The Trials and Tribulations of Structure Assisted Design of KCa Channel Activators

Figures

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  Figure 1. Design scheme of KCa2.2 selective activators and Rosetta models of the top 10 binding poses with the lowest energy of template compounds in the interface between CaM (pink) and the CaM-BD (light green) of KCa3.1 and KCa2.2. The docking model of SKA-74 (A) and SKA-76 (B) showed that the ten lowest binding energy scored models exhibit good structural convergence in KCa2.2 but not in KCa3.1 suggesting selectivity for KCa2.2 over KCa3.1.

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  Figure 2. Rosetta ligand docking models of the lowest energy-binding poses of SKA-130 (A, B) and SKA-230 (C, D) in the interface between CaM (pink) and CaM-BD (light green) of KCa3.1 and KCa2.2. Hydrogen bonds and van der Waals interactions are indicated by purple and black lines. (A, B) Molecular docking suggests that SKA-130 converged well and formed several van der Waals contacts in KCa2.2 but not in KCa3.1. (C, D) Both KCa3.1 and KCa2.2 models exhibited good structural convergence. The molecular docking model showed that while SKA-230 only formed 2 hydrogen bonds in KCa3.1, it formed four hydrogen bonds in KCa2.2 suggesting selectivity for KCa2.2.

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  Figure 3. General scheme for the synthesis of SKA-74, SKA-75, SKA-76 (A) and SKA-111 derivatives (B).

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  Figure 4. Whole-cell KCa3.1 and KCa2.2 responses elicited by 10 µM of activator and normalized to 10 µM of the mixed activator SKA-31. Experiments were performed by automated electrophysiology with 250 nM of free internal Ca2+. In each experiment the new activator was tested first, washed out with 2 saline additions, and then SKA-31 was applied as positive control for normalization of the response. (A) Screening of SKA-74 derivatives. (B) Screening of SKA-75 and SKA-76 derivatives. The bar graphs show means ± SD of the slope conductance measured between −85 and −65 mV (n = 2–7 cells). Chemical structures are shown below the bar graphs.

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  Figure 5. Whole-cell KCa3.1 and KCa2.2 responses elicited by 10 µM of activator and normalized to 10 µM of the mixed activator SKA-31. Experiments were performed by automated electrophysiology with 250 nM of free internal Ca2+. In each experiment the new activator was tested first, washed out with 2 saline additions, and then SKA-31 was applied as positive control for normalization of the response. (A) Screening of SKA-111 derivatives. (B) Screening of aromatically substituted SKA-111 derivatives. The bar graphs show means ± SD of the slope conductance measured between −85 and −65 mV (n = 2–7 cells). Chemical structures are shown below the bar graphs.

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  Figure 6. Docking model of SKA-111 in the full-length KCa3.1 structure and mutational strategy. (A) Bottom and side view of the full-length KCa3.1 cryo-EM structure following Rosetta refinement in the Ca2+ free closed state (pdb: 6cnm) and Open state I (pdb: 6cnn). The channel is shown in gray, the CaM C-lobe in purple and the CaM N-lobe in yellow. Next to open state 1 we show a zoom out of the lowest energy docking pose of SKA-111 in the interface between the CaM N-lobe and the S45A helix interface. Hydrogen bonds are shown in black, van der Waals interactions are visualized in purple. For clarity, not all side chains of CaM residues within contact range of SKA-111 are explicitly shown. Please note the channel residues S181, A184 and L185. (B) Alignment of the S45A helix sequence in KCa2.1, KCa2.2, KCa2.3 and KCa3.1. Residues that were mutated are highlighted in yellow and the EC50 values for SKA-111 shown next to each mutant (for confidence intervals see Figures 7 and 8 ).

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  Figure 7. Mutations of S181 and L185 in the S45A helix disturb SKA-111 activity but not calcium gating. (A) Representative whole-cell WT and mutant KCa3.1 currents with an intracellular free calcium concentration of 250 nM in the presence and absence of SKA-111. (B) Concentration–response for SKA-111 induced current activation: WT (EC50 = 146 nM, 95% CI: 99–193 nM), T212F-V272F (EC50 = 153 nM, 95% CI: 114–182 nM, P = 0.1017), S181A (EC50 = 1.012 µM, 95% CI: 0.780–1.244 µM, P < 0.0001), A184F (EC50 = 1.326 µM, 95% CI: 1.205–1.447 µM, P < 0.0001), L185A (EC50 = 0.993 µM, 95% CI: 0.903–1.083 µM, P < 0.0001), S181A-L185A (EC50 = 2.654 µM, 95% CI: 2.619–2.624 µM, P < 0.0001). Whole-cell KCa3.1 currents were elicited by voltage-ramps from −120 to + 40 mV with an intracellular free calcium concentration of 250 nM. Data points are mean ± S.D. from 3–5 independent cells/recordings. The reported P values are for an extra sum-of-squares F test (GraphPad Prism5; GraphPad Software, La Jolla, CA) to compare the curves of KCa3.1 mutants to WT. (C) Inside-out calcium concentration-response curves for WT KCa3.1 (EC50 = 437 nM, 95% CI: 353–521 nM, nH = 1.98) and the S181A-L185A double mutant (EC50 = 392 nM,…

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  Figure 8. Rosetta Model of the wild type KCa3.1 channel in open state 1 with serine in position 372 (A) and of the S372R mutant (B). Hydrogen bonds are visualized in black. (C) Overlay of the Rosetta models of the S45A helix/CaM N-lobe interface pocket in the wild type (yellow) and the S372R mutant shown in space fill. (D) Concentration-response curve for SKA-111 induced current activation: WT (EC50 = 146 nM, 95% CI: 99–193 nM), S372R (EC50 = 6.860 µM, 95% CI: 6.788–6.932 µM, P < 0.0001), S181A-L185A-S372R (EC50 = 8.876 µM, 95% CI: 8.652–9.100 µM, P < 0.0001). Data points are mean ± S.D. from 3–5 independent cells/recordings.

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  Figure 9. Interactions between SKA-111 and residues in the KCa3.1 S45A helix (A) and the CaM N-lobe (B) interface pocket. Hydrogen bonds are indicated by black lines, van der Waals interactions are indicated by purple lines. (C) The KCa3.1-CaM channel complex seen from the intracellular side. The S45A helix/CaM N-lobe interface colored dark blue. Two other, potential binding sites for small molecules are highlighted with orange or sky-blue circles. The channel is shown in gray, the CaM C-lobe in purple and the CaM N-lobe in yellow.