Ramírez D , Bedoya M , Kiper AK , Rinné S , Morales-Navarro S , Hernández-Rodríguez EW , Sepúlveda FV , Decher N , González W .

11180604 Fondo Nacional de Desarrollo Científico y Tecnológico, 1140624 Fondo Nacional de Desarrollo Científico y Tecnológico, 1191133 Fondo Nacional de Desarrollo Científico y Tecnológico, DE1482-4/1 Deutsche Forschungsgemeinschaft, MiNICAD Millennium Nucleus of Ion Channels Associated Diseases

	Figure 1. Binding site of different drugs in K2P channels. (A) A1899 blocker interacting with TASK-1 at the central cavity [13]. (B) Local anesthetic bupivacaine allosterically inhibiting TASK-1 channels interacting in the lateral fenestration [14]. (C) Norfluoxetine interacting with TREK-2 in the lateral fenestrations [10]. (D) PK-THPP blocker interacting in the central cavity with TASK-3 (reported in this study). Two K+ ions were associated to the TASK homology models in positions S2 and S4 of the selectivity filter and two water molecules at sites S1 and S3 [13,14, current study].
	Figure 2. PK-THPP biding site. (A) Chemical structure of PK-THPP, 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine (yellow), piperidin-4-yl-butan-1-one (blue), biphenyl (green). Yellow and green moieties are separated by a carbonyl group (B) Dose-response curve of PK-THPP on human TASK-3. Block was analyzed at the end of the test pulse of +40 mV. (C) TASK-3 mutant channels with a reduced sensitivity to PK-THPP are marked with asterisks. All values are expressed as means ± standard error (S.E., bars). The significance of the differences between the mutants and the WT result was assessed using two-tailed Student’s t-tests. Asterisks indicate significance: * p < 0.05; ** p < 0.01; *** p < 0.001. Other differences did not reach statistical significance. For all oocyte experiments, n = 3–7 cells. Residues identified as THPP binding site hits have significant levels of p < 0.01 and p < 0.001, except T93 (p < 0.05); this is important from the modelling point of view. (D) Residues of PK-THPP binding site are facing the pore and the fenestrations as it is shown in TASK-3 models in the down (T3tre2OO) and up (T3tre1CC) states (lateral view from the pore). Hits (Chain A: T93, L122 and Q126; Chain B: G231, G236, A237, L247 and T248) are represented in licorice with individual colors. L239 hit, which could play a fundamental role in the up-to-down state transition is represented using yellow Van der Waals spheres. For better visualization, only transmembrane (TM) segments M2-chain A and M4-chain B are represented.
	Figure 3. TASK-3 modeling. (A) Left: HOLE representation of the pore for the T3tre2OO model. Right: Graphs show the average diameter and the standard error of mean (SEM) of the pore in T3tre1CC, T3tre2OO and T3twiOO models for the last 10ns MDs (10 structures from each MDs → 1 frame per ns were taken). The central cavity is located at the bottom of the selectivity filter (−20 Å to 0 Å in the z-axis). The z-axis is lined by the ion-conducting pore. Within the central cavity, the bottleneck of models with open fenestrations is at z-axis = −15.5 Å (size 4.32 ± 0.013) in T3tre2OO and at z-axis = −15 Å (size 3.76 ± 0.263) in T3twiOO. Meanwhile, at z-axis = −15.5 Å, the pore size in T3tre1CC is 12.96 ± 0.061, red line. (B) Left: HOLE representation of the fenestrations (F1 left fenestration, F2 right fenestration) for the T3tre2OO model. Right: Plot of the radius of the TASK-3 fenestrations for each MD. The bottleneck diameters (red line) of F1 and F2 are at the following positions in the x-axis in each model: T3tre1CC, F1 x-axis = −11 Å (size 1.34 ± 0.178) and F2 x-axis = 10 Å (size 1.50 ± 0.153); T3tre2OO, F1 x-axis = −8 Å (size 3.54 ± 0.093) and F2 x-axis = 7.5 Å (size 3.6 ± 0.035); T3twiOO: F1 x-axis = −8 Å (size 3.84 ± 0.096) and F2 x-axis = 8 Å (size 4.66 ± 0.052). The x-axis is perpendicular to the ion-conducting pore and is lined by the access to the hydrophobic core of the lipid bilayer from the pore central cavity. HOLE color code used is as follows: Blue, radius > 1.15 Å; green, radius between 0.6–1.15 Å. Subunits A (orange) and B (red) of the homology model of TASK-3 channel based on TREK-2 (T3tre2OO) are shown in cartoon representation in each left figure.
	Figure 4. TASK-3–THPP series structural-activity correlation. (A) Correlation plots obtained for THPP series interacting with TASK-3 models in down-state (T3tre1CC) and up-state (T3twiOO and T3tre2OO). The compound docking poses with the best relation between the relative binding free energy (ΔGBind) per model and their experimental biological activity (pIC50), expressed as Ln (100/IC50), were used for comparison. Five compounds of the THPP series (dots) were selected to understand how slight structural modifications result in significant differences in the binding affinity against TASK-3. They are PK-THPP (black), 17b (green), 20b (gray), 21 (blue) and 22 (red). (B) Chemical structures of the five studied ligands of THPP series. The low affinity compound 17b exhibits a different moiety (blue, unsubstituted pyrrolidine) than the high affinity compounds from 20b to 23 (red, substituted piperidine). Consequently, the shared seven-point pharmacophore RRAHRHA (from the left to the right and upper side of the molecules) where R (aromatic ring, orange dot), A (H-Bond acceptor group, red dot) and H (hydrophobic group, green dot) shared by the high affinity compounds is not present completely in the low affinity compound 17b, which does not exhibit the last A group.
	Figure 5. Orientation of the PK-THPP poses within the significant clusters. Significant cluster-10 (blue), −23 (magenta), −28 (gray) and −33 (red) are represented in sticks. K+ ions at S4 are shown in sphere representation and TASK-3 models in cartoon representation. Right, the structure nearest the centroid in each significant cluster per model is shown. Hits within 5 Å of the poses are shown in stick representation as well as T93 residue. For better visualization, TM3 and TM4 are not shown. Open fenestration in T3twiOO and T3tre2OO models are shown as green dotted surface.
	Figure 6. Redefined binding mode of PK-THPP in TASK-3. (A) Left, PK-THPP interacting with TASK-3. Right, zoomed binding site with key residues represented as stick and the hydrogen bond with T93 shown explicitly. (B) Contact frequencies of PK-THPP with T3tre2OO residues at 3 Å. Bars indicate the contact frequency along the 250ns MDs. (C) Interactions between the residues of T3tre2OO and PK-THPP are categorized into hydrophobic, water bridges and hydrogen bonds. The stacked bar charts are normalized over the course of the unrestrained MDs. The interactions were calculating using the Simulation Interaction Diagram tool included in the Schrödinger Suite. (D) Root mean square fluctuation (RMSF, black line) characterizing the internal atom fluctuations of PK-THPP during the 250ns MDs. The numbers for the atoms are presented in the x-axis and in the structure.

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