Targeting F508del-CFTR to develop rational new therapies for cystic fibrosis

Abstract

The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First, the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl(-) channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR. We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development. These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR.

Figure 1

Single-channel activity of wild-type and F508del-CFTR. Representative recordings of wild-type and F508del-CFTR Cl⁻ channels in excised inside-out membrane patches from C127 cells expressing recombinant CFTR. ATP (1 mmol/L) and PKA (75 nmol/L) were continuously present in the intracellular solution, voltage was clamped at -50 mV, and a large Cl⁻ concentration gradient was imposed across the membrane patch ([Cl⁻]_Ext=10 mmol/L; [Cl⁻]_Int=147 mmol/L). Dashed lines indicate where the channels are closed and downward deflections correspond to channel openings. Beneath each of the prolonged 20 s recordings, the last 1 s of the record is shown on an expanded scale. Other details are as described in Cai and Sheppard. Modified, with permission, from Cai and Sheppard.

Figure 2

The organization of the ATP-binding sites in CFTR. The simplified model shows the molecular architecture of ATP-binding site 1 (site 1) and ATP-binding site 2 (site 2) in an open CFTR Cl⁻ channel. Each ATP-binding site is formed by the Walker A and B motifs (labeled A and B, respectively) of one NBD and the LSGGQ motif of the other NBD. Site 2 contains a canonical LSGGQ motif, whereas site 1 contains a non-canonical LSGGQ motif (LSHGH). Site 2 also contains a catalytic base (E1371) at the distal end of the Walker B motif, but this residue is absent in site 1 (S573). The location of the CF mutation F508del on the surface of NBD1 opposite intracellular loop 4 (ICL4) is shown by an asterisk. Abbreviations: MSD, membrane-spanning domain; NBD, nucleotide-binding domain; P, phosphorylation of the RD; P_i, inorganic phosphate; RD, regulatory domain. In and out denote the intra- and extracellular sides of the membrane, respectively. See text for further information. Modified, with permission, from Hwang and Sheppard.

Figure 3

Chemical structures of some CFTR correctors identified by HTS. Abbreviations: Corr-4a, N-[2-(5-Chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]bithiazolyl-2′-yl]-benzamide; VRT-325, 4-Cyclohexyloxy-2-{1-[4-(4-methoxy-benzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline; RDR1, 5-(4-nitrophenyl)-2-furaldehyde 2-phenylhydrazone.

Figure 4

Chemical structures of some CFTR potentiators identified by HTS. Abbreviations: ΔF508_act-02, 2-(2-chlorobenzamido)-4,5,6,7-tetrahydro-3H-indene-1-carboxamide; VRT-532, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol; VX-770, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide. For comparison, the chemical structure of genistein is shown.