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 highest-scoring 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 ∼200-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.

Fig. 1.

A, structure of GlyH-101 in licorice representation with a transparent molecular surface. In the licorice representation, oxygen atoms are red, bromine brown, nitrogen blue, carbon gray, and hydrogen white. The molecular surface is colored according to the electrostatic potential energy. The scale bar shows the correspondence between the colors and electrostatic potential energy values. B, I-V curves in the absence and presence of 1, 10, and 50 μM GlyH-101. Wild-type CFTR channels were expressed in Xenopus laevis oocytes, and transmembrane currents were measured by using two-electrode, voltage-clamp methods. C, voltage dependence of the GlyH-101 EC₅₀ for the wt CFTR, consistent with the Woodhull model (Woodhull, 1973; Tikhonov and Magazanik, 1998). See Materials and Methods for details. The EC₅₀ at 0 mV was 1.1 ± 0.11 μM (n = 4), and the apparent electrical distance (from the outside) sensed by the blocker was 0.35 ± 0.013 (n = 4).

Fig. 2.

Predicted binding mode for GlyH-101. A, view from the side of transmembrane domain 1 (green). B, view from the side of transmembrane domain 2 (gray). C, top view from the extracellular side. Phe337 is displayed in pink, Thr338 in red, Ser341 in yellow, Phe342 in purple, and Ile1131 in blue. Our model of the CFTR shows that both Phe337 and Thr338 lie close to the negatively charged GlyH-101 when it is bound in the pore. Phe337, Thr338, and Ile1131 were each mutated to cysteine in silico (Maestro; Schrödinger), and the distances between the sulfur atom of the thiol group and each of the six carbon atoms in the benzene ring of GlyH-101 were measured. The average distances to the benzene ring from the sulfur atom were 5.9 Å for position 337 and 7.3 Å for position 338. Both values are less than the Debye length (9.2 Å) in frog Ringer's solution. The distance from position 1131 was 11.5 Å. The position of Ser341 indicates that it might be protected from electrically neutral reagents through steric hindrance provided by bound GlyH-101.

Fig. 3.

State-dependent reactivity of the F337C CFTR with [Au(CN)₂]⁻. A, exposure of an oocyte to 30-s and 1-min pulses of 600 μM [Au(CN)₂]⁻ resulted in reductions of conductance. Covalent labeling of the F337C CFTR was almost complete after a cumulative exposure time of 2 min. Isop, isoproterenol. B, before activation of the F337C CFTR with isoproterenol and IBMX, 600 μM [Au(CN)₂]⁻ was applied to the oocyte for 18 min. The conductance was partially reduced, as demonstrated by the additional increase in conductance with the application of 1 mM KCN. The subsequent application of [Au(CN)₂]⁻ almost completely abolished F337C CFTR conductance. C and D, time courses of the decreases in normalized conductance as a result of F337C (C) and T338C (D) modifications with [Au(CN)₂]⁻. Data points represent mean ± S.E.M. (n = 3). For the F337C CFTR, the abscissa represents cumulative [Au(CN)₂]⁻ exposure time. For the T338C CFTR, the abscissa represents cumulative [Au(CN)₂]⁻ exposure (exposure time × [Au(CN)₂]⁻ concentration used). The reaction rate for the F337C CFTR before activation of the channels was ∼20 times slower than the rate after activation. The rate for the T338C CFTR was almost 30 times slower before activation. The lines represent the best fits of single-exponential functions to the conductance data. The half-life of each single-exponential fit is shown. The second-order reaction rate constants for the F337C CFTR before and after activation were 1.5 and 26 M⁻¹ s⁻¹, respectively. The second-order reaction rate constants for the T338C CFTR before and after activation were 1.2 × 10² and 3.0 × 10³ M⁻¹ s⁻¹, respectively.

Fig. 4.

Time courses of reactions of R334C (an engineered cysteine at position 334) with 10 μM [Au(CN)₂]⁻ (A) or 250 nM MTSET⁺ (B). Data points represent mean ± S.E.M. (n = 3). The abscissa represents the cumulative reagent exposure time. The lines represent the best fits of single-exponential functions to the conductance data. The half-life of each single-exponential fit is shown. For both [Au(CN)₂]⁻ and MTSET⁺, the reaction rate for the R334C CFTR before activation of the channels was faster than the rate after activation. The second-order reaction rate constants for [Au(CN)₂]⁻ before and after activation were 8.9 × 10² and 2.6 × 10² M⁻¹ s⁻¹, respectively. The second-order reaction rate constants for MTSET⁺ before and after activation were 7.2 × 10⁴ and 3.9 × 10⁴ M⁻¹ s⁻¹, respectively.

Fig. 5.

GlyH-101 protection of T338C (an engineered cysteine at position 338) from modification by [Au(CN)₂]⁻. A, exposure of an oocyte to 30-s and 1-min pulses of 5 μM [Au(CN)₂]⁻ resulted in reductions of conductance. Covalent labeling of the T338C CFTR was almost complete after a cumulative exposure time of 2 min. Isop, isoproterenol. B, the oocyte was exposed to 5 μM [Au(CN)₂]⁻ in the presence of 10 μM GlyH-101. The blockade attributable to covalent labeling was less than 50% after a cumulative exposure time of 2 min. The application of GlyH-101 was started 30 s before the application of [Au(CN)₂]⁻, to ensure maximal blockade of the channels during the [Au(CN)₂]⁻ reaction. The GlyH-101 application was continued 30 s after the end of the [Au(CN)₂]⁻ application. C and D, F337C (C) and T338C (D) CFTR channels were protected by 10 μM GlyH-101 from reactions with [Au(CN)₂]⁻. Data points represent mean ± S.E.M. (n = 3). The F337C CFTR was reacted with 600 μM [Au(CN)₂]⁻ and the T338C CFTR was reacted with 5 μM [Au(CN)₂]⁻ in the presence and absence of 10 μM GlyH-101. The lines represent the best fits of single-exponential functions to the normalized conductance data. The half-life of each single-exponential fit is shown. E and F, the rates of reaction of [Au(CN)₂]⁻ with F337C (E) and T338C (F) CFTRs decreased as the concentration of GlyH-101 was increased. Data points represent mean ± S.E.M. (n = 3). Solid lines, best fits of sigmoidal dose-response curves to the reaction rate data. Dashed lines, dose-response curves for GlyH-101 blockade of the cysteine-substituted constructs observed in this study. The reaction rate constant observed in the absence of GlyH-101 was used as the maximal value of the sigmoidal curve. The EC₅₀ and the minimal rate constant for the F337C CFTR were 1.6 μM and 0.08 mM⁻¹ s⁻¹, respectively; values for the T338C CFTR were 3.3 μM and 0.02 μM⁻¹ s⁻¹. The EC₅₀(0) values for GlyH-101 blockade for the F337C and T338C CFTRs were 1.8 ± 0.063 (n = 3) and 3.7 ± 0.27 μM (n = 3), respectively.

Fig. 6.

GlyH-101 protection of T338C (an engineered cysteine at position 338) from MTSES⁻ but not from MTSET⁺. A, the T338C CFTR was reacted with 5 μM MTSES⁻ in the presence and absence of 10 μM GlyH-101. Data points represent mean ± S.E.M. (n = 3). The lines represent the best fits of single-exponential functions to the conductance data. The half-life of each single-exponential fit is shown. The MTSES⁻ reaction rate was almost 3 times slower in the presence of GlyH-101. The second-order reaction rate constants for MTSES⁻ in the presence and absence of 10 μM GlyH-101 were 1.2 × 10³ and 3.3 × 10³ M⁻¹ s⁻¹, respectively. B, the T338C CFTR was reacted with 50 μM MTSET⁺ in the presence and absence of 10 μM GlyH-101. Data points represent mean ± S.E.M. (n = 3). The presence of GlyH-101 had no impact on the MTSET⁺ reaction rate. The second-order reaction rate constants for MTSET⁺ in the presence and absence of GlyH-101 were 1.8 × 10² M⁻¹ s⁻¹.

Fig. 7.

A, time courses of the reactions of the I1131C CFTR with 1 mM MTSES⁻ in the presence and absence of 10 μM GlyH-101. Data points represent mean ± S.E.M. (n = 3). Covalent labeling of the I1131C CFTR with MTSES⁻ resulted in increases in conductance. The lines represent the best fits of single-exponential functions to the conductance data. The half-life of each single-exponential fit is shown. The second-order reaction rate constants in the presence and absence of 10 μM GlyH-101 were 28 and 20 M⁻¹ s⁻¹, respectively. The EC₅₀ at 0 mV for GlyH-101 blockade for the I1131C CFTR was 0.86 ± 0.016 μM (n = 3). B, time courses of the reactions of the S341C CFTR with 1 mM NEM in the presence and absence of 10 μM GlyH-101. Data points represent mean ± S.E.M. (n = 3). Covalent labeling of the S341C CFTR with NEM resulted in reductions in conductance. The lines represent the best fits of single-exponential functions to the conductance data. The half-life of each single-exponential fit is shown. The second-order reaction rate constants in the presence and absence of 10 μM GlyH-101 were 5.3 and 16 M⁻¹ s⁻¹, respectively. The EC₅₀ at 0 mV for GlyH-101 blockade for the S341C CFTR was 0.89 ± 0.056 μM (n = 3).

Fig. 8.

F342A CFTR transmembrane currents. F342A CFTR channels were expressed in Xenopus laevis oocytes, and transmembrane currents were measured at clamped voltages by using a two-electrode, voltage-clamp technique. A, the voltage dependence of the GlyH-101 EC₅₀ for the F342A CFTR is consistent with the Woodhull model (Woodhull, 1973; Tikhonov and Magazanik, 1998), in a manner similar to that of wt CFTR (Fig. 1). Data points represent mean ± S.E.M. (n = 3). The EC₅₀ at 0 mV was 5.2 ± 0.83 nM (n = 3), and the apparent electrical distance was 0.35 ± 0.019 (n = 3). B, the channels were activated with a stimulating cocktail containing isoproterenol and IBMX. Six current traces were recorded in the absence of GlyH-101 and are shown superimposed. At the beginning of each trace, the voltage was held at 0 mV for 0.5 s; it was then stepped to −110, −80, −50, −20, +10, or +40 mV for 1.5 s. After this step, the voltage was held at 0 mV for 1 s. C, GlyH-101 was applied to the same oocyte as used in B. Six traces were recorded in the presence of 100 nM GlyH-101 and are shown superimposed. At the beginning of each trace, the voltage was held at 0 mV for 0.5 s; it was then stepped to −110, −80, −50, −20, +10, or +40 mV for 2 s. After this step, the voltage was held at +40 mV for 5 s. D, the rates of single-exponential time courses shown in B were plotted against the concentration of GlyH-101. The relaxation rate (the reciprocal of the time constant for the single-exponential relaxation) exhibited a linear dependence on the GlyH-101 concentration. As described by eq. 3, the second-order binding rate constant k′_on and the off-rate k_off could be determined from the best-fit line. E, the second-order binding rate constant k′_on values (mean ± S.E.M.; n = 3) were plotted against the transmembrane voltage. The data were fit by using the equation k′_on(V) = k′_on(0)exp(δ_bFV/RT), where δ_b is the voltage dependence constant for k′_on and F, R, and T have their usual meanings (Tikhonov and Magazanik, 1998); k′_on(0) was estimated to be 2.3 × 10⁷ M⁻¹ s⁻¹. F, the off-rates (mean ± S.E.M.; n = 3) were plotted against the transmembrane voltage. The data were fit by using the equation k_off(V) = k_off(0)exp(−δFV/RT), where δ is the voltage dependence constant for k_off and F, R, and T have their usual meanings (Tikhonov and Magazanik, 1998); k_off(0) was estimated to be 0.088 s⁻¹.

Fig. 9.

Homology model of the CFTR based on MsbA (Mornon et al., 2009). A, top view from the extracellular side. Arg334, Phe337, and Thr338 are shown as van der Waals representations. The carbon atoms of Thr334 are colored green, and Phe337 and Thr338 are colored pink and yellow, respectively. B, close-up view of the structure shown in A, around Thr334. The CFTR molecule is shown as a cartoon representation, with its molecular surface indicated by mesh. Transmembrane domain 1 is shown in cyan and transmembrane domain 2 in orange. C, view with Thr334, shown in B, mutated to cysteine with Maestro 9.1 (Schrödinger).