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Probing the molecular basis of hERG drug block with unnatural amino acids.

ABSTRACT: Repolarization of the cardiac action potential is primarily mediated by two voltage-dependent potassium currents: I Kr and I Ks . The voltage-gated potassium channel that gives rise to I Kr, Kv11.1 (hERG), is uniquely susceptible to high-affinity block by a wide range of drug classes. Pore residues Tyr652 and Phe656 are critical to potent drug interaction with hERG. It is considered that the molecular basis of this broad-spectrum drug block phenomenon occurs through interactions specific to the aromatic nature of the side chains at Tyr652 and Phe656. In this study, we used nonsense suppression to incorporate singly and doubly fluorinated phenylalanine residues at Tyr652 and Phe656 to assess cation-? interactions in hERG terfenadine, quinidine, and dofetilide block. Incorporation of these unnatural amino acids was achieved with minimal alteration to channel activation or inactivation gating. Our assessment of terfenadine, quinidine, and dofetilide block did not reveal evidence of a cation-? interaction at either aromatic residue, but, interestingly, shows that certain fluoro-Phe substitutions at position 652 result in weaker drug potency.

SUBMITTER: Macdonald LC

PROVIDER: S-EPMC5762913 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Probing the molecular basis of hERG drug block with unnatural amino acids.

Macdonald Logan C LC Kim Robin Y RY Kurata Harley T HT Fedida David D

Scientific reports 20180110 1

Repolarization of the cardiac action potential is primarily mediated by two voltage-dependent potassium currents: I Kr and I Ks . The voltage-gated potassium channel that gives rise to I Kr, Kv11.1 (hERG), is uniquely susceptible to high-affinity block by a wide range of drug classes. Pore residues Tyr652 and Phe656 are critical to potent drug interaction with hERG. It is considered that the molecular basis of this broad-spectrum drug block phenomenon ...[more]

PMID: 29321549

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Project description:A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5-10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F'-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F'-[K(DipR)5]) alone and F'-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F'-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F'-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F'-[K(DipR)5] alone or F'-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

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Probing the molecular basis of hERG drug block with unnatural amino acids.

Publications

Probing the molecular basis of hERG drug block with unnatural amino acids.

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