Molecular Determinants of Pentamidine-Induced hERG Trafficking Inhibition

Dennis, Adrienne T.; Wang, Lu; Wan, Haochuan; Nassal, Drew; Deschênes, Isabelle; Ficker, Eckhard

doi:10.1124/mol.111.075135

Cited by 59 publications

(35 citation statements)

References 55 publications

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“…1A and B), which illustrates that trafficking from the ER to the Golgi was impaired. In fact, increasing evidence shows that drugs affect hERG channels by interfering with their trafficking [5,12]. Numerous studies have shown that misfolding of hERG channels is most often the cause of defects in trafficking.…”

Section: Discussionmentioning

confidence: 99%

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Berberine Induces hERG Channel Deficiency through Trafficking Inhibition

Zhang

Zhi

Huang

et al. 2014

Cell Physiol Biochem

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Aims: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the IKr, which plays an essential role in repolarization of action potentials. hERG channels are targeted by various pro-arrhythmic drugs. Berberine (BBR) was previously found to acutely inhibit hERG currents and prolong action potential duration. The present study aimed to determine long-term effects of BBR on the expression of 135kDa/155kDa hERG and the mechanism. Methods and Results: hERG expression was assessed by western blot. Mature hERG (155 kDa) was reduced, whereas ER-located hERG (135 kDa) was increased by BBR. This indicated that hERG was restricted to the ER and that BBR disrupted channel trafficking. To determine the mechanism of trafficking inhibition, we performed western blot and immunoprecipitation to test folding of hERG by assessing interaction between hERG and Hsp90/Hsp70. Both the expression of Hsp90 and its interaction with hERG were strongly decreased by BBR. These data suggest that BBR reduces channel folding to induce trafficking inhibition. Western blot and confocal imaging were used to further detect whether the unfolded protein response (UPR) was activated. Active ATF6, a marker of the UPR, was activated by BBR. Calnexin and calreticulin, chaperones that are activated by ATF6 to assist channel folding, were also elevated and increasingly colocalized with hERG. These data also demonstrate that the UPR was activated. Immunoprecipitation and western blot assays were performed after BBR treatment to examine ubiquitination and degradation, common endpoints of the UPR. We found that the ER-restricted hERG was ubiquitinized and degraded in the lysosomes and proteasomes. Conclusion: Our study demonstrates that BBR induces hERG channel deficiency by inhibiting channel trafficking after incubation for 24h. Trafficking inhibition activated the UPR, and the ER-restricted hERG was ubiquitinized and degraded in lysosomes and proteasomes.

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Section: Discussionmentioning

confidence: 99%

“…For example, dofetilide and cisapride are proarrhythmic due to their acute blockade of hERG channels [2,3]. Arsenic trioxide, pentamidine and desipramine cause LQTS by altering hERG expression [4,5,6]. Importantly, most of these drugs alter hERG expression by interfering with its trafficking.…”

Section: Introductionmentioning

confidence: 99%

Berberine Induces hERG Channel Deficiency through Trafficking Inhibition

Zhang

Zhi

Huang

et al. 2014

Cell Physiol Biochem

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“…Although a drug’s EC 50 value for chaperoning is strongly correlated with its ligand binding affinity (Kd) and/or its ability to activate/inhibit its protein target [53,94–96] chaperoning EC 50s are generally, but not always [97], 100 fold higher (i.e., less potent) than those required for functional activation/inhibition [80,81,95]. The lower potency of chaperoning presumably derives from lower affinity interactions with immature intermediates, relative to high-affinity interactions with native, mature conformations [71].…”

Section: Pharmacology Of Pcsmentioning

confidence: 99%

Pharmacological chaperoning: A primer on mechanism and pharmacology

Leidenheimer

Ryder

2014

Pharmacological Research

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Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs is considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated.

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“…ER analysis is now routinely used to predict the QT effect in the targeted patient population, including clinical scenarios with doses and formulations not directly evaluated in the TQT study and QT effects in specific populations and under certain conditions (e.g., drug interactions) with increased exposure of the drug [13][14][15][16][17][18][19]. Extensive experience with QT-prolonging drugs demonstrate that the effect on the QT interval is directly related to plasma levels of the drug or main metabolites, with few exceptions (e.g., QT prolongation inhibition of hERG protein trafficking, which is delayed in relation to peak plasma levels [20][21][22]). In our view, it therefore makes sense to focus on QT effects in relation to plasma concentration of the drug, rather than by timepoint without consideration of the pharmacology of the drug, and a wider role for ER analysis in the assessment of drug-induced ECG effects seems justified.…”

Section: Introductionmentioning

confidence: 99%

Implications of the IQ-CSRC Prospective Study: Time to Revise ICH E14

et al. 2015

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Exposure-response (ER) analysis has evolved as an important tool to evaluate the effect of a drug on cardiac repolarization, as reflected in the QTc interval. It has been suggested that careful electrocardiogram (ECG) evaluation in 'first-in-human' studies using ER analysis could replace or serve as an alternative to the E14 'thorough QT' study. This commentary shares and discusses the results of a recently conducted study with the objective to evaluate this approach. Six drugs with a well-characterized QT effect, five of which are known QT prolongers, were evaluated in a study design similar to a conventional single-ascending-dose study. Each drug was given to nine healthy subjects (six for placebo) in two dose levels, which for the positive drugs (ondansetron, quinine, dolasetron, moxifloxacin, and dofetilide) were chosen with the intent to cause 10-12 ms and 15-20 ms QTc prolongation. Replicate 12-lead ECGs were extracted from continuous recordings pre-dose and serially after dosing and paired with drug concentration determinations. The ER criteria for the identification of a QT effect, a statistically significant positive ER slope and an effect above 10 ms, were met with all five positive drugs, and an effect exceeding 10 ms could be excluded at the supratherapeutic dose of the negative drug, levocetirizine. The study results thereby provided evidence to support that careful QT assessment in early phase clinical studies can be used as an alternative to the thorough QT study. Clinical and regulatory implications, as well as limitations of this approach, are discussed in the commentary.

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Molecular Determinants of Pentamidine-Induced hERG Trafficking Inhibition

Cited by 59 publications

References 55 publications

Berberine Induces hERG Channel Deficiency through Trafficking Inhibition

Berberine Induces hERG Channel Deficiency through Trafficking Inhibition

Pharmacological chaperoning: A primer on mechanism and pharmacology

Implications of the IQ-CSRC Prospective Study: Time to Revise ICH E14

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