Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

Abstract

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ∼tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

A Novel Approach to Recovery of Function of Mutant Proteins by Slowing Down Translation

“…To study the effect of translation inhibition on the functional surface expression of F508del-CFTR, we used FRT cells stably expressing recombinant F508del-CFTR. CFTR activity was measured by two independent methods, an Ussing chamber assay (25) and a conductance assay in a 24-well format (26), both of which measure CFTR-dependent transepithelial Cl Ϫ transport. The Ussing chamber assay was designed to measure this transport in the presence of a chloride ion gradient.…”

The Therapeutic Potential of Small-molecule Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…By contrast, Knapp et al 97 posited the imaginative idea of a single binding site that alters its conformation during the processing of F508del-CFTR and its delivery to the cell surface. Finally, Kalid et al 98 adopted in silico structure-based screening to search for CFTR correctors, potentiators and dual-acting small molecules. Based on analyses of homology modeling, three binding sites for small-molecules were identified on F508del-CFTR: (i) the NBD1:NBD2 interface, (ii) the NBD1:ICL4 cavity formed by deletion of F508 and (iii) the interface of NBD1 and NBD2 with ICL1, 2 and 4 ( Fig.…”

“…6). 98 From 100,000 chemicals screened in silico and 496 selected for functional testing, two CFTR corrector-potentiators were identified.…”

Ligand-based design, in silico ADME-Tox filtering, synthesis and biological evaluation to discover new soluble 1,4-DHP-based CFTR activators

“…Small-molecule therapy for CF-causing mutations is thought to have considerable promise [4] but to the best of our knowledge only one structure-based virtual screening strategy [5] and no ligand-based approach for the discovery of new CFTR modulators is reported in the literature (some QSAR and SAR studies were performed a posteriori after synthesis and biological testing of compounds [6][7][8][9]). …”

“…This approach could be extremely useful for CFTR potentiators because the lack of a high resolution 3D structure of the full length protein prevents a clear strategy for designing compounds that act by direct interaction with CFTR to be defined [5]). …”