In silico analysis of the dynamic regulation of cardiac electrophysiology by Kv 11.1 ion-channel trafficking.

Cardiac electrophysiology is regulated by continuous trafficking and internalization of ion channels occurring over minutes to hours. Kv 11.1 (also known as hERG) underlies the rapidly activating delayed-rectifier K+ current (IKr ), which plays a major role in cardiac ventricular repolarization. Experimental characterization of the distinct temporal effects of genetic and acquired modulators on channel trafficking and gating is challenging. Computer models are instrumental in elucidating these effects, but no currently available model incorporates ion-channel trafficking. Here, we present a novel computational model that reproduces the experimentally observed production, forward trafficking, internalization, recycling and degradation of Kv 11.1 channels, as well as their modulation by temperature, pentamidine, dofetilide and extracellular K+ . The acute effects of these modulators on channel gating were also incorporated and integrated with the trafficking model in the O'Hara-Rudy human ventricular cardiomyocyte model. Supraphysiological dofetilide concentrations substantially increased Kv 11.1 membrane levels while also producing a significant channel block. However, clinically relevant concentrations did not affect trafficking. Similarly, severe hypokalaemia reduced Kv 11.1 membrane levels based on long-term culture data, but had limited effect based on short-term data. By contrast, clinically relevant elevations in temperature acutely increased IKr due to faster kinetics, while after 24 h, IKr was decreased due to reduced Kv 11.1 membrane levels. The opposite was true for lower temperatures. Taken together, our model reveals a complex temporal regulation of cardiac electrophysiology by temperature, hypokalaemia, and dofetilide through competing effects on channel gating and trafficking, and provides a framework for future studies assessing the role of impaired trafficking in cardiac arrhythmias. KEY POINTS: Kv 11.1 channels underlying the rapidly activating delayed-rectifier K+ current are important for ventricular repolarization and are continuously shuttled from the cytoplasm to the plasma membrane and back over minutes to hours. Kv 11.1 gating and trafficking are modulated by temperature, drugs and extracellular K+ concentration but experimental characterization of their combined effects is challenging. Computer models may facilitate these analyses, but no currently available model incorporates ion-channel trafficking. We introduce a new two-state ion-channel trafficking model able to reproduce a wide range of experimental data, along with the effects of modulators of Kv 11.1 channel functioning and trafficking. The model reveals complex dynamic regulation of ventricular repolarization by temperature, extracellular K+ concentration and dofetilide through opposing acute (millisecond) effects on Kv 11.1 gating and long-term (hours) modulation of Kv 11.1 trafficking. This in silico trafficking framework provides a tool to investigate the roles of acute and long-term processes on arrhythmia promotion and maintenance.

Virtual screening of DrugBank database for hERG blockers using topological Laplacian-assisted AI models.

The human ether-a-go-go (hERG) potassium channel (Kv11.1) plays a critical role in mediating cardiac action potential. The blockade of this ion channel can potentially lead fatal disorder and/or long QT syndrome. Many drugs have been withdrawn because of their serious hERG-cardiotoxicity. It is crucial to assess the hERG blockade activity in the early stage of drug discovery. We are particularly interested in the hERG-cardiotoxicity of compounds collected in the DrugBank database considering that many DrugBank compounds have been approved for therapeutic treatments or have high potential to become drugs. Machine learning-based in silico tools offer a rapid and economical platform to virtually screen DrugBank compounds. We design accurate and robust classifiers for blockers/non-blockers and then build regressors to quantitatively analyze the binding potency of the DrugBank compounds on the hERG channel. Molecular sequences are embedded with two natural language processing (NLP) methods, namely, autoencoder and transformer. Complementary three-dimensional (3D) molecular structures are embedded with two advanced mathematical approaches, i.e., topological Laplacians and algebraic graphs. With our state-of-the-art tools, we reveal that 227 out of the 8641 DrugBank compounds are potential hERG blockers, suggesting serious drug safety problems. Our predictions provide guidance for the further experimental interrogation of DrugBank compounds' hERG-cardiotoxicity.

Physicochemical QSAR analysis of hERG inhibition revisited: towards a quantitative potency prediction.

In an earlier study (Didziapetris R & Lanevskij K (2016). J Comput Aided Mol Des. 30:1175-1188) we collected a database of publicly available hERG inhibition data for almost 6700 drug-like molecules and built a probabilistic Gradient Boosting classifier with a minimal set of physicochemical descriptors (log P, pKa, molecular size and topology parameters). This approach favored interpretability over statistical performance but still achieved an overall classification accuracy of 75%. In the current follow-up work we expanded the database (provided in Supplementary Information) to almost 9400 molecules and performed temporal validation of the model on a set of novel chemicals from recently published lead optimization projects. Validation results showed almost no performance degradation compared to the original study. Additionally, we rebuilt the model using AFT (Accelerated Failure Time) learning objective in XGBoost, which accepts both quantitative and censored data often reported in protein inhibition studies. The new model achieved a similar level of accuracy of discerning hERG blockers from non-blockers at 10 µM threshold, which can be conceived as close to the performance ceiling for methods aiming to describe only non-specific ligand interactions with hERG. Yet, this model outputs quantitative potency values (IC50) and is not tied to a particular classification cut-off. pIC50 from patch-clamp measurements can be predicted with R2 ≈ 0.4 and MAE < 0.5, which enables ligand ranking according to their expected potency levels. The employed approach can be valuable for quantitative modeling of various ADME and drug safety endpoints with a high prevalence of censored data.

Nitidine chloride inhibits fibroblast like synoviocytes-mediated rheumatoid synovial inflammation and joint destruction by targeting KCNH1.

OBJECTIVE: Nitidine chloride (NC), a natural small molecular compound from traditional Chinese herbal medicine zanthoxylum nitidum, has been shown to exhibit anti-tumor effect. However, its role in autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we investigate the effect of NC in controlling fibroblast-like synoviocytes (FLS)-mediated synovial inflammation and joint destruction in RA and further explore its underlying mechanism(s). METHODS: FLSs were separated from synovial tissues obtained from patients with RA. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression was measured using quantitative RT-PCR. ELISA was used to measure the levels of cytokines and MMPs. Cell proliferation was detected using EdU incorporation. Migration and invasion were evaluated by Boyden chamber assay. RNA sequencing analysis was used to identify the target of NC. Collagen-induced arthritis (CIA) model was used to evaluate the in vivo effect of NC. RESULTS: NC treatment reduced the proliferation, migration, invasion, and lamellipodia formation but not apoptosis of RA FLSs. We also demonstrated the inhibitory effect of NC on TNF-α-induced expression and secretion of IL-6, IL-8, CCL-2, MMP-1 and MMP-13. Furthermore, we identified KCNH1, a gene that encodes ether-à-go-go-1 channel, as a novel targeting gene of NC in RA FLSs. KCNH1 expression was increased in FLSs and synovial tissues from patients with RA compared to healthy controls. KCNH1 knockdown or NC treatment decreased the TNF-α-induced phosphorylation of AKT. Interestingly, NC treatment ameliorated the severity of arthritis and reduced synovial KCNH1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that NC treatment inhibits aggressive and inflammatory actions of RA FLSs by targeting KCNH1 and sequential inhibition of AKT phosphorylation. Our findings suggest that NC might control FLS-mediated rheumatoid synovial inflammation and joint destruction, and be a novel therapeutic agent for RA.

Regional heritability mapping identifies several novel loci (STAT4, ULK4, and KCNH5) for primary biliary cholangitis in the Japanese population.

While the advent of GWAS more than a decade ago has ushered in remarkable advances in our understanding of complex traits, the limitations of single-SNP analysis have also led to the development of several other approaches. Simulation studies have shown that the regional heritability mapping (RHM) method, which makes use of multiple adjacent SNPs jointly to estimate the genetic effect of a given region of the genome, generally has higher detection power than single-SNP GWAS. However, thus far its use has been mostly limited to agricultural settings, and its potential for the discovery of new genes in human diseases is yet to be fully exploited. In this study, by applying the RHM method to primary biliary cholangitis (PBC) in the Japanese population, we identified three novel loci (STAT4, ULK4, and KCNH5) at the genome-wide significance level, two of which (ULK4 and KCNH5) have not been found associated with PBC in any population previously. Notably, these genes could not be detected by using conventional single-SNP GWAS, highlighting the potential of the RHM method for the detection of new susceptibility loci in human diseases. These findings thereby provide strong empirical evidence that RHM is an effective and practical complementary approach to GWAS in this context. Also, liver tissue mRNA microarray analysis revealed higher gene expression levels in ULK4 in PBC patients (P < 0.01). Lastly, we estimated the common SNP heritability of PBC in the Japanese population (0.210 ± 0.026).

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Increased Expression of Kv10.2 in the Hippocampus Attenuates Valproic Acid-Induced Autism-Like Behaviors in Rats.

The role of potassium channels provides suggestive evidence for the etiology of autism. The voltage-gated potassium channel Kv10.2 (KCNH5) is widely expressed in the brain. However, the inherent relationship between Kv10.2 and autism is still unclear. Herein, a rat valproic acid (VPA)-induced autism spectrum disorder model was established. The expression level of Kv10.2 was obviously decreased in the hippocampus of VPA rats. Kv10.2 was mainly localized in neurons. Subsequently, a recombinant lentivirus expressing Kv10.2 was used to upregulate the expression of Kv10.2 in the hippocampus of VPA-exposed rats. The results were promising as injection of the Kv10.2 lentivirus in the hippocampus relieved anxiety and stereotypical behavior, and improved the social and exploratory abilities of rats that were prenatally exposed to VPA. In addition, spectral analysis of electroencephalogram data revealed that animals exposed to VPA exhibited increased high-frequency activity compared with the control rats, and this activity recovered to a certain extent after upregulation of Kv10.2 expression by lentivirus injection. These results suggest that changes in Kv10.2 may play an important role in the etiology of autism, thus providing a promising direction for further research on autism.

Tetrandrine, a novel inhibitor of ether-à-go-go-1 (Eag1), targeted to cervical cancer development.

Mortality-to-incidence ratios in patients with cancer are extremely high, positioning cancer as a major cause of death worldwide. Ether-à-go-go-1 (Eag1) is an ion channel that plays important roles in tumour proliferation, malignant transformation, invasion, metastasis, recurrence, and prognosis. Therefore, identifying potent and specific Eag1 channel inhibitors is crucial. In this study, we identified the first natural inhibitor of Eag1, the traditional Chinese medicine agent tetrandrine, and explored the underlying mechanism. Tetrandrine directly interacted with Eag1 and inhibited the currents in a concentration-dependent manner (IC50 of 69.97 ± 5.2 µM), and the amino acids Ile 550 , Thr 552 , and Gln 557 in the Eag1 C-linker domain were critical for tetrandrine's inhibitory effect. Moreover, tetrandrine reduced the proliferation of HeLa cells and Chinese hamster ovary (CHO) cells stably expressing Eag1 in a concentration-dependent manner. Finally, tetrandrine (30 mg/kg/day) inhibited tumor growth in mice, demonstrating a 64.21% inhibitory rate of HeLa cell-transplanted tumors. These results suggest that tetrandrine is a potent and selective Eag1 channel inhibitor, and could act as a leading compound in the development of therapies for Eag1 ion channel dysfunction-induced diseases.

Identification of small-molecule ion channel modulators in C. elegans channelopathy models.

Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies.

Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 µM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo.

Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC-microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2±26.3nM and 144.8±35.1nM, respectively. In dog ventricular cardiomyocytes, DHE dose-dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10µM DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12±10% (0.05mg/kg/5min) and 60±26% (0.5mg/kg/5min), and induced Torsade de Pointes arrhythmias (TdP, 0.5mg/kg/5min) in 2 rabbits. In cAVB dogs, 0.33mg/kg/5min DHE increased QT duration by 48±10% (P<0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), methanolic extracts of Evodia, DHE and hortiamine dose-dependently prolonged APD. At 3µM DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC-CMs and dose-dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression.

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 µM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 µM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and Ikr blocker.

Administration of Non-Torsadogenic human Ether-à-go-go-Related Gene Inhibitors Is Associated with Better Survival for High hERG-Expressing Glioblastoma Patients.

PURPOSE: Glioblastoma is the most malignant primary brain tumor, with a median survival of less than 2 years. More effective therapeutic approaches are needed to improve clinical outcomes. EXPERIMENTAL DESIGN: Glioblastoma patient-derived cells (GPDC) were isolated from patient glioblastomas and implanted in mice to form xenografts. IHC was performed for human Ether-à-go-go-Related Gene (hERG) expression and tumor proliferation. Sphere-forming assays with the hERG blocker E-4031 were performed on a high and low hERG-expressing lines. A glioblastoma tissue microarray (TMA; 115 patients) was used to correlate hERG expression with patient survival. Clinical data were analyzed to determine whether patient survival was affected by incidental administration of hERG inhibitory drugs and the correlative effect of patient glioblastoma hERG expression levels. RESULTS: hERG expression was upregulated in glioblastoma xenografts with higher proliferative indices. High hERG-expressing GPDCs showed a reduction in sphere formation when treated with hERG inhibitors compared with low hERG-expressing GPDCs. Glioblastoma TMA analysis showed worse survival for glioblastoma patients with high hERG expression versus low expression-43.5 weeks versus 60.9 weeks, respectively (P = 0.022). Furthermore, patients who received at least one hERG blocker had a better survival rate compared with patients who did not (P = 0.0015). Subgroup analysis showed that glioblastoma patients with high hERG expression who received hERG blockers had improved survival (P = 0.0458). There was no difference in survival for low hERG-expressing glioblastoma patients who received hERG blockers (P = 0.4136). CONCLUSIONS: Our findings suggest that hERG is a potential glioblastoma survival marker, and that already approved drugs with non-torsadogenic hERG inhibitory activity may potentially be repurposed as adjuvant glioblastoma therapy in high hERG-expressing glioblastoma patients. Clin Cancer Res; 23(1); 73-80. ©2016 AACRSee related commentary by Arcangeli and Becchetti, p. 3.

Early drug development: assessment of proarrhythmic risk and cardiovascular safety.

INTRODUCTION: hERG assays and thorough ECG trials have been mandated since 2005 to evaluate the QT interval and potential proarrhythmic risk of new chemical entities. The high cost of these studies and the shortcomings inherent in these binary and limited approaches to drug evaluation have prompted regulators to search for more cost effective and mechanistic paradigms to assess drug liability as exemplified by the CiPA initiative and the exposure response ICH E14(R3) guidance document. Areas covered: This review profiles the changing regulatory landscape as it pertains to early drug development and outlines the analyses that can be performed to characterize preclinical and early clinical cardiovascular risk. Expert commentary: It is further acknowledged that the narrow focus on the QT interval needs to be expanded to include a more comprehensive evaluation of cardiovascular risk since unanticipated off target effects have led to the withdrawal of multiple drugs after they had been approved and marketed.

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs.

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K+ current (IKr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific IKr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na+ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca2+ current (ICaL) caused by a decrease in Ca2+-dependent inactivation (CDI). Acute exposure to sparfloxacin, an IKr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack IKr due to E4031 pretreatment. Sparfloxacin reduced peak ICaL but increased late ICaL by slowing its inactivation. In contrast, ketoconazole, an IKr blocker without prolongation of QT interval and TdP produced reduction of both peak and late ICaL, suggesting the role of increased late ICaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca2+ with Ba2+ abolished the sparfloxacin effects on ICaL. In addition, sparfloxacin modulated ICaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native IKr, demonstrated similar increase in late ICaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late ICaL. Thus, drugs that cause delayed ICaL inactivation and IKr blockage may have more adverse effects than those that selectively block IKr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and IKr antagonist potencies.

Discovery of 5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide (TAK-259) as a Novel, Selective, and Orally Active α1D Adrenoceptor Antagonist with Antiurinary Frequency Effects: Reducing Human Ether-a-go-go-Related Gene (hERG) Liabilities.

A novel structural class of iminopyridine derivative 1 was identified as a potent and selective human α1D adrenoceptor (α1D adrenergic receptor; α1D-AR) antagonist against α1A- and α1B-AR through screening of an in-house compound library. From initial structure-activity relationship studies, we found lead compound 9m with hERG K(+) channel liability. To develop analogues with reduced hERG K(+) channel inhibition, a combination of site-directed mutagenesis and docking studies was employed. Further optimization led to the discovery of (R)-9s and 9u, which showed antagonistic activity by a bladder strip test in rats with bladder outlet obstruction, as well as ameliorated cystitis-induced urinary frequency in rats. Ultimately, 9u was selected as a clinical candidate. This is the first study to show the utility of iminopyridine derivatives as selective α1D-AR antagonists and evaluate their effects in vivo.

Inhibition of Schistosoma mansoni ether-a-go-go related gene-encoded potassium channels leads to hypermotility and impaired egg production.

The purpose of this work was to investigate the effect of ether-a-go-go related gene (ERG) potassium channel inhibition on Schistosoma mansoni. Use of dofetilide to block the schistosome ERGs resulted in a striking 'corkscrew' effect. The worms were unable to control their motility; they were hypermotile. The treated worms produced abnormal eggs, some of which consisted of little more than a spine. One of the S. mansoni ERGs (SmERGs), Smp_161140, was chosen for further study by RNAi. The transcript was knocked down to 50% compared to the controls. These RNAi-treated worms demonstrated seizure-like movements. In S. mansoni, as in other organisms, ERG channels seem to play a role in regulating muscle excitability. This work shows that egg production can be greatly reduced by effectively targeting muscle coordination in these important parasites.

Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.