Electrogenic reaction step and phospholipid translocation pathway of the mammalian P4-ATPase ATP8A2.

ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.

Stimulation of Ca2+ -ATPase Transport Activity by a Small-Molecule Drug.

The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small-molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP-dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50 =6.0±0.3 µM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases.

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

Label-Free Bioelectrochemical Methods for Evaluation of Anticancer Drug Effects at a Molecular Level.

Cancer is a multifactorial family of diseases that is still a leading cause of death worldwide. More than 100 different types of cancer affecting over 60 human organs are known. Chemotherapy plays a central role for treating cancer. The development of new anticancer drugs or new uses for existing drugs is an exciting and increasing research area. This is particularly important since drug resistance and side effects can limit the efficacy of the chemotherapy. Thus, there is a need for multiplexed, cost-effective, rapid, and novel screening methods that can help to elucidate the mechanism of the action of anticancer drugs and the identification of novel drug candidates. This review focuses on different label-free bioelectrochemical approaches, in particular, impedance-based methods, the solid supported membranes technique, and the DNA-based electrochemical sensor, that can be used to evaluate the effects of anticancer drugs on nucleic acids, membrane transporters, and living cells. Some relevant examples of anticancer drug interactions are presented which demonstrate the usefulness of such methods for the characterization of the mechanism of action of anticancer drugs that are targeted against various biomolecules.

Niosomal Formulation of a Lipoyl-Carnosine Derivative Targeting TRPA1 Channels in Brain.

The transient receptor potential akyrin type-1 (TRPA1) is a non-selective cation channel playing a pivotal role in pain sensation and neurogenic inflammation. TRPA1 channels expressed in the central nervous system (CNS) have a critical role in the modulation of cortical spreading depression (CSD), which is a key pathophysiological basis of migraine pain. ADM_09 is a recently developed lipoic acid-based TRPA1 antagonist that is able to revert oxaliplatin-induced neuropathic pain and inflammatory trigeminal allodynia. In this context, aiming at developing drugs that are able to target TRPA1 channels in the CNS and promote an antioxidant effect, permeability across the blood-brain barrier (BBB) represents a central issue. Niosomes are nanovesicles that can be functionalized with specific ligands selectively recognized by transporters expressed on the BBB. In this work, the activity of ADM_09 on neocortex cultures was studied, and an efficient formulation to cross the BBB was developed with the aim of increasing the concentration of ADM_09 into the brain and selectively delivering it to the CNS rapidly after parenteral administration.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic.

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

Superparamagnetic iron oxide nanoparticles (SPIONs) modulate hERG ion channel activity.

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as diagnostic agents in magnetic resonance imaging (MRI), for drug delivery vehicles and in hyperthermia treatment of tumors. Although the potential benefits of SPIONs are considerable, there is a distinct need to identify any potential cellular damage associated with their use. Since human ether à go-go-related gene (hERG) channel, a protein involved in the repolarization phase of cardiac action potential, is considered one of the main targets in the drug discovery process, we decided to evaluate the effects of SPIONs on hERG channel activity and to determine whether the oxidation state, the dimensions and the coating of nanoparticles (NPs) can influence the interaction with hERG channel. Using patch clamp recordings, we found that SPIONs inhibit hERG current and this effect depends on the coating of NPs. In particular, SPIONs with covalent coating aminopropylphosphonic acid (APPA) have a milder effect on hERG activity. We observed that the time-course of hERG channel modulation by SPIONs is biphasic, with a transient increase (∼20% of the amplitude) occurring within the first 1-3 min of perfusion of NPs, followed by a slower inhibition. Moreover, in the presence of SPIONs, deactivation kinetics accelerated and the activation and inactivation I-V curves were right-shifted, similarly to the effect described for the binding of other divalent metal ions (e.g. Cd2+ and Zn2+). Finally, our data show that a bigger size and the complete oxidation of SPIONs can significantly decrease hERG channel inhibition. Taken together, these results support the view that Fe2+ ions released from magnetite NPs may represent a cardiac risk factor, since they alter hERG gating and these alterations could compromise the cardiac action potential.

A Comparative Study of Phosphatidylcholine versus Phosphatidylserine-Based Solid Supported Membranes for the Preparation of Liposome-Rich Interfaces.

Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.

Drug Interactions With the Ca2+-ATPase From Sarco(Endo)Plasmic Reticulum (SERCA).

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is an intracellular membrane transporter that utilizes the free energy provided by ATP hydrolysis for active transport of Ca2+ ions from the cytoplasm to the lumen of sarco(endo)plasmic reticulum. SERCA plays a fundamental role for cell calcium homeostasis and signaling in muscle cells and also in cells of other tissues. Because of its prominent role in many physiological processes, SERCA dysfunction is associated to diseases displaying various degrees of severity. SERCA transport activity can be inhibited by a variety of compounds with different chemical structures. Specific SERCA inhibitors were identified which have been instrumental in studies of the SERCA catalytic and transport mechanism. It has been proposed that SERCA inhibition may represent a novel therapeutic strategy to cure certain diseases by targeting SERCA activity in pathogens, parasites and cancer cells. Recently, novel small molecules have been developed that are able to stimulate SERCA activity. Such SERCA activators may also offer an innovative and promising therapeutic approach to treat diseases, such as heart failure, diabetes and metabolic disorders. In the present review the effects of pharmacologically relevant compounds on SERCA transport activity are presented. In particular, we will discuss the interaction of SERCA with specific inhibitors and activators that are potential therapeutic agents for different diseases.

Molecular Insights into hERG Potassium Channel Blockade by Lubeluzole.

BACKGROUND/AIMS: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. METHODS: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. RESULTS: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. CONCLUSIONS: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Monitoring Interactions Inside Cells by Advanced Spectroscopies: Overview of Copper Transporters and Cisplatin.

BACKGROUND: Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum- based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation. METHODS: The use of powerful spectroscopic, spectrometric and computational methods has allowed a deep structural insight into the mode of interaction of platinum drugs with the metal-binding domains of the transporter proteins. RESULTS: This review article focuses on the mode in which platinum drugs can compete with copper ion for binding to transport proteins and consequent structural and biological effects. Three types of transporters are discussed in detail: copper transporter 1 (Ctr1), the major responsible for Cu+ uptake; antioxidant-1 copper chaperone (Atox1), responsible for copper transfer within the cytoplasm; and copper ATPases (ATP7A/B), responsible for copper export into specific subcellular compartments and outside the cell. CONCLUSION: The body of knowledge summarized in this review can help in shaping current chemotherapy to optimize the efficacy of platinum drugs (particularly in relation to resistance) and to mitigate adverse effects on copper metabolism.

Lipoyl-Homotaurine Derivative (ADM_12) Reverts Oxaliplatin-Induced Neuropathy and Reduces Cancer Cells Malignancy by Inhibiting Carbonic Anhydrase IX (CAIX).

Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM_12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells' aggressiveness.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA.

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Discovery of a new mexiletine-derived agonist of the hERG K+ channel.

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in the rapid component (IKr) of cardiac action potential repolarization phase. A large number of structurally different compounds block hERG and cause a high risk of arrhythmias. Among the drugs that block hERG channel, a few compounds have been identified as hERG channel activators. Such compounds may be useful, at least in theory, for the treatment of long term QT syndrome. Here we describe a new activator of hERG channel, named MC450. This compound is a symmetric urea, derived from (R)-mexiletine. Using patch-clamp recordings, we found that MC450 increased the activation current of hERG channel, with an EC50 of 41±4µM. Moreover MC450 caused a depolarizing shift in the voltage dependence of inactivation from -64.1±1.2mV (control), to -35.9±1.4mV, whereas it had no effect on the voltage dependence of activation. Furthermore, MC450 slowed current inactivation and the effect of MC450 was attenuated by the inactivation-impaired double mutant G628C/S631C.

Effect of cisplatin on the transport activity of PII-type ATPases.

Cisplatin (cis-diamminedichlorido-Pt(ii)) is extensively used as a chemotherapeutic agent against various types of tumors. However, cisplatin administration causes serious side effects, including nephrotoxicity, ototoxicity and neurotoxicity. It has been shown that cisplatin can interact with P-type ATPases, e.g., Cu+-ATPases (ATP7A and ATP7B) and Na+,K+-ATPase. Cisplatin-induced inhibition of Na+,K+-ATPase has been related to the nephrotoxic effect of the drug. To investigate the inhibitory effects of cisplatin on the pumping activity of PII-type ATPases, electrical measurements were performed on sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Na+,K+-ATPase embedded in vesicles/membrane fragments adsorbed on a solid-supported membrane. We found that cisplatin inhibits SERCA and Na+,K+-ATPase only when administered without a physiological reducing agent (GSH); in contrast, inhibition was also observed in the case of Cu+-ATPases in the presence of 1 mM GSH. Our results indicate that cisplatin is a much stronger inhibitor of SERCA (with an IC50 value of 1.3 µM) than of Na+,K+-ATPase (with an IC50 value of 11.1 µM); moreover, cisplatin inhibition of Na+,K+-ATPase is reversible, whereas it is irreversible in the case of SERCA. In the absence of a physiological substrate, while Cu+-ATPases are able to translocate cisplatin, SERCA and Na+,K+-ATPase do not perform ATP-dependent cisplatin displacement.

Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B.

ATP7A and ATP7B are Cu+ -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu+ ions across the membrane from delivery to acceptor proteins without establishing a free Cu+ gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper. © 2016 IUBMB Life, 69(4):218-225, 2017.

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues.

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.

Antimony-Phosphomolybdate ATPase Assay.

Hydrolytic activity is an important functional parameter of enzymes like adenosinetriphosphatases (ATPases). It is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. Here, we describe a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate and may be valuable in biochemical and biomedical investigations of ATPase enzymes as well as in high-throughput drug screening. This method has been successfully applied to native and recombinant ATPases.

Electrophysiological Measurements on Solid Supported Membranes.

The solid supported membrane (SSM) represents a convenient model system for a biological membrane with the advantage of being mechanically so stable that solutions can be rapidly exchanged at the surface. The SSM consists of a hybrid alkanethiol-phospholipid bilayer supported by a gold electrode. Proteoliposomes, membrane vesicles, or membrane fragments containing the transport protein of interest are adsorbed on the SSM surface and are subjected to a rapid substrate concentration jump. The substrate concentration jump activates the protein and the charge displacement concomitant with its transport activity is recorded as a current transient. Since this technique is well suited for the functional characterization of electrogenic membrane transporters, it is expected to become a promising platform technology for drug screening and development.

Inhibition of hERG potassium channel by the antiarrhythmic agent mexiletine and its metabolite m-hydroxymexiletine.

Mexiletine is a sodium channel blocker, primarily used in the treatment of ventricular arrhythmias. Moreover, recent studies have demonstrated its therapeutic value to treat myotonic syndromes and to relieve neuropathic pain. The present study aims at investigating the direct blockade of hERG potassium channel by mexiletine and its metabolite m-hydroxymexiletine (MHM). Our data show that mexiletine inhibits hERG in a time- and voltage-dependent manner, with an IC50 of 3.7 ± 0.7 µmol/L. Analysis of the initial onset of current inhibition during a depolarizing test pulse indicates mexiletine binds preferentially to the open state of the hERG channel. Looking for a possible mexiletine alternative, we show that m-hydroxymexiletine (MHM), a minor mexiletine metabolite recently reported to be as active as the parent compound in an arrhythmia animal model, is a weaker hERG channel blocker, compared to mexiletine (IC50 = 22.4 ± 1.2 µmol/L). The hERG aromatic residues located in the S6 helix (Tyr652 and Phe656) are crucial in the binding of mexiletine and the different affinities of mexiletine and MHM with hERG channel are interpreted by modeling their corresponding binding interactions through ab initio calculations. The simulations demonstrate that the introduction of a hydroxyl group on the meta-position of the aromatic portion of mexiletine weakens the interaction of the drug xylyloxy moiety with Tyr652. These results provide further insights into the molecular basis of drug/hERG interactions and, in agreement with previously reported results on clofilium and ibutilide analogs, support the possibility of reducing hERG potency and related toxicity by modifying the aromatic pattern of substitution of clinically relevant compounds.