Computational chemistry as a means to identify improved E1 ubiquitin activating enzyme inhibitors.

<p>The PYR-41 binding site was modeled and suitability as a “druggable” target assessed by SiteMap [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163615#pone.0163615.ref026" target="_blank">26</a>]. Features of the predicted binding site (insert) describe a molecular target appropriate for drug optimization. The right panel shows docking of PYR-41 to the E-1 activating enzyme.</p

Vorinostat increases CFTR band B and augments ion channel activity in combination with lumacaftor.

<p>(A) Western blot of cell lysates treated with vorinostat from HeLa (left panel) or HEK 293 (right panel) cells. (B) Short circuit current measurements in CFBE monolayers treated with vorinostat, lumacaftor or a combination of the two compounds. After establishment of a Cl^- gradient and addition of amiloride (100 μM), monolayers were treated with forskolin (20 μM) and ivacaftor (VX-770, 10 μM), followed by administration of CFTR Inhibitor 172 (10 μM). N = 4 filters per condition. Error bars represent SEM with p values indicated.</p

Cellular processes that influence the F508del CFTR pool.

<p>Cellular processes that influence the F508del CFTR pool.</p

Fluorescence based total cellular CFTR measurement.

<p>(A) Cartoon depicts fluorescence based analysis in the microtiter plate assay. (B) Examples of positive hits from a screen of ~10,000 compounds using libraries including Pharmakon-1600, Enamine_30K, Enzo_FDA_01, and Selleck_FDA_01. Each value shown is >3 standard deviations above mean for all compounds tested.</p

CFTR expression in airway epithelial cells.

<p>(A, B) Levels of mRNA (A), and protein (B) in human bronchial epithelial cells expressing wild type (WT) or F508del CFTR. Cells were grown on plastic, as polarizing cell monolayers on permeable supports using an air/liquid (A/L) interface, or on the same supports with overlying liquid (liquid/liquid, L/L). Mature, fully glycosylated CFTR (Band C), and the ER localized glycoform (Band B) are depicted for air/liquid interface culture, which is most representative of physiologic conditions. (C, D) Steady state levels of CFTR mRNA (C) and protein (D) in primary airway epithelial cells grown at an air/liquid interface and isolated from non-CF or F508del homozygous individuals. mRNA levels were normalized against CFBE cells expressing WT CFTR (A) or non-CF human primary airway cells (C) in fold difference. N ≥ 3 samples per condition. Representative western blots in (B) and (D) are shown.</p

Verification of the hits using western analysis and transepithelial conductance measurements.

<p>(A) Western blots of cell lysates from HeLa expressing F508del CFTR treated for 24 hours with 10 μM primary hit compounds identified to increase total CFTR fluorescence. (B) Transepithelial conductance measurements after 24 hours incubation with selected compounds alone (10 μM), or combined with lumacaftor (3 μM). (C) Chemical structure of vorinostat.</p

The F508del CFTR Band B pool available for correction can be increased by blocking of ubiquitination.

<p>(A, B) Western blot of HeLa, CFBE, or HEK 293 lysates treated with PYR-41 (Selleck Chemicals). (C) Short circuit current (Isc) from CFBE monolayers treated with PYR-41, C18, or a combination of both compounds monitored by modified Ussing chamber analysis. N = 4 filters per condition. Error bars represent SEM with p values indicated. The change in Isc for each perturbation is shown. Values were normalized against combined Isc changes in the DMSO control, taken as 100%. (D) Effects of PYR-41 plus C18 in HEK 293 expressing the CFTR E92K mutation.</p

High content microscopy identifies compounds that increase the F508del CFTR pool.

<p>(A) Cartoon depicts subcellular regions of interest (ROI) for image analysis in a high throughput screen of small molecules. With this method, compounds can be identified that influence either plasma membrane or total cellular expression of F508del CFTR, with representative immunofluorescence image also shown. (B) Fluorescence intensity distribution within key regions of interest in vehicle treated samples within a high content screen of 2,000 compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository. (C) PubChem identifiers for a subset of small molecules that augment total cellular and/or surface expression of F508del CFTR. Each value shown is >3 standard deviations above mean fluorescence intensity for all compounds tested.</p