hERG Activity Research Articles

Cardiotoxicity assessment of 31 herbal formulae by activity of hERG potassium channel in HEK 293 cells

Objectives: Drug-induced blockade of the human ether-à-go-go related gene (hERG) potassium ion channel causes acquired long QT syndrome, which is known to cause cardiac arrhythmias and be fatal. To establish safety evidence of herbal formulae, we evaluated the effects of 31 herbal formulae on hERG channel activity.Methods: The current through hERG channel was measured by changing the membrane voltage before and after treatment with 31 herbal formulae in HEK 293 cell overexpressing hERG channel using a whole-cell patch clamp system. The current-voltage curves and the activity curves were fitted, and the hERG activity and 50% inhibitory concentration (IC50) according to each herbal formula were calculated.Results: Chokyungjongok-tang, Oncheong-eum, and Cheongsangbangpung-tang strongly inhibited the hERG activity, with IC50 values of 67.67, 141.2, and 296.3 μg/mL, respectively. Yeonkyopaedok-san, Eunkyo-san, Ukgan-san gajinphibanha, Daegunjoong-tang (except Oryzae gluten), Insamyangyoung-tang, Banhahubak-tang, SokyungHwalhyul-tang, Jodeung-san, Hyeonggaeyeongyo-tang, and Bangkeehwangkee-tang weakly inhibited hERG activity, with IC50 values ranging from 400 to 1000 μg/mL. The other 18 herbal formulae showed very weak hERG activity inhibition of less than 50% at the highest concentration (1000 μg/mL).Conclusion: This study provided safety information on cardiotoxicity by cardiac arrhythmia risk assessment of herbal formulae, and is expected to be a reference data for predicting the safety and risk of herbal formulae.

High-Throughput Chemical Screening and Structure-Based Models to Predict hERG Inhibition.

Simple SummaryCardiovascular disease is the leading cause of death for people of most ethnicities in the United States. The human ether-a-go-go-related gene (hERG) potassium channel plays a pivotal role in cardiac rhythm regulation, and cardiotoxicity associated with hERG inhibition by drug molecules and environmental chemicals is a major public health concern. An evaluation of the effect of environmental chemicals on hERG channel function can help inform the potential public health risks of these compounds. To assess the cardiotoxic effect of diverse drugs and environmental compounds, the Tox21 federal research program has screened a collection of 9667 chemicals for inhibitory activity against the hERG channel. A set of molecular descriptors covering physicochemical and structural properties of chemicals, self-organizing maps, and hierarchical clustering were applied to characterize the chemicals inhibiting hERG. Machine learning approaches were applied to build robust statistical models that can predict the probability of any new chemical to cause cardiotoxicity via this mechanism.Chemical inhibition of the human ether-a -go-go-related gene (hERG) potassium channel leads to a prolonged QT interval that can contribute to severe cardiotoxicity. The adverse effects of hERG inhibition are one of the principal causes of drug attrition in clinical and pre-clinical development. Preliminary studies have demonstrated that a wide range of environmental chemicals and toxicants may also inhibit the hERG channel and contribute to the pathophysiology of cardiovascular (CV) diseases. As part of the US federal Tox21 program, the National Center for Advancing Translational Science (NCATS) applied a quantitative high throughput screening (qHTS) approach to screen the Tox21 library of 10,000 compounds (~7871 unique chemicals) at 14 concentrations in triplicate to identify chemicals perturbing hERG activity in the U2OS cell line thallium flux assay platform. The qHTS cell-based thallium influx assay provided a robust and reliable dataset to evaluate the ability of thousands of drugs and environmental chemicals to inhibit hERG channel protein, and the use of chemical structure-based clustering and chemotype enrichment analysis facilitated the identification of molecular features that are likely responsible for the observed hERG activity. We employed several machine-learning approaches to develop QSAR prediction models for the assessment of hERG liabilities for drug-like and environmental chemicals. The training set was compiled by integrating hERG bioactivity data from the ChEMBL database with the Tox21 qHTS thallium flux assay data. The best results were obtained with the random forest method (~92.6% balanced accuracy). The data and scripts used to generate hERG prediction models are provided in an open-access format as key in vitro and in silico tools that can be applied in a translational toxicology pipeline for drug development and environmental chemical screening.

Identification of novel bioactive compounds from Olea europaea by evaluation of chemical compounds in the OliveNet™ library: in silico bioactivity and molecular modelling, and in vitro validation of hERG activity

BackgroundAs highlighted in the OliveNet™ library, Olea europaea consists of a diverse collection of chemical compounds. We have classified over 600 compounds into 13 main classes and 47 subclasses. Various compounds, including oleuropein and hydroxytyrosol, have been investigated for their potential beneficial effects in multiple human pathologies. However, the vast majority of compounds remain largely unexplored and approximately 50% are currently non-commercially available. MethodHere, we utilise conventional software to characterise the absorption, distribution, metabolism, excretion, and toxicity profile of OliveNet™ compounds. Molecular docking was performed for assessment of P-glycoprotein (P-gp) inhibition and interactions with the human ether-à-go-go-related gene (hERG) channel. Potential hERG ion channel inhibition was calibrated using in vitro patch clamp assays and steered molecular dynamics (SMD) simulations were used to examine membrane permeability of a subset of compounds. ResultsOur findings indicated that 313 out of 675 olive compounds were predicted to be absorbed by the gastrointestinal tract. Hydroxytyrosol required the least amount of force to pass through the lipid bilayer compared to elenolic acid diglucoside in SMD simulations. Based on the ADMET and molecular docking data, the hERG inhibitory activities of verbascoside, oleuropein, and hydroxytyrosol were investigated using patch clamp assays and they were identified as non-inhibitors. ConclusionsWhile the favourable properties of well-known compounds were confirmed, we identified oleuropein aglycone decarboxymethyl dialdehyde acetal form, decarboxymethyl elenolic acid dialdehyde, acetal of decarboxymethyl elenolic acid dialdehyde, methyl malate-β-hydroxytyrosol ester, hydroxytyrosil elenolate, D-(+)-erythro-1-(4-hydroxy-3-methoxy)-214-phenyl-1,2,3-propantriol, (+)-1-acetoxypinoresinol-4″-O-methyl ether, and 3-[1-(hydroxymethyl)-(E)-1-propenyl] glutaric acid as potential candidates for synthesis and further evaluation.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

Development of improved SRC-3 inhibitors as breast cancer therapeutic agents.

Steroid receptor coactivators (SRCs) possess specific and distinct oncogenic roles in the initiation of cancer and in its progression to a more aggressive disease. These coactivators interact with nuclear receptors and other transcription factors to boost transcription of multiple genes, which potentiate cancer cell proliferation, migration, invasion, tumor angiogenesis and epithelial-mesenchymal transition (EMT). Targeting SRCs using small molecule inhibitors (SMIs) is a promising approach to control cancer progression and metastasis. By high-throughput screening analysis, we recently identified SI-2 as a potent SRC SMI. To develop therapeutic agents, SI-10 and SI-12, the SI-2 analogs are synthesized that incorporate the addition of F atoms to the SI-2 chemical structure. As a result, these analogs exhibit a significantly prolonged plasma half-life, minimal toxicity and improved hERG activity. Biological functional analysis showed that SI-10 and SI-12 treatment (5-50 nM) can significantly inhibit viability, migration and invasion of breast cancer cells in vitro and repress the growth of breast cancer PDX organoids. Treatment of mice with 10 mg/kg/day of either SI-10 or SI-12 was sufficient to repress the growth of xenograft tumors derived from MDA-MB-231 and LM2 cells. Furthermore, in spontaneous and experimental metastasis mouse models developed from MDA-MB-231 and LM2 cells, respectively, SI-10 and SI-12 effectively inhibited the progression of breast cancer lung metastasis. These results demonstrate that SI-10 and SI-12 are promising therapeutic agents and are specifically effective in blocking tumor metastasis, a key point in tumor progression to a more lethal state that results in patient mortality in the majority of cases.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 μM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

HERG Channel-Related Cardiotoxicity Assessment of 13 Herbal Medicines

Objectives: As the use of herbal medicinal products (HMPs) increases worldwide, systematic verification of the safety of HMPs is required. The induction of cardiotoxicity is one of the major factors in post-approval withdrawal of medicinal products, and drug-induced cardiotoxicity assessment is emerging as an important step in drug development. In the present study, we evaluated human ether-à-go-go-related gene (hERG) potassium channel-related cardiotoxicity to predict the risk of cardiac arrhythmia in thirteen herbal medicines known to have cardiac toxicity. Methods: We measured the inhibition rate of hERG potassium channel activity of 13 medicinal herbal extracts in hERG-expressing HEK 293 cells using an automated patch-clamping system. Quinidine was used as a positive control for inhibition of hERG activity. Results: Extracts of Evodiae Fructus, Strychni Semen, and Corydalis Tuber potently inhibited the activity of hERG, and IC<sub>50</sub> values were 3.158, 19.87, and 41.26 <i>μ</i>g/mL, respectively. Cnidi Fructus, Ephedra Herba, Lithospermi Radix, Polygoni Multiflori Radix, Visci Ramulus et Folium, Asiasari Radix et Rhizoma, and Scolopendra weakly inhibited hERG activity, and the IC<sub>50</sub> value for each herbal medicine was more than 400 <i>μ</i>g/mL. Aconiti Kusnezoffii Tuber and two types of Aconiti Lateralis Radix Preparata (Po and Yeom) had weak inhibitory activity against hERG, and the IC<sub>50</sub> values were more than 700 <i>μ</i>g/mL. The IC<sub>50</sub> value of quinidine against hERG was 1.021 <i>μ</i>M. Conclusion: Evodiae Fructus, Strychni Semen, and Corydalis Tuber acted as potent inhibitors against hERG. These herbal medicines may cause cardiac arrhythmia through QT prolongation, so care should be taken when taking them.

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243.

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.

CardioTox net: a robust predictor for hERG channel blockade based on deep learning meta-feature ensembles

MotivationEther-a-go-go-related gene (hERG) channel blockade by small molecules is a big concern during drug development in the pharmaceutical industry. Blockade of hERG channels may cause prolonged QT intervals that potentially could lead to cardiotoxicity. Various in-silico techniques including deep learning models are widely used to screen out small molecules with potential hERG related toxicity. Most of the published deep learning methods utilize a single type of features which might restrict their performance. Methods based on more than one type of features such as DeepHIT struggle with the aggregation of extracted information. DeepHIT shows better performance when evaluated against one or two accuracy metrics such as negative predictive value (NPV) and sensitivity (SEN) but struggle when evaluated against others such as Matthew correlation coefficient (MCC), accuracy (ACC), positive predictive value (PPV) and specificity (SPE). Therefore, there is a need for a method that can efficiently aggregate information gathered from models based on different chemical representations and boost hERG toxicity prediction over a range of performance metrics.ResultsIn this paper, we propose a deep learning framework based on step-wise training to predict hERG channel blocking activity of small molecules. Our approach utilizes five individual deep learning base models with their respective base features and a separate neural network to combine the outputs of the five base models. By using three external independent test sets with potency activity of IC50 at a threshold of 10 upmum, our method achieves better performance for a combination of classification metrics. We also investigate the effective aggregation of chemical information extracted for robust hERG activity prediction. In summary, CardioTox net can serve as a robust tool for screening small molecules for hERG channel blockade in drug discovery pipelines and performs better than previously reported methods on a range of classification metrics.

Abstract 296: Structure-based and property-based drug design of AZD5305, a highly selective PARP1 inhibitor and trapper

Abstract Since the approval of olaparib in 2014 for BRCA mutated (BRCAm) ovarian cancer, many PARP inhibitors have been developed and have seen widespread success. However, as a class, these drugs are not without adverse events which have limited their ability to be combined with chemotherapy. Most first generation PARP inhibitors were developed and optimized before the concept of PARP1-DNA trapping was discovered as the mechanism by which PARP inhibitors exert their synthetic lethal effects on BRCAm cells. Moreover, the first generation PARP inhibitors were not optimized for selectivity across the PARP family potentially driving undesirable side effects, including intestinal toxicity from tankyrase inhibition or hematological toxicity from PARP2 inhibition. With this in mind, we set out to discover a best-in-class, second generation PARP inhibitor that was highly selective for PARP1 over the other 16 members of the PARP family, as well as a highly potent PARP1-DNA trapper. PARP1 and PARP2 have a highly similar amino acid sequence, and most of the residues around the nicotinamide binding site are identical. However, there are some key residue differences in the helical domain which serves a regulator of the nicotinamide binding pocket. The publication of NMS-P118 in 2015 by Nerviano Medical Sciences showed that a highly selective PARP1 inhibitor could be found. This work inspired us to screen an extensive list of previously reported PARP inhibitors for selectivity against PARP2 and we found that FR257516 met the selectivity criteria as previously reported, but lacked the ability to trap PARP1 to DNA and hence lacked any activity in a cell colony formation assay in DLD-1 BRCA2-/- cells. Using parallel chemistry to generate diverse analogs, X-ray crystallography to enable structure-based design, and exploration of multiple nicotinamide mimetic cores, we were able to generate lead compound AZ4554, which was a PARP1 selective PARP1-DNA trapper with potent activity in BRCAm cells. Using concepts of property-based drug design, we were able to optimize lead compound AZ4554 into candidate drug AZD5305, making key improvements in secondary pharmacology, including reducing hERG activity, and intrinsic clearance in human microsomes through the introduction of polar atoms to lower logD without compromising permeability or oral bioavailability. AZD5305 is a highly selective binder of PARP1 over PARP2 and other PARP enzymes by fluorescence polarization, surface plasmon resonance, and single molecule spectroscopy. It is highly potent against DLD-1 BRCA2-/- cells, while sparing isogenic BRCA WT cells. The secondary pharmacology of AZD5305 is remarkably clean, with hERG activity >40 µM. AZD5305 has a very favorable pre-clinical PK profile, low predicted human dose, and has shown efficacy in an MDA-MB-436 mouse xenograft model. Citation Format: Sudhir Hande, Amber Balazs, Sébastien L. Degorce, Kevin Embrey, Avipsa Ghosh, Sonja J. Gill, Anders Gunnarsson, Giuditta Illuzzi, Jordan Lane, Carrie Larner, Elisabetta Leo, Andrew Madin, Lisa McWilliams, Mark J. O'Connor, Jonathan Orme, Fiona Pachl, Martin Packer, Andy Pike, Philip Rawlins, Marianne Schimpl, Anna D. Staniszewska, Andrew Zhang, Xiaolan Zheng, Jeffrey W. Johannes. Structure-based and property-based drug design of AZD5305, a highly selective PARP1 inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 296.

Creation of a new class of radiosensitizers for glioblastoma based on the mibefradil pharmacophore.

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.

Discovery of Ethyl Ketone-Based Highly Selective HDACs 1, 2, 3 Inhibitors for HIV Latency Reactivation with Minimum Cellular Potency Serum Shift and Reduced hERG Activity

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

An Asymmetric C-Type Inactivated Structure of HERG Channel and Its Binding to Chemically Diverse Drugs

The fast C-type inactivation displayed by the voltage-activated potassium channel hERG plays a critical role in the repolarization of cardiac cells, and malfunction caused by non-specific binding of drugs or naturally occurring missense mutations affecting inactivation can lead to pathologies. From a pharmacological standpoint, the C-type inactivated state of hERG could bind a large set of chemically diverse drugs. Therefore, understanding the structural basis of C-type inactivation in hERG could help screening compounds for their impact on hERG activity, an indispensable step for drug development. In present study, long-timescale molecular dynamics simulation, free energy landscape calculations, and electrophysiological experiments are combined to address the structural and functional impacts of several disease-associated mutations. Our data suggest that C-type inactivation in hERG is associated with an asymmetrical constricted-like conformation of the selectivity filter, identifying F627 side-chain and the hydrogen bond between Y616 and N627 are key determinants. The energetics of inactivation in hERG, Shaker, and KcsA shows that the C-type inactivation rate depends on the degree of opening of the intracellular gate via the filter-gate allosteric coupling, providing a molecular explanation of the fast inactivation in hERG channel. After the selectivity filter constricts asymmetrically, it is observed that the key residue in the binding site, Y652 in all four subunits, shows high structural flexibility. The snapshots were clustered based on the conformational similarity of Y652, and typical conformations were selected to perform ensemble docking with multiple chemically diverse drugs. The docking results show there are different C-type inactivated conformations more favorable for drugs binding compared with the first experimental conductive structure, demonstrating that the high flexibility of Y652 after constriction is the structural basis for the binding of chemically diverse drug to the hERG channel.

Cardiac hERG K+ Channel as Safety and Pharmacological Target.

The human ether-á-go-go related gene (hERG, KCNH2) encodes the pore-forming subunit of the potassium channel responsible for a fast component of the cardiac delayed rectifier potassium current (IKr). Outward IKr is an important determinant of cardiac action potential (AP) repolarization and effectively controls the duration of the QT interval in humans. Dysfunction of hERG channel can cause severe ventricular arrhythmias and thus modulators of the channel, including hERG inhibitors and activators, continue to attract intense pharmacological interest. Certain inhibitors of hERG channel prolong the action potential duration (APD) and effective refractory period (ERP) to suppress premature ventricular contraction and are used as class III antiarrhythmic agents. However, a reduction of the hERG/IKr current has been recognized as a predominant mechanism responsible for the drug-induced delayed repolarization known as acquired long QT syndromes (LQTS), which is linked to an increased risk for "torsades de pointes" (TdP) ventricular arrhythmias and sudden cardiac death. Many drugs of different classes and structures have been identified to carry TdP risk. Hence, assessing hERG/IKr blockade of new drug candidates is mandatory in the drug development process according to the regulatory agencies. In contrast, several hERG channel activators have been shown to enhance IKr and shorten the APD and thus might have potential antiarrhythmic effects against pathological LQTS. However, these activators may also be proarrhythmic due to excessive shortening of APD and the ERP.

Functional Characterization of KCNH2 genetic variants, encoding hERG potassium channel, as a clinically-relevant information for type 2 LQTS syndrome

hERG potassium channels are essential for normal cardiac electrical activity. Mutations in the hERG gene (KCNH2) cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. More than 1000 mutations in hERG sequence are described in databases and most of them remains to be characterized. The objective of the work is to determine the consequences of the variants identified in LQT2 patients on the expression and function of the hERG channel. An optimized sequence hERG fused to pHluorin tag was synthesized. By amplifying mutated overlapping fragments, and using the Gibson assembly strategy, we constructed hERG-pHluorin plasmids with missense variations. CHO cells were transfected by electroporation with the Maxcyte system or Fugene method, and studied after 40 hours. Confocal images and flow cytometry analysis were used to evaluate the percentage of transfected cells and to quantify the membrane channel expression, through pH sensitive pHluorin tag. Conventional or automated patch clamp were used to evaluate the hERG activity. In the present work, we constructed hERG plasmids carrying 48 variations identified in patients in France. Higher efficiency of transfection was observed with electroporation as compared to the Fugene method. The activity of hERG-pHluorin was validated in manual patch-clamp, the pHluorin tag does not interfere with channel activity. Characterization of several mutations in conventional patch clamp showed difference in current amplitude. Automated patch-clamp data was compared to manual patch-clamp, as an initial validation for high throughput screening of all constructed mutations. High-throughput characterization of KCNH2 genetic variants is relevant to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information will be indicated in a patient database, as a basis of personalized medicine.

Design and synthesis of novel desfluoroquinolone-aminopyrimidine hybrids as potent anti-MRSA agents with low hERG activity

Despite the fact that the introduction of a fluorine atom at the C-6 position has resulted in the evolution of fluoroquinolones, fluoroquinolone-induced cardiac toxicity has drawn considerable attention. In this context, desfluoroquinolone-based hybrids with involvement of C-7 aminopyrimidine functional group were designed and synthesized. The biological results showed majority of these hybrids still demonstrated potent anti-MRSA activity with MIC values between 0.38 and 1.5 μg/mL, despite the lack of the typical C-6 fluorine atom. Particularly, the most active B14 exhibited activities at submicromolar concentrations against a panel of MRSA strains including vancomycin-intermediate strains, levofloxacin-resistant isolates, and linezolid-resistant isolates, etc. As expected, it also displayed highly selective toxicity toward bacterial cells and low hERG inhibition. Further resistance development study indicated MRSA is unlikely to acquire resistance against B14. The docking study revealed that two hydrogen bonds were formed between the C-7 substituent and the surrounding DNA bases, which might contribute to overcome resistance by reducing the dependence on the magnesium-water bridge interactions with topoisomerase IV. These results indicate a promising strategy for developing new antibiotic quinolones to combat multidrug resistance and cardiotoxicity.

Optimization of a series of potent, selective and orally bioavailable SYK inhibitors

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Crispri Ion Channel Gene Modulation in Human iPSC-Cardiomyocytes

Cardiotoxicity is a major limiting factor of drugs going to market. Current means to assess cardiotoxicity are outdated as compounds are only screened for hERG (KCNH2) channel sensitivity. The sole measurement of hERG activity is restrictive as there are multiple modes in which a drug may induce off-target effects and potentiate arrhythmia. Therefore, there is a need for models measuring multiple channel effects on cardiomyocytes. Our objective was to functionally validate loss-of-function gene perturbations in cardiac electrophysiology to potentiate a high-throughput platform for cardiotoxicity studies. We employed CRISPRi, a means for highly specific gene modulation, commercial pre-differentiated human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM), and an all-optical dynamic cardiac electrophysiology system (OptoDyCE) which simultaneously records voltage and calcium in high-throughput (96-well) format. Pre-differentiated cardiomyocytes were transduced with sgRNAs subcloned into a lentiviral vector containing an eGFP fluorescent marker. sgRNAs were designed around the transcription start site of Kir2.1(KCNJ2) and KCNH2. Five days post-transduction, hiPSC-CMs were transfected with a dox-inducible dCas9-KRAB, containing a mCherry fluorescent marker. A near-infrared voltage-sensitive dye, BeRST, was used to monitor action potential duration (APD) and responses were recorded using the OptoDyCE system. sgRNAs targeting KCNJ2, resulted in about a 15-40% decrease in basic cycle length. Additionally, sgRNAs, targeting KCNH2, exhibited about a 10-40% increase in APD at 90% (APD90) in spontaneous recordings and showed about a 5-20% increase in APD90 from hiPSC-CMs subjected to pacing at 0.5Hz. Considering the role of these two K+ channels in modulating heart rate and repolarization properties, these results were within expected outcomes. Our data corroborates the potential of a CRISPRi-based platform for precise gene modulation of ion channels with all-optical cardiac electrophysiology technology for high throughput functional interrogation in already differentiated hiPSC-CMs for human cardiac disease modelling, cell therapies and drug discovery.

An Update on the Structure of hERG.

The human voltage-sensitive K+ channel hERG plays a fundamental role in cardiac action potential repolarization, effectively controlling the QT interval of the electrocardiogram. Inherited loss- or gain-of-function mutations in hERG can result in dangerous “long” (LQTS) or “short” QT syndromes (SQTS), respectively, and the anomalous susceptibility of hERG to block by a diverse range of drugs underlies an acquired LQTS. A recent open channel cryo-EM structure of hERG should greatly advance understanding of the molecular basis of hERG channelopathies and drug-induced LQTS. Here we describe an update of recent research that addresses the nature of the particular gated state of hERG captured in the new structure, and the insight afforded by the structure into the molecular basis for high affinity drug block of hERG, the binding of hERG activators and the molecular basis of hERG's peculiar gating properties. Interpretation of the pharmacology of natural SQTS mutants in the context of the structure is a promising approach to understanding the molecular basis of hERG inactivation, and the structure suggests how voltage-dependent changes in the membrane domain may be transmitted to an extracellular “turret” to effect inactivation through aromatic side chain motifs that are conserved throughout the KCNH family of channels.