Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

High-throughput screening has led to the identification of small-molecule blockers of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, but the structural basis of blocker binding remains to be defined. We developed molecular models of the CFTR channel on the basis of homology to the bacterial transporter Sav1866, which could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker such as N-(2-naphthalenyl)- [(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101), which is thought to act by entering the channel from the extracellular side. As a more-stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual, ligand-docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highestscoring docked position was near two pore-lining residues, Phe337 and Thr338, and the rates of reactions of anionic, thiol-directed reagents with cysteines substituted at these positions were slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (Phe342) was replaced with alanine, the apparent affinity of the blocker was increased ϳ200-fold. A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

“…Figure S3 a shows the superposition of our model on this crystal structure, which yields an RMSD of 1.6 Å. Panel b shows that residues lining the pore at the membrane-spanning domain (MSD), observed by the end of 50 ns simulations, agree in general with the CFTR model built by Norimatsu and collaborators [37], [38] which was also confirmed by cysteine scanning experiments [37], [39]. Likewise, the pore radius profile evaluated for our wild-type structural model ( Figure S3 c solid curve , with the gray band displaying the fluctuations observed in 50 ns simulations) is qualitatively consistent with that observed by Norimatsu and coworkers [37] for the MSD.…”

Localizing a gate in CFTR

“…Besides MTS reagents (17,21), few probes were reported to interact with the channel from the external side. Recently, GlyH-101, a high-affinity CFTR inhibitor (39), was reported to block the pore by lodging in the external vestibule close to positions 337 and 338 in TM6 (40). Interestingly, in the current study, we found that externally applied bulky Texas red MTSEA can modify the cysteine placed at position 338 (Fig.…”

“…− applied before and after cAMP-dependent activation of wholecell CFTR channels, Norimatsu et al reported a similar closedstate preference of accessibility for 334C (40). However, the state-dependent accessibility pattern of 337C in the current study strays from their results.…”

Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP

“…Electrophysiology, computational studies and chemical modifications suggest GlyH-101 binds near Phe-337 in the extracellular CFTR pore (near binding site A in Fig. 1) and blocks the pore directly (Muanprasat et al, 2004;Sonawane et al, 2008;Norimatsu et al, 2012). Electrophysiology and mutagenesis studies suggest that CFTR inh -172 stabilizes the CFTR closed channel Caci et al, 2008;Kopeikin et al, 2010) by binding at or near Arg-347 in the MSD at a location roughly equidistant to the extracellular and cytoplasmic membrane surfaces.…”