Direct Binding of the Corrector VX-809 to Human CFTR NBD1: Evidence of an Allosteric Coupling between the Binding Site and the NBD1:CL4 Interface

Abstract

Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of -strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (T), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative T shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR.

Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R

“…Binding to this site, or sites located nearby, might improve the stability of this fragile region which is, like the ICL4:NBD1 interface, more sensitive to any change than other parts of the protein (Zhang and Chen, 2016;Callebaut et al, 2017b). This hypothesis requires to be further explored, especially as other possible VX-809-binding sites have been proposed (Okiyoneda et al, 2013;Hudson et al, 2017).…”

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

“…Although, the binding site for VX‐809 has not been fully defined, regions of a putative pocket were implicated in biochemical studies (ie, MSD1,) or in silico studies (NBD1 and the coupling helices extending from MSD1 and MSD2,). Although some of the residues highlighted in previous studies are conserved between the human and zebrafish proteins (ie, R170, F374, L375, E403, and R1070), others are not (i.e, E402, V510, E474, G1069).…”

“…On the other hand, F508del-mCftr, with 78% sequence identity with the human protein, does show rescue of its processing defect with VX-809, arguing that comparative studies of the zebrafish, mouse, and hCFTR proteins may inform the mechanism of action of this corrector. 62 Although, the binding site for VX-809 has not been fully defined, regions of a putative pocket were implicated in biochemical studies (ie, MSD1, 35,40 ) or in silico studies (NBD1 and the coupling helices extending from MSD1 and MSD2, 59,[63][64][65]. Although some of the residues highlighted in previous studies are conserved between the human and zebrafish proteins (ie, R170, F374, L375, E403, and R1070), others are not (i.e, E402, V510, E474, G1069).…”

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator

“…The corrector compound, VX809 or Lumacaftor, which has been approved for clinical use in humans, produces functional expression of F508del-hCFTR at ϳ14% of the level of WT-hCFTR (60). There are conflicting reports in the literature concerning whether Lumacaftor provides effective shortterm stabilization of hCFTR channel function in vivo on the time scale of minutes (31,37,53,63). Furthermore, one recent paper demonstrated significant thermodynamic destabilization of isolated hNBD1 in DSC experiments conducted in the presence of 1 mM Lumacaftor (63).…”

“…There are conflicting reports in the literature concerning whether Lumacaftor provides effective shortterm stabilization of hCFTR channel function in vivo on the time scale of minutes (31,37,53,63). Furthermore, one recent paper demonstrated significant thermodynamic destabilization of isolated hNBD1 in DSC experiments conducted in the presence of 1 mM Lumacaftor (63). Although additional research will be required to resolve the controversies related to the interaction of Lumacaftor with hCFTR, it is clear that this drug does not fully correct the thermal stability defect caused by the F508del mutation in full-length hCFTR (31), and it does not support any proper channel gating at 37°C in temperature-dependent electrophysiology assays like those employed in this paper (31), while dTTP does (Fig.…”