Drug-likeness of linear pentamidine analogues and their impact on the hERG K+ channel - correlation with structural features

Abstract

This work presents drug-likeness and the cardiotoxicity profiles of six potent pentamidine analogs 1-6 and three new compounds 7-9 as chemotherapeutics for therapy of Pneumocystis jiroveci pneumonia. A combination of experimental and computational approaches was used in the cardiotoxicity examination. The hERG trafficking and functionality of the hERG currents were tested by western blot analyses, immunofluorescent staining procedures, and patch-clamp electrophysiological assays. Cardiotoxicity combined with blocking the hERG K⁺ channel was predicted, and then simulated by docking to the CSM-TM model 732 protein. Location of pentamidines in the proximity of Leu622, Thr623, Ser649, Tyr652, Ala653, and Phe656, and the high energies of interactions were in accordance with probable blocking of the hERG channel. However, in the biochemical experiments, no significant changes in I _hERG densities and a minor effect on hERG maturation were observed. Predicted metabolic transformation of pentamidines with S atoms in the aliphatic linker leads to oxidation of one S atom, but those with the phenyl sulfanilide moiety can be oxidized to chinones. The tested pentamidines characterized by the presence of sulfur atoms or sulfanilide groups, have favorable drug-likeness parameters and are promising lead structures in the development of new potent chemotherapeutics against PJP.

Fig. 1. Chemical formulas of pentamidine analogs 1–9 used in this study.

Fig. 2. Synthetic route of new pentamidine analogs 7–9. Reagents and conditions: (a) NMP, 60 °C, 1 h; (b) 50% NH₂OH/ EtOH, r.t., 24 h; (c) HCOOH/10%PdC/AcOH, reflux, 3 h, then aq. HCl.

Fig. 3. Products of metabolism of pentamidine and 1–9 derivatives.

Fig. 4. Two views of predicted docking pose in the cavity of hERG K⁺ channel for compounds: 4 (cyan), 5 (fuchsia) and 6 (green). For clarity, subunits of the hERG K⁺ channel were removed and key residues (Thr623, Ser624, Tyr652, and Phe656) interacting with the ligands are visible. Upper part: projection of the compounds from the wall side seen perpendicular. Lower part: projection of the compounds from the upper side of the channel.

Fig. 5. Binding modes of compounds 4, 5 and 6 in the hERG K⁺ channel optimized by MD.

Fig. 6. Absence of I_hERG inhibition in response to treatment with 4–9 (1 μM). (A) Representative traces of I_hERG treated with 6 (1 μM) under baseline and 15 min perfusion. Stimulation protocol is shown at the right panel. (B) I–V relationship curves of step (left panels) and tail (right panels) current densities following treatment with 4–9 at baseline, 3.5 min, 7 min and 15 min. (C, D) Summary of step current (C) and tail current (D) densities, determined at +10 mV, under control (untreated) condition and following treatment with 4–9. Summarized data are normalized by their baseline. All data are presented as the mean ± SD. N-values for baseline, 3.5, 7 and 15 min. Measurements are as follows: 4: 17, 10, 9, 6; 5: 8, 7, 7, 6; 6: 17, 8, 8, 6; 7: 10, 10, 9, 8; 8: 10, 9, 8, 8; 9: 9, 9, 9, 5.

Fig. 7. The maturation of hERG is affected differently by pentamidine and its analogues 1–9 in HEK-hERG cells. (A) Western blot results of HEK-hERG cells exposed to pentamidine (10 μM), dofetilide (1 μM) and 1–9 (1 and 10 μM) for 24 h. Total protein ponceau staining was used as a loading control. (B) Summarized data of the ratios of mature and immature hERG expression in HEK-hERG cells after 24 h treatment with pentamidine (10 μM), dofetilide (1 μM) or analogues. ‡ indicated P < 0.05 vs. control, § P < 0.05 vs. pentamidine, # P < 0.05 vs. dofetilide. Control protein ratios were designated as 1. (C) Two concentration comparisons in the maturation ratio of hERG. * P < 0.05 between 1 and 10 μM treatment. Data are presented in mean ± SD. N-values for are as follows: control: 26; pentamidine: 27; dofetilide: 24; 1 (1, 10 μM): 10, 10; 2: 13, 10; 3: 4, 4; 4: 4, 4; 5: 7, 10; 6: 4, 3; 7: 4, 4; 8: 4, 4; 9: 4, 3.

Fig. 8. Dose–response effects of 1, 2 and 5 on hERG maturation. (A) Dose-dependent effect on hERG expression after 24 h treatment with 1, 2 and 5 (0.5–20 μM), pentamidine (10 μM) or dofetilide (1 μM). In the lower panels, ponceau staining reveals equal loading. (B) Summarized results of (A). Data are presented as the ratios of mature and immature hERG. ‡ indicates P < 0.05 vs. control, § P < 0.05 vs. pentamidine, # P < 0.05 vs. dofetilide. N-values for are as follows: control: 29; pentamidine: 27; dofetilide: 27; 1 (all concentrations): 6; 2 (all concentrations): 6; 5: (all concentrations) 3.

Fig. 9. Expression of hERG at the membrane in control (untreated) and HEK-hERG cells treated for 24 h with pentamidine (10 μM), dofetilide (1 μM), 1, 2 or 5 (10 μM). The scale bar represents 20 μm.