Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome

Figures

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  FIGURE 1. Overview of the KATP channel and the investigated inhibiting compounds. (A) Two opposing subunits of the KIR6.2 channel pore are shown in orange, one SUR1 subunit is shown in green. The two mutation sites L164 and Q52 are shown in purple and pink, respectively. The circles provide close-ups of the mutation sites. Since Q52 is located on the surface of KIR6.2 close to SUR1 and the orientation of the SUR1 subunits is different in the 6C3O and 6C3P structure, the close-ups of Q52 are shown for both structures. The loop L0, which comprises residues important for KIR-SUR coupling, is shown in light green for the 6C3P structure, while it is not modelled in the 6C3O structure. (B) Chemical structure of betaxolol. (C) Chemical structure of levobetaxolol. (D) Chemical structure of travoprost. (E) Chemical structure of latanoprost.

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  FIGURE 2. Pharmacological validation of Q52R and L164P KIR6.2 mutations. IC50 curves of KIR6.2/SUR2a inward (at −80 mV), left panels, and outward (at +50 mV), right panels, current inhibition in response to increasing concentrations of (A) BaCl2 and (B) glibenclamide. Wildtype KIR6.2/SUR2a serves as control. Data were fitted with Hill equation to estimate the IC50 values. Data are shown as mean ± SEM. N depicts number of measurements on independent inside-out patches.

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  FIGURE 3. Inhibition of Q52R and L164P KIR6.2/SUR2a dependent current by betaxolol and levobetaxolol. IC50 curves of KIR6.2/SUR2a inward (at −80 mV), left panels, and outward (at +50 mV), right panels, current inhibition in response to increasing concentrations of (A) betaxolol and (B) levobetaxolol. Wildtype KIR6.2/SUR2a serves as control. Data were fitted with Hill equation to estimate the IC50 values. Data are shown as mean ± SEM. N depicts number of measurements on independent inside-out patches.

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  FIGURE 4. Coarse-grained MD simulation with the KIR6.2 channel pore and betaxolol. (A) Overview of a typical simulation system after 1 µs simulation. Different subunits of the KIR6.2 channel are shown as grey and lilac spheres, respectively. Bound betaxolol molecules are shown as cyan spheres. Water molecules are illustrated as dots, while lipid molecules are omitted for clarity. (B) Close-up of the exterior of M1, where a betaxolol molecule is bound near the PIP2 binding site. PIP2 molecules are shown as orange spheres. (C) Close-up of the transmembrane cavity. (D) Close-up of the G-loop region.

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  FIGURE 5. Putative betaxolol binding sites and stability investigated in atomistic MD simulations with the KIR6.2 channel pore. (A) Overview of the binding sites of betaxolol to the KIR channel pore. Different KIR subunits are shown in cartoon representation and colored in dark and light grey. The different betaxolol molecules are shown in teal. (B–D) Distances between betaxolol and the KIR6.2 channel in 9 replica of 350ns-long MD simulations. The differently colored lines represent the distances of the docked betaxolol molecules to the respective binding sites in the different runs. (E–G) Close-ups of the betaxolol binding sites at TM-cavity (E), PIP2 binding site (F), G-loop gate (G) seen at the end of run1 of the all-atom MD simulations. The drug is shown as teal sticks, with hydrogen bonds shown as green dotted lines.

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  FIGURE 6. Evaluation of KIR6.2/SUR2a dependent currents in response to dinoprostone, latanoprost and travoprost. (A) IC50 curves of WT KIR6.2/SUR2a inward (at −80 mV), left panel, and outward (at +50 mV), right panel, current inhibition in response to increasing concentrations of dinoprostone or latanoprost. (B) IC50 curves of WT, Q52R and L164P KIR6.2/SUR2a inward (at −80 mV), left panel, and outward (at +50 mV), right panel, current inhibition in response to increasing concentrations of travoprost. Data were fitted with Hill equation to estimate the IC50 values. Data are shown as mean ± SEM. N depicts number of measurements on independent inside-out patches.

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  FIGURE 7. Binding sites of travoprost and latanoprost found in a total of 60 μs coarse-grained MD simulations with the 6C3O structure. The smaller figure on the left shows the overall structure of the 6C3O channel, the location of the lipid bilayer membrane is indicated with a white bar. For the sake of clarity, the structure is divided in different zones, which are shown in different colors (blue = extracellular part as well as the outer lipid layer of membrane, green = inner lipid layer of membrane, red = cytoplasmic domain near cell membrane, yellow = cytoplasmic domain). The figure above the overall structure shows the orientation of the SUR subunits in a top-down view of the channel. The different sub figures show close-ups of different interaction sites of the drugs to the channel. Travoprost is shown in shades of green, latanoprost in shades of orange and yellow. In each of the close-ups, all four subunits of the KIR channel are shown colored in white. For SUR, only one subunit is shown. The TM0 of SUR colored in middle grey, while the rest of SUR is colored in dark grey.

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  FIGURE 8. Binding sites of travoprost and latanoprost found in a total of 60 μs coarse-grained MD simulations with the 6C3P structure. The smaller figure on the left shows the overall structure of the 6C3P channel, the location of the lipid bilayer membrane is indicated with a white bar. For the sake of clarity, the structure is divided in different zones, which are shown in different colors (blue = extracellular part as well as the outer lipid layer of membrane, green = inner lipid layer of membrane, red = cytoplasmic domain near cell membrane, yellow = cytoplasmic domain). The figure above the overall structure shows the orientation of the SUR subunits in a top-down view of the channel. The different sub figures show close-ups of different interaction sites of the drugs to the channel. Travoprost is shown in shades of green, latanoprost in shades of orange and yellow. In each of the close-ups, all four subunits of the KIR channel are shown colored in white. For SUR, only one subunit is shown. The TM0 of SUR colored in middle grey, while the rest of SUR is colored in dark grey.

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  FIGURE 9. Evaluation of KIR6.2/SUR2a dependent currents in response to ethosuximide, fluvastatin and pazopanib. IC50 curves of WT KIR6.2/SUR2a inward (at −80 mV), left panel, and outward (at +50 mV), right panel, current inhibition in response to increasing concentrations drugs. Data were fitted with Hill equation to estimate the IC50 values. Data are shown as mean ± SEM. N depicts number of measurements on independent inside-out patches.