Rational design, synthesis, and evaluation of novel polypharmacological compounds targeting NaV1.5, KV1.5, and K2P channels for atrial fibrillation

Figures

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  Figure 1. Structures of LA compounds that block KV1.5, TASK-1 and/or NaV1.5 channels. From the right to the left of the compounds are shown the aromatic ring, an intermediate amide linker, and a hydrophilic domain.

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  Figure 2. General scheme of design and selection of compounds for synthesis. A, Derivatives of bromoacetic acid. B, (S)-2-bromo-propionic acid derivatives. Ar, aromatic amines. R1 and R2, aliphatic amines.

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  Figure 3. Synthesis of the set of compounds 6 and 7.

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  Figure 4. Structure of compounds 6a-6f and 7a-7d and their yield.

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  Figure 5. The inhibitory activity of compounds 6a-6f and 7a-7d were evaluated in TASK-1 and KV1.5 channels using the TEVC technique at +40 mV and a concentration of 100 μM. For TASK-1; n = 4 (6e, 7a, 7b, 7c), n = 3 (6a, 6b, 6c, 6d, 7d), n = 15 for 6f and for KV1.5; n = 5 (except in 6a and 7b, where n = 4). Data are presented as Mean ± SD. Statistical significance is represented by letters indicating the individual effects of the compounds in each channel (gray letters for TASK-1 and blue letters for KV1.5). There are no significant differences between drug blockades in a certain channel when they share the same letter. The combined effect, comparing compound blockades in both channels with another independent compound acting in both channels as well, is indicated with an asterisk.

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  Figure 6. Inhibitory effects of compounds 6e, 6f, 7a, 7b and 7c on TASK-1, KV1.5 and NaV1.5 channels. A, analyses of channel inhibition by 100 μM 6e, 6f, 7a, 7b and 7c. For TASK-1 and KV1.5, currents were analyzed at +40 mV and for NaV1.5 at −30 mV. Data are presented as Mean ± SD. Statistical significance is represented by letters indicating the individual effects of the compounds in each channel (gray letters for TASK-1, blue letters for KV1.5, and red letters for NaV1.5). There are no significant differences for drug block in a certain channel when they share the same letter. The combined effect, comparing compound blockades in the three channels with another independent compound acting in the three channels as well, is indicated with an asterisk. NaV1.5; 6e n = 20, 6f n = 9, 7a n = 3, 7b n = 4, 7c n = 3; for TASK-1 and KV1.5, see Figure 5. Representative current traces before and after application of 100 μM of the compound 6f (B) and compound 7a (C).

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  Figure 7. IC50 of compound 6f in TASK-1, KV1.5 and NaV1.5 channels. A, representative current traces before and after application of increasing concentrations of compound 6f for TASK-1, KV1.5 and NaV1.5. B, dose-response curves were used to calculate the half-maximal inhibition (IC50 values) of compound 6f for TASK-1, KV1.5 and NaV1.5 channels. Block was analyzed at +40 mV. For NaV1.5, the block was determined at −30 mV. For TASK-1 and NaV1.5, a concentration range from 0.01 μM to 1000 μM was used; for KV1.5, it was 1 μM to 1000 μM. n = 6 to 15.

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  Figure 8. The activity of compound 6f on different channels was analyzed at100 μM by TEVC in X. laevis oocytes. All data is presented as mean ± SD. The experiments were replicated as follows; TASK-1 n = 4, KV1.5 n = 5, NaV1.5 n = 9, Kir2.1 n = 3, TREK-1 n = 4 and TASK-4 n = 3.

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  Figure 9. Comparative analysis of patch-clamp recordings of single atrial cardiomyocytes isolated from tissue samples of patients in sinus rhythm and with atrial fibrillation. A–E, Atrial action potential (AP) recordings from sinus rhythm (SR) patient samples (n = 13 cells/N = 5 patients). A, representative AP traces from patch-clamp recordings performed on isolated human atrial cardiomyocytes under basal conditions (Baseline, black) and 8 to 10 min after application of 12.5 μM compound 6f (red). B–E, recorded changes in AP indices (AP amplitude, maximum upstroke velocity, APD50 and APD90) relative to baseline after application of compound 6f at concentrations of 1 μM, 6.25 μM and 12.5 μM. F–J, AP recordings from atrial fibrillation (AF) patient samples (n = 7 cells/N = 5 patients). F, representative AP traces under basal conditions (Baseline, black) and 8 to 10 min after application of 12.5 μM compound 6f (red). G–J, recorded changes in AP indices (AP amplitude, maximum upstroke velocity, APD50 and APD90) relative to baseline after application of compound 6f at concentrations of 1 μM, 6.25 μM and 12.5 μM. Data are given as mean ± standard error of the mean. Individual columns were…

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  Figure 10. Common pharmacophore to 6f and vernakalant. A, Compounds 6f (yellow) and vernakalant (black) are shown in stick representation. Green and red spheres stand for hydrophobic and hydrogen acceptor groups, respectively. The Orange halo represents an aromatic ring. B and C, Pharmacophoric features represented in 6f and vernakalant, respectively, using the same colors as in (A). Notice that the NH atoms of the amide linker (-CONH-) are not present in vernakalant.

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  Figure 11. Impact of three concentrations (1, 10 and 100 μM) of compound 6f on single cell and whole-atria properties. A, AP traces of N = 45 atrial cardiomyocyte models at 2 Hz in control conditions and after the application of compound 6f at 100 μM. B, rate-dependent variation of the APD (ΔAPD). “N” indicates the number of atrial cardiomyocyte models able to propagate the stimulus according to the concentration applied and the pacing rate. C, concentrations-dependent increase in the effective refractory period (ΔERP) compared to control conditions in the N = 45 atrial cardiomyocyte models. D, AF cardioversion and prevention efficacy (i.e., percentage of AF episodes cardioverted or prevented) in the population of 45 virtual-atria models. E, AF duration in the 45 virtual-atria models considering that AF episodes are analyzed for 7 s of activity.

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  Figure 12. Consecutives snapshots of the transmembrane voltage (Vm) for a representative AF episode in control conditions and after the virtual administration of 10 and 100 μM of compound 6f. RA-LA: Right and left atrium; SCV-IVC: Superior and inferior cava vein; rPV-lPV: Right and left pulmonary veins.