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.

Fig. 1

Modular organization of CFTR. CFTR is composed of two membrane spanning domains (MSD1 and MSD2), each comprised of six transmembrane segments connected by intracellular loops, as well as two nucleotide binding domains (NBD1 and NBD2) and a regulatory domain (R domain)

Fig. 2. Known CFTR modulators

Fig. 3. Generic virtual screening workflow

Fig. 4

NBD1:NBD2 interface binding site. a MOLCAD [36] surface showing the shape and size of the interface-site. NBD1 is shown in red; NBD2 is shown in green. b Binding site residues (stick representation) and binding site interaction regions generated with SiteMap. Yellow hydrophobic region, blue H-bond donor region; and red: H-bond acceptor region

Fig. 5

Comparison of the ICL4:NBD1 interaction network in the wt and F508del models of the CFTR intracellular domains. a Overlay of wt (cyan) and F508del (orange) models. The deletion of F508 and the resulting alteration of the loop conformation affect the interaction of NBD1 with ICL4. b The F508 network of aromatic interactions in the wt model. c Loss of aromatic interactions in the F508del model

Fig. 6

F508del binding site (NBD1:ICL4 interface). a MOLCAD surface of the interface site. b Binding site residues (stick representation) and binding site interaction regions generated with SiteMap. Yellow hydrophobic region, blue H-bond donor region, and red H-bond acceptor region

Fig. 7. In silico generation of the R1070W mutant in the F508del model suggests that a tryptophan in this position may restore interactions lost in the F508del mutant

Fig. 8

Multi-domain interface site. a MOLCAD surface of the interface site. b Binding site residues (stick representation) and binding site interaction regions generated with SiteMap. Yellow hydrophobic region, blue H-bond donor region; and red H-bond acceptor region

Fig. 9

Hits from the NBD1:2 interface site and selected analogs. Vendor catalog numbers in parentheses

Fig. 10

Hits from the F508del site and selected analogs. Vendor catalog numbers in parentheses

Fig. 11

Hits from the multi-domain interface site and selected analogs. Vendor catalog numbers in parentheses

Fig. 12

Band C Western blot analysis. HeLa cells were transiently transfected with wt CFTR or F508del-CFTR. One day after transfection cells were incubated at 30 °C with vehicle (DMSO), 20 μM EPX-106817 or 10 μM corrector 4a (C4) for 24 h after which cells were collected and whole cell lysates analyzed by Western blot. Compared to DMSO, EPX-106817 and C4 reproducibly increased the amount of Band C observed for both wt and F508del-CFTR transfected cells