to penetrate cell membranes and translocate different cargoes into cells. Although recently the topic of many research articles, to our best knowledge no single systematic study of CPPs has been carried out as yet, meaning information can only by gathered piece by piece from different sources. We therefore decided to start analytical screening of CPP specificity in cell lines. We used 22 different CPPs, which have all been published before, and present the first analytical screen in 4 selected cell lines (MDCK, HEK293, HeLa, and Cos-7). Furthermore, we examined the influence of different conditions, such as protease inhibitors, incubation conditions, endocytosis inhibitors, temperature, and cytotoxicity. We clearly demonstrate that the 22 CPPs can be classified into 3 groups based on their internalization properties, even after trypsinization. Moreover, we show that additional agents, which should increase cellular uptake or dissolve endosomal/lysosomal entrapped CPPs, only have low effects. Our intensive screening under standardized conditions provides the opportunity to compare cellular uptake of CPPs, an important step for the use of CPPs as peptidic vectors in the medical field.

Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids. This review focuses on CPP-delivery of several classes of oligonucleotides (ONs), namely antisense oligonucleotides, splice switching oligonucleotides (SSOs) and siRNAs. Two main strategies have been used to transport ONs with CPPs: covalent conjugation (which is more appropriate for charge-neutral ON analogues) and non-covalent complexation (which has been used for siRNA delivery essentially). Chemical synthesis, mechanisms of cellular internalization and various applications will be reviewed. A comprehensive coverage of the enormous amount of published data was not possible. Instead, emphasis has been put on strategies that have proven to be effective in animal models of important human diseases and on examples taken from the authors' own expertise.