Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

<p>Abstract</p> <p>Background</p> <p>Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.</p> <p>Methods</p> <p>We evaluated by short-circuit current (<it>I</it>_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.</p> <p>Results</p> <p>Neither hNE nor EPI-hNE4 treatments did modify <it>I</it>_scin control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased <it>I</it>_scby 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate <it>I</it>_sc, an effect which was blocked by EPI-hNE4.</p> <p>Conclusions</p> <p>These results indicate that hNE does activate ENaC and transepithelial Na⁺transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.</p

SMAC mimetic Debio 1143 synergizes with taxanes, topoisomerase inhibitors and bromodomain inhibitors to impede growth of lung adenocarcinoma cells

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance

Hepatitis C virus-mediated mitochondrial dysfunctions are prevented and rescued by cyclophilin inhibition

Alisporivir (Debio-025) is an analogue of cyclosporine A andrepresents the prototype of a new class of non-immunosuppressivecyclophilin inhibitors. In vitro and in vivo studies have shownthat alisporivir inhibits hepatitis C virus (HCV) replication andongoing clinical trials are exploring its therapeutic potential inpatients with chronic hepatitis C. Recent data suggest that theantiviral effect is mediated by inhibition of cyclophilin A whichis an essential host factor in the HCV life cycle. However, alisporiviralso inhibits mitochondrial permeability transition by bindingto cyclophilin D. As HCV is known to affect mitochondrialfunction, we explored the effect of alisporivir on HCV proteinmediatedmitochondrial dysfunction. By the use of inducible celllines, which allow to investigate the effects of HCV polyproteinexpression independent from viral RNA replication and whichrecapitulate the major alterations of mitochondrial bioenergeticsobserved in infectious cell systems, we show that alisporivir preventsHCV protein-mediated cytochrome c redistribution,decrease of cell respiration, collapse of mitochondrial membranepotential, overproduction of reactive oxygen species and mitochondrialcalcium overload. Strikingly, some of the HCV-mediatedmitochondrial dysfunctions could even be rescued byalisporivir. These observations provide new insights into thepathogenesis of HCV-related liver disease and reveal an additionalmechanism of action of alisporivir that is likely beneficialin the treatment of chronic hepatitis C

Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1

Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein-protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein-protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance