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Ferroptosis is an emerging target in rheumatoid arthritis (RA). We previously reported that transient receptor potential melastatin 7 (TRPM7) expression is correlated with RA cartilage destruction and demonstrated that TRPM7 mediates ferroptosis in chondrocytes. Here, we further determined the role and mechanism of (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593), a TRPM7 inhibitor, in chondrocyte ferroptosis of RA. We established in vitro models of ferroptosis in human chondrocytes (C28/I2 cells) by using ferroptosis inducer Erastin. The results showed that NS8593 could protect C28/I2 cells from ferroptosis by inhibiting TRPM7 channel, which was manifested by restoring cell viability, reducing cytotoxicity, affecting the expression of ferroptosis marker protein, and restoring redox balance to alleviate Erastin-induced oxidative stress injury. Mechanistically, the Heme oxygenase-1 (HO-1) axis responded to Erastin stimulation, which resulted in TRPM7-mediated chondrocyte ferroptosis, NS8593 could reduce the expression of HO-1 by inhibiting TRPM7 channel. Moreover, NS8593 alleviated articular cartilage destruction and inhibited chondrocyte ferroptosis in AA rats. In conclusion, NS8593 mitigated articular cartilage damage and chondrocyte ferroptosis through the TRPM7/HO-1 pathway, suggesting that NS8593 may be a potential novel drug for the treatment of RA., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

COVID-19 has spread around the world and caused serious public health and social problems. Although several vaccines have been authorized for emergency use, new effective antiviral drugs are still needed. Some repurposed drugs including Chloroquine, Hydroxychloroquine and Remdesivir were immediately used to treat COVID-19 after the pandemic. However, the therapeutic effects of these drugs have not been fully demonstrated in clinical studies. In this paper, we found an antimalarial drug, Naphthoquine, showed good broad-spectrum anti-coronavirus activity. Naphthoquineinhibited HCoV-229E, HCoV-OC43 and SARS-CoV-2 replication in vitro, with IC 50 = 2.05 ± 1.44 μM, 5.83 ± 0.74 μM, and 2.01 ± 0.38 µM, respectively. Time-of-addition assay was also performed to explore at which stage Naphthoquine functions during SARS-CoV-2 replication. The results suggested that Naphthoquine may influence virus entry and post-entry replication. Considering the safety of Naphthoquine was even better than that of Chloroquine, we think Naphthoquine has the potential to be used as a broad-spectrum drug for coronavirus infection.

Background: Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone., Methods: An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice., Results: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 μM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection., Conclusion: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis., (© 2021. The Author(s).)

Liver fibrosis is one of the most common pathological consequences of chronic liver diseases (CLD). To develop effective antifibrotic strategies, a novel class of 1-(substituted phenyl)-1,8-naphthalidine-3-carboxamide derivatives were designed and synthesized. By means of the collagen type I α 1 (COL1A1)-based screening and cytotoxicity assay in human hepatic stellate cell (HSC) line LX-2, seven compounds were screened out from total 60 derivatives with high inhibitory effect and relatively low cytotoxicity for further COL1A1 mRNA expression analysis. It was found that compound 17f and 19g dose-dependently inhibited the expression of fibrogenic markers, including α-smooth muscle actin (α-SMA), matrix metalloprotein 2 (MMP-2), connective tissue growth factor (CTGF) and transforming growth factor β1 (TGFβ1) on both mRNA and protein levels. Further mechanism studies indicated that they might suppress the hepatic fibrogenesis via inhibiting both PI3K/AKT/Smad and non-Smad JAK2/STAT3 signaling pathways. Furthermore, 19g administration attenuated hepatic histopathological injury and collagen accumulation, and reduced fibrogenesis-associated protein expression in liver tissues of bile duct ligation (BDL) rats, showing significant antifibrotic effect in vivo. These findings identified 1,8-naphthalidine derivatives as potent anti-hepatic fibrosis agents, and provided valuable information for further structure optimization., (Copyright © 2021. Published by Elsevier Ltd.)

In this study, we investigated natural vascular scaffolding (NVS) treatment on vascular functionality using freshly isolated human popliteal arteries in vitro. Arteries were exposed to intraluminal NVS treatment consisting of a compound (4 amino-1,8-naphthalimide) photoactivated by a 450-nm light-emitting light fiber placed inside the artery. This procedure results in covalent linking between the extracellular matrix proteins to achieve a larger vessel diameter post-angioplasty and minimizing elastic recoil. Immediately following NVS treatment, rings were cut from the treated arteries and mounted in organ baths for contractility testing in response to U46619 and sodium nitroprusside. We also investigated the effect of NVS treatment on IL-6 cytokine release from vascular rings following a 4-h organoculture post-NVS treatment. Based on our results, we conclude that exposure of the vessels to NVS treatment does not adversely affect the contractile responsiveness of the vascular smooth muscle and exerts no pro-inflammatory effect. Graphical abstract.

Ventricular arrhythmia (VA) is the major cause of death in patients with left ventricular (LV) hypertrophy and/or acute ischemia. We hypothesized that apamin, a blocker of small-conductance Ca 2+ -activated K + (SK) channels, alters Ca 2+ handling and exhibits anti-arrhythmic effects in ventricular myocardium. Spontaneous hypertensive rats were used as a model of LV hypertrophy. A dual optical mapping of membrane potential ( V m ) and intracellular calcium (Ca i ) was performed during global hypoxia (GH) on the Langendorff perfusion system. The majority of pacing-induced VAs during GH were initiated by triggered activities. Pretreatment of apamin (100 nmol/L) significantly inhibited the VA inducibility. Compared with SK channel blockers (apamin and NS8593), non-SK channel blockers (glibenclamide and 4-AP) did not exhibit anti-arrhythmic effects. Apamin prevented not only action potential duration (APD 80 ) shortening (-18.7 [95% confidence interval, -35.2 to -6.05] ms vs. -2.75 [95% CI, -10.45 to 12.65] ms, P = 0.04) but also calcium transient duration (CaTD 80 ) prolongation (14.52 [95% CI, 8.8-20.35] ms vs. 3.85 [95% CI, -3.3 to 12.1] ms, P < 0.01), thereby reducing CaTD 80 - APD 80 , which denotes "Ca i / V m uncoupling" (33.22 [95% CI, 22-48.4] ms vs. 6.6 [95% CI, 0-14.85] ms, P < 0.01). The reduction of Ca i / V m uncoupling was attributable to less prolonged Ca 2+ decay constant and suppression of diastolic Ca i increase by apamin. The inhibition of VA inducibility and changes in APs/CaTs parameters caused by apamin was negated by the addition of ouabain, an inhibitor of Na + /K + pump. Apamin attenuates APD shortening, Ca 2+ handling abnormalities, and Ca i / V m uncoupling, leading to inhibition of VA occurrence in hypoxic hypertrophied hearts. NEW & NOTEWORTHY We demonstrated that hypoxia-induced ventricular arrhythmias were mainly initiated by Ca 2+ -loaded triggered activities in hypertrophied hearts. The blockades of small-conductance Ca 2+ -activated K + channels, especially "apamin," showed anti-arrhythmic effects by alleviation of not only action potential duration shortening but also Ca 2+ handling abnormalities, most notably the "Ca 2+ /voltage uncoupling."

Calcium-activated potassium (K Ca ) channels contribute to multiple neuronal properties including spike frequency and afterhyperpolarizing potentials (AHPs). K Ca channels are classified as K Ca 1.1, K Ca 2, or K Ca 3.1 based on single-channel conductance and pharmacology. Ca 2+ -dependent AHPs in vertebrates are categorized as fast, medium, or slow. Fast and medium AHPs are generated by K Ca 1.1 and K Ca 2 channels, respectively. The K Ca subtype responsible for slow AHPs is unclear. Prolonged, Ca 2+ -dependent AHPs have been described in several leech neurons. Unfortunately, apamin and other K Ca blockers often prove ineffective in the leech. An alternative approach is to utilize K Ca modulators, which alter channel sensitivity to Ca 2+ . Vertebrate K Ca 2 channels are targeted selectively by the positive modulator CyPPA and the negative modulator NS8593. Here we show that AHPs in identified motor and mechanosensory leech neurons are enhanced by CyPPA and suppressed by NS8593. Our results indicate that K Ca 2 channels underlie prolonged AHPs in these neurons and suggest that K Ca 2 modulators may serve as effective tools to explore the role of K Ca channels in leech physiology.

Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma (HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several HCC cell lines, such as Huh-1 cells. Phosphorylated p62 ( p -p62) inhibits the protein-protein interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between Keap1 and p -p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and synthesised novel K67 derivatives by modification of the substituent at the 4-position of the two benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1- p -p62 PPI to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the viability of Huh-7 cells, which is a non-resistant HCC cell line. The isopropoxy derivative also increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg 2+ and divalent metal cations such as Ca 2+ , Zn 2+ , Cd 2+ , Mn 2+ , and Ni 2+ . TRPM7 channels are inhibited by cytosolic Mg 2+ , rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg 2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg 2+ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC 50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg 2+ ] o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg 2+ depletion. A total of 1.4 mM [Mg 2+ ] o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg 2+ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni 2+ , Zn 2+ , Cd 2+ , Co 2+ , Mn 2+ , Sr 2+ , and Ba 2+ requires TRPM7 channel activity. Mg 2+ loading modestly reduced cytotoxicity of Zn 2+ , Co 2+ , Ni 2+ , and Mn 2+ but not of Cd 2+ . Channel blocker NS8593 reduced Co 2+ and Mn 2+ but not Cd 2+ or Zn 2+ cytotoxicity and interfered with Mg 2+ loading as evaluated by TRPM7 channel basal activity. Ba 2+ and Sr 2+ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg 2+ entry requires TRPM7 channels.

Oocyte activation inefficiency is one of the reasons for female infertility and Ca 2+ functions play a critical role in the regulation of oocyte activation. We used various inhibitors of Ca 2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca 2+ ATPases (SERCAs, the main Ca 2+ pumps which decrease the intracellular Ca 2+ level by refilling Ca 2+ into the sarcoplasmic reticulum), transient receptor potential (TRP) ion channel subfamily member 7 (TRPM7, a Ca 2+ /Mg 2+ -permeable non-selective cation channel), T-type Ca 2+ channels and calcium channel Orai1, to investigate their roles in [Ca 2+ ] i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca 2+ channels were important for initiation and maintenance of [Ca 2+ ] i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.

We report the synthesis, photophysical characterization, and biological evaluation of four DNA-binding ruthenium(II) polypyridyl 4-nitro- and 4-amino-1,8-naphthalimide conjugates. A meta arrangement around the ring connecting the 1,8-naphthalimide to a bipyridine ligand creates a cleft, the result of which renders the shape of the complex complementary to that of DNA. We have demonstrated that each complex exhibits water solubility and a distinctive set of photophysical properties that has allowed the nature of their interaction with DNA to be probed by various ground- and excited-state titrations. Furthermore, by varying the ancillary ligands, we also demonstrate their ability to act as DNA photocleavers, where all compounds have been found to cleave supercoiled DNA with high efficiency. Detailed cellular uptake experiments revealed that the conjugates accumulate in the cytoplasm and nucleus of HeLa cells, showing characteristic red metal-to-ligand charge-transfer emission, and also exhibit photoactivated cytotoxicity within the cells upon irradiation at 450 nm. A comparison between the meta and para arrangements of the 1,8-naphthalimide moiety relative to the Ru(II) center suggests increased DNA binding in the case of the meta arrangement; however, bipyridine-4-amino-1,8-naphthalimide conjugates appear to show superior phototoxicity in comparison to their 4-nitro derivatives.

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Background: Naphthoquine (NQ) is a suitable partner anti-malarial for the artemisinin-based combination therapy (ACT), which is recommended to be taken orally as a single-dose regimen. The metabolism of NQ was mainly mediated by CYP2D6, which is well-known to show gender-specific differences in its expression. In spite of its clinical use, there is limited information on the pharmacokinetics of NQ, and no data are available for females. In this study, the effect of gender on the pharmacokinetics and antiplasmodial efficacy of NQ in rodents was evaluated. The underlying factors leading to the potential gender difference, i.e., plasma protein binding and metabolic clearance, were also evaluated., Methods: The pharmacokinetic profiles of NQ were investigated in healthy male or female rats after a single oral administration of NQ. The antiplasmodial efficacy of NQ was studied in male or female mice infected with Plasmodium yoelii. The recrudescence and survival time of infected mice were also recorded after drug treatment. Plasma protein binding of NQ was determined in pooled plasma collected from male or female mice, rat or human. In vitro metabolism experiments were performed in the liver microsomes of male or female mice, rat or human., Results: The results showed that the gender of rats did not affect NQ exposure (AUC 0-t and C max ) significantly (P > 0.05). However, a significant (P < 0.05) longer t 1/2 was found for NQ in male rats (192.1 ± 47.7), compared with female rats (143.9 ± 27.1). Slightly higher but not significant (P > 0.05) antiplasmodial activity was found for NQ in male mice (ED 90 , 1.10 mg/kg) infected with P. yoelii, compared with female mice (ED 90 , 1.67 mg/kg). The binding rates of NQ to plasma protein were similar in males and females. There was no metabolic difference for NQ in male and female mice, rat or human liver microsomes., Conclusions: These results indicated that the pharmacokinetic profiles of NQ were similar between male and female rats, except for a longer t 1/2 in male rats. The difference was not associated with plasma protein binding or hepatic metabolic clearance. Equivalent antiplasmodial activity was found for NQ in male and female mice infected with P. yoelii. This study will be helpful for the rational design of clinical trials for NQ.

TRPM7 belongs to the Transient Receptor Potential Melastatin family of ion channels and is a divalent cation-conducting ion channel fused with a functional kinase. TRPM7 plays a key role in a variety of diseases, including neuronal death in ischemia, cancer, cardiac atrial fibrillation, malaria invasion. TRPM7 is aberrantly over-expressed in lung, liver and heart fibrosis. It is also overexpressed after renal ischemia-reperfusion, an event that induces kidney injury and fibrosis. However, the role of TRPM7 in kidney fibrosis is unclear. Using the unilateral ureteral obstruction (UUO) mouse model, we examined whether TRPM7 contributes to progressive renal damage and fibrosis. We find that TRPM7 expression increases in UUO kidneys. Systemic application of NS8593, a known TRPM7 inhibitor, prevents kidney atrophy in UUO kidneys, retains tubular formation, and reduces TRPM7 expression to normal levels. Cell proliferation of both tubular epithelial cells and interstitial cells is reduced by NS8593 treatment in UUO kidneys, as are TGF-β1/Smad signaling events. We conclude that TRPM7 is upregulated during inflammatory renal damage and propose that pharmacological intervention targeting TRPM7 may prove protective in progressive kidney fibrosis.

Aims: This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism., Main Methods: ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca 2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca 2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system., Key Findings: High K + (80 mM K + ) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca 2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca 2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs)., Significance: Our results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca 2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator., (Copyright © 2019. Published by Elsevier Inc.)

Reaction phenotyping using human liver microsomes or hepatocytes with chemical inhibitors is one of the most commonly applied methods to assess the fraction metabolized (f m ) of drug candidates by enzymes. The f m information is critical to understanding the risk of victim drug-drug interactions in the clinic. Inhibitor selectivity is essential in order to generate reliable data and irreversible inhibitors are often preferred over reversible inhibitors to minimize the impact of inhibitor depletion. Although many selective cytochrome P450 (CYP) inhibitors are available, the identification of selective CYP2B6 inhibitors has been challenging due to cross inhibition to the other enzymes. In this study, dasotraline was evaluated as a selective inactivator of CYP2B6 under reaction phenotyping conditions with human hepatocytes. The results show that dasotraline is a very selective inactivator for CYP2B6 with minimal inhibition to other enzymes. A concentration of 0.1 μM dasotraline is recommended for reaction phenotyping with a hepatocyte cell density of 0.5 million cells/ml or 0.5 μM for 2 million cells/ml, when using a 15 minute preincubation, as well as the protocol of inactivator removal before the addition of substrates., (© 2019 John Wiley & Sons, Ltd.)

Aims: Acute myocardial infarction (AMI) is associated with intracellular Ca2+ build-up. In healthy ventricles, small conductance Ca2+-activated K+ (SK) channels are present but do not participate in repolarization. However, SK current is increased in chronic myocardial infarction and heart failure, and recently, SK channel inhibition was demonstrated to reduce arrhythmias in AMI rats. Hence, we hypothesized that SK channel inhibitors (NS8593 and AP14145) could reduce arrhythmia development during AMI in a porcine model., Methods and Results: Twenty-seven pigs were randomized 1:1:1 to control, NS8593, or AP14145. Haemodynamic and electrophysiological parameters [electrocardiogram (ECG) and monophasic action potentials (MAP)] were continuously recorded. A balloon was placed in the mid-left anterior descending artery, blinded to treatment. Infusion lasted from 10 min before occlusion until 30 min after. Occlusion was maintained for 1 h, followed by 2 h of reperfusion. Upon occlusion, cardiac output dropped similarly in all groups, while blood pressure remained stable. Heart rate decreased in the NS8593 and AP14145 groups. QRS duration increased upon occlusion in all groups but more prominently in AP14145-treated pigs. Inhibition of SK channels did not affect QT interval. Infarct MAP duration shortened comparably in all groups. Ventricular fibrillation developed in 4/9 control-, 4/9 AP14145-, and 2/9 NS8593-treated pigs. Ventricular tachycardia was rarely observed in either group, whereas ventricular extrasystoles occurred comparably in all groups., Conclusion: Inhibition of SK channels was neither beneficial nor detrimental to ventricular arrhythmia development in the setting of AMI in this porcine model., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Schistosoma haematobium and Schistosoma mansoni infections have broadly overlapping geographical distributions. Praziquantel is the only treatment for human schistosomiasis, so drug tolerance and/or resistance are major concerns. Artemisinin-naphthoquine phosphate (CO-ArNp), an artemisinin-based combination therapy endorsed by the World Health Organization as a gold standard therapy for malaria, has also been identified as a promising treatment for S. mansoni. In this in vitro study, we tested the effect of 1-40 μg/ml CO-ArNp on S. haematobium worms, and inspected tegumental changes by using scanning electron microscopy (SEM), aiming to determine if this combination therapy has a broad-spectrum antischistosomal activity. Incubation of S. haematobium adults with 20 or 30 μg/ml CO-ArNp caused 100% mortality of worms within 72 or 48 h, respectively. SEM examination showed extensive tegumental alterations such as oedema, constriction, shortening and loss of spines, fissuring, sloughing and perforation, resulting in exposure of the underlying basal lamina, mainly in treated male schistosomes. Besides the well-established potent efficacy, bioavailability, tolerability and safety of the antimalarial artemisinin-naphthoquine phosphate combined therapy, these results may also suggest its possible utilization as a new broad-spectrum antischistosomal agent.

Introduction: A 'holy grail' of treatment options for attention-deficit hyperactivity disorder (ADHD) has been an agent with low abuse potential and peak-trough clinical effects, providing sustained therapeutic benefits throughout the day. One such agent, dasotraline, a dopamine and norepinephrine reuptake inhibitor agent, was recently reviewed by the FDA. Areas covered: The authors completed a timely drug review using a PubMed literature search using words 'Dasotraline, ADHD' 'stimulant, abuse' 'atomoxetine, ADHD.' FDA fact sheets of available medications were reviewed for comparison of safety and tolerability data. The authors reviewed preclinical, efficacy, and safety trials of dasotraline in ADHD: two phase 1, one phase 2, and several phase 3 trials have established efficacy in reducing ADHD symptoms. Expert opinion: Due to its stable plasma concentrations with once-daily dosing, dasotraline could have sustained treatment benefits for ADHD, with low abuse potential and a stable therapeutic response over a 24-h period.

To examine whether TRPM7, a member of the melastatin family of transient receptor potential channels, is a physiological pathway for Mg 2+ entry in mammalian cells, we studied the effect of TRPM7 regulators on cytoplasmic free Mg 2+ concentration ([Mg 2+ ] i ) of rat ventricular myocytes. Acutely isolated single cells were AM-loaded with the fluorescent indicator furaptra, and [Mg 2+ ] i was estimated at 25 °C. After [Mg 2+ ] i was lowered by soaking the cells with a high-K + and Mg 2+ -Ca 2+ -free solution, [Mg 2+ ] i was recovered by extracellular perfusion of Ca 2+ -free Tyrode's solution that contained 1 mM Mg 2+ . The initial rate of increase in [Mg 2+ ] i was analyzed as the Mg 2+ influx rate. The Mg 2+ influx rate was increased by the TRPM7 activator, naltriben (2-50 μM), in a concentration-dependent manner with a half maximal effective concentration (EC 50 ) of 24 μM. This EC 50 value is similar to that reported for the activation of recombinant TRPM7 overexpressed in HEK293 cells. Naltriben (50 μM) caused little change in basal [Mg 2+ ] i (~ 0.9 mM) in Ca 2+ -free Tyrode's solution, but significantly raised [Mg 2+ ] i to 1.31 ± 0.03 mM in 94 min after the removal of extracellular Na + . Re-introduction of extracellular Na + lowered [Mg 2+ ] i back to the basal level even in the presence of naltriben. Application of 10 μM NS8593, an inhibitor of TRPM7, significantly lowered [Mg 2+ ] i to 0.72 ± 0.03 mM in 50-60 min independent of extracellular Na + . The results suggest that Mg 2+ entry through TRPM7 significantly contributes to physiological Mg 2+ homeostasis in mammalian heart cells.

Synthetic cannabinoids are a class of novel psychoactive substances that exhibit high affinity at the cannabinoid type-1 (CB 1 ) receptor and produce effects similar to those of Δ-9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis. Illicit drug manufacturers are continually circumventing laws banning the sale of synthetic cannabinoids by synthesizing novel structures and doing so with little regard for the potential impact on pharmacological and toxicological effects. Synthetic cannabinoids produce a wide range of effects that include cardiotoxicity, seizure activity, and kidney damage, and they can cause death. Six synthetic cannabinoids, recently detected in illicit preparations, MMB-FUBINACA, MDMB-FUBINACA, CUMYL-PICA, 5F-CUMYL-PICA, NNEI, and MN-18 were assessed for: 1) receptor binding affinity at the human CB 1 and human CB 2 receptors, 2) function in [ 35 S]GTP γ S and cAMP signaling, and 3) THC-like effects in a mouse drug discrimination assay. All six synthetic cannabinoids exhibited high affinity for human cannabinoid receptors type-1 and type-2 and produced greater maximal effects than THC in [ 35 S]GTP γ S and cAMP signaling. Additionally, all six synthetic cannabinoids substituted for THC in drug discrimination, suggesting they probably possess subjective effects similar to those of cannabis. Notably, MDMB-FUBINACA, a methylated analog of MMB-FUBINACA, had higher affinity for CB 1 than the parent, showing that minor structural modifications being introduced can have a large impact on the pharmacological properties of these drugs. This study demonstrates that novel structures being sold and used illicitly as substitutes for cannabis are retaining high affinity at the CB 1 receptor, exhibiting greater efficacy than THC, and producing THC-like effects in models relevant to subjective effects in humans., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

In the process of recent hit-to-lead studies, not only in industry but also in academia, early evaluation of metabolic properties has been one of the key aspects supporting a higher probability of success in drug discovery. In this review, we introduce the development of chemical seeds targeting the Kelch-like ECH-associated protein-1 (Keap1) as an example of an academic hit-to-lead study considering metabolic stability. Keap1 regulates the function of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces various antioxidative or detoxification proteins. An inhibitor of protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 is expected to be a novel target for drug discovery. However, Nrf2 is also activated in several cancers, such as human hepatocellular carcinoma, and causes chemoresistance, which is mediated by phosphorylated p62/Sqstm1 (p-p62), an autophagy-related protein that also undergoes a PPI with Keap1. In this case, an Nrf2 suppressor could be used to attenuate drug resistance. We discovered inhibitors against the Nrf2-Keap1 PPI and p-p62-Keap1 PPI using high-throughput screening and established the synthetic routes for the hit compounds and their derivatives. Furthermore, we assessed the metabolic stability of both of the PPI inhibitors in human liver microsomes and identified the metabolic sites.

Both big (BKCa) and small (SKCa) conductance Ca-sensitive K channels are present in mammalian cardiac cell mitochondria (m). We used pharmacological agonists and antagonists of BKCa and SKCa channels to examine the importance of endogenous opening of these channels and the relative contribution of either or both of these channels to protect against contractile dysfunction and reduce infarct size after ischemia reperfusion (IR) injury through a mitochondrial protective mechanism. After global cardiac IR injury of ex vivo perfused Guinea pig hearts, we found the following: both agonists NS1619 (for BKCa) and DCEB (for SKCa) improved contractility; BKCa antagonist paxilline (PAX) alone or with SKCa antagonist NS8593 worsened contractility and enhanced infarct size; both antagonists PAX and NS8593 obliterated protection by their respective agonists; BKCa and SKCa antagonists did not block protection afforded by SKCa and BKCa agonists, respectively; and all protective effects by the agonists were blocked by scavenging superoxide anions (O2) with Mn(III) tetrakis (4-benzoic acid) porphyrin (TBAP). Contractile function was inversely associated with global infarct size. In in vivo rats, infusion of NS8593, PAX, or both antagonists enhanced regional infarct size while infusion of either NS1619 or DCEB reduced infarct size. In cardiac mitochondria isolated from ex vivo hearts after IR, combined SKCa and BKCa agonists improved respiratory control index and Ca retention capacity compared with IR alone, whereas the combined antagonists did not alter respiratory control index but worsened Ca retention capacity. Although the differential protective bioenergetics effects of endogenous or exogenous BKCa and SKCa channel opening remain unclear, each channel likely responds to different sensing Ca concentrations and voltage gradients over time during oxidative stress-induced injury to individually or together protect cardiac mitochondria and myocytes.

We provide evidence for location and function of a small conductance, Ca 2+ -activated K + (SK Ca ) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SK Ca agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SK Ca antagonists worsened IR injury. Intravenous infusion of a SK Ca channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca 2+ calmodulin binding domain is required for Ca 2+ sensing to induce mK + influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK + influx was driven by adding CaCl 2 . Moreover, there was a greater fall in membrane potential (ΔΨ m ), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SK Ca channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SK Ca channel opening remains unclear. SK Ca channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa 2+ overload., (Published by Elsevier B.V.)

A screen for inhibitors of Vibrio cholerae motility identified the compound 3-amino 1,8-naphthalimide (3-A18NI), a structural analog of the cholera drug virstatin. Similar to virstatin, 3-A18NI diminished cholera toxin production. In contrast, 3-A18NI impeded swimming and/or swarming motility of V. cholerae and V. parahemolyticus suggesting that it could target the chemotaxis pathway shared by the polar and lateral flagellar system of vibrios. 3-A18NI did not inhibit the expression of V. cholerae major flagellin FlaA or the assembly of its polar flagellum. Finally, 3-A18NI enhanced V. cholerae colonization mimicking the phenotype of chemotaxis mutants that exhibit counterclockwise-biased flagellum rotation., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The Keap1-Nrf2 system is involved not only in biological defense but also in malignancy progression and chemoresistance. The ubiquitin-binding protein p62/Sqstm1 (p62), which is highly expressed in several cancers, competes with Nrf2 for Keap1 binding, leading to activation of Nrf2-mediated gene expression and survival of cancer cells. We had previously identified an inhibitor for the Keap1-phosphorylated-p62 (p-p62) protein-protein interaction (PPI), the acetonyl naphthalene derivative K67. In this study, we established facile synthetic routes for K67 and derivatives with various side chains on the C-2 position of naphthalene ring. K67 possessed high selectivity in the inhibition of Keap1-p-p62. Other derivatives showed potent Keap1-Nrf2 and Keap1-p-p62 PPI inhibitory activities, though the selectivity between the two activities was lower than K67., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the present study, the influence of the recently synthesized styrylquinolinium dye 4-{(E)-2-[4-(dimethylamino)naphthalen-1-yl]ethenyl}-1-methylquinolinium iodide (DANSQI) on the intracellular processes as well as the electrical outputs of Candida melibiosica 2491 yeast-based biofuel cell was investigated. The addition of nanomolar quantities of DANSQI to the yeast suspension results in an increase of the current outputs right after the startup of the biofuel cells, associated with an electrooxidation of the dye on the anode. After that, the formed cation radical of the dye penetrates the yeast cells, provoking a set of intracellular changes. Studies of the subcellular anolyte fractions show that 1μM dye increased the peroxisomal catalase activity 30-times (1.15±0.06Unit/mg protein) and over twice the mitochondrial cytochrome c oxidase activity (92±5Unit/mg protein). The results obtained by electrochemical and spectrophotometric analyses let to the supposition that the dye acts as subcellular shuttle, on account of its specific intramolecular charge transfer properties. The transition between its benzoid, quinolyl radical and ion forms and their putative role for the extracellular and intracellular charge transfer mechanisms are discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of PRMT5 was observed in human CML LSCs. Silencing PRMT5 with shRNA or blocking PRMT5 methyltransferase activity with the small-molecule inhibitor PJ-68 reduced survival, serial replating capacity, and long-term culture-initiating cells (LTC-ICs) in LSCs from CML patients. Further, PRMT5 knockdown or PJ-68 treatment dramatically prolonged survival in a murine model of retroviral BCR-ABL-driven CML and impaired the in vivo self-renewal capacity of transplanted CML LSCs. PJ-68 also inhibited long-term engraftment of human CML CD34+ cells in immunodeficient mice. Moreover, inhibition of PRMT5 abrogated the Wnt/β-catenin pathway in CML CD34+ cells by depleting dishevelled homolog 3 (DVL3). This study suggests that epigenetic methylation modification on histone protein arginine residues is a regulatory mechanism to control self-renewal of LSCs and indicates that PRMT5 may represent a potential therapeutic target against LSCs.

Cerebellar Purkinje cells (PCs) are primarily affected in many spinocerebellar ataxias (SCA). In this study we investigated functional activity of PCs in transgenic mouse model of SCA2, a polyglutamine neurodegenerative hereditary disorder. In our studies we used extracellular single-unit recording method to compare spontaneous activity of PCs in age-matched wild-type mice and SCA2-58Q transgenic mice. We discovered that the fraction of PCs with bursting and an irregular pattern of spontaneous activity dramatically increases in aged SCA2-58Q mice compared with wild-type littermates. Small-conductance calcium-activated potassium (SK) channels play an important role in determining firing rate of PCs. Indeed, we demonstrated that intraperitoneal (IP) injection of SK channel inhibitor NS8593 induces an irregular pattern of PC activity in wild-type mice. Furthermore, we demonstrated that IP injection of SK channel-positive modulator chlorzoxazone (CHZ) decreases spontaneous firing rate of cerebellar PCs. Finally, we have shown that IP injections with CHZ normalize firing activity of cerebellar PCs from aging SCA2-58Q mice. We propose that alterations in PC firing patterns is one of potential causes of ataxic symptoms in SCA2 and in other SCAs and that positive modulators of SK channels can be used to normalize activity of PCs and alleviate ataxic phenotype in patients with SCA., (Copyright © 2016 the American Physiological Society.)

Emergence and spread of antibiotic-resistant Acinetobacter baumannii have become a major public health concern. This study was designed to investigate the efficacy of Holarrhena antidysenterica extract and its major steroidal alkaloid conessine as resistance-modifying agents (RMAs) on the susceptibility of A. baumannii to novobiocin and rifampicin. A significant synergistic activity of both the extract and conessine in combination with either novobiocin or rifampicin with fractional inhibitory concentration index ≤0.5 was demonstrated. Fluorescent dyes and different efflux pump inhibitors were used to further investigate the synergism. Increase in the uptake of 1-N-phenylnaphthylamine in the bacterial cells treated with the extract and conessine was not observed indicating that both substances did not act as permeabilizers. With regard to efflux pump inhibition, no accumulation in ethidium bromide (EtBr) was noticed suggesting that the AdeABC pump was not involved. In contrast, accumulation in Pyronin Y was significantly increased (p < 0.05) demonstrating that the synergism was due to interference with the AdeIJK pump. Study on frequencies of the spontaneous mutational resistance to the extract in combination with antibiotics demonstrated attenuation in drug-resistant organisms. Thus, H. antidysenterica extract and conessine as RMAs may offer a combinatory therapy to restore antibiotic susceptibility in the extensively drug-resistant A. baumannii.

Stenotrophomonas maltophilia harbors six lytic transglycosylases (LTs): mltA, mltB1, mltB2, mltD1, mltD2, and slt LT deletion increased susceptibility of S. maltophilia to aminoglycosides (AGs) and macrolides, and the underlying mechanisms were investigated. The expression of AG-modifying enzymes and efflux pumps was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Susceptibility to 1-N-phenylnaphthylamine, vancomycin, SDS, and bile salts was measured to assess outer membrane permeability. In conclusion, increased outer membrane permeability contributes to LT deletion-mediated increase in aminoglycoside and macrolide susceptibility., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bi-functional protein comprising an ion channel moiety covalently linked to a protein kinase domain. Currently, the prevailing view is that a decrease in the cytosolic Mg(2+) concentration leads to activation of divalent cation-selective TRPM7 currents. TRPM7 plays a role in immune responses, hypotension, tissue fibrosis, and tumor progression and, therefore, represents a new promising therapeutic target. Because of the dearth of pharmacological tools, our mechanistic understanding of the role of TRPM7 in physiology and pathophysiology still lags behind. Therefore, we have recently carried out a high throughput screen for small-molecule activators of TRPM7. We have characterized the phenanthrene naltriben as a first stimulatory agonist of the TRPM7 channel. Surprisingly, the effect of naltriben on TRPM7 was found to be unaffected by the physiological levels of cytosolic Mg(2+). Here, we demonstrate that mibefradil and NNC 50-0396, two benzimidazole relatives of the TRPM7 inhibitor NS8593, are positive modulators of TRPM7. Using Ca(2+) imaging and the patch-clamp technique, we show that mibefradil activates TRPM7-mediated Ca(2+) entry and whole-cell currents. The response to mibefradil was fast, reversible, and reproducible. In contrast to naltriben, mibefradil efficiently activates TRPM7 currents only at physiological intracellular Mg(2+) concentrations, and its stimulatory effect was fully abrogated by high internal Mg(2+) levels. Consequently, a TRPM7 variant harboring a gain-of-function mutation was insensitive to further mibefradil activation. Finally, we observed that the effect of mibefradil was selective for TRPM7 when various TRP channels were tested. Taken together, mibefradil acts as a Mg(2+)-regulated agonist of the TRPM7 channel and, hence, uncovers a new class of TRPM7 agonists.

Background and Objectives: Dasotraline is a novel inhibitor of dopamine and norepinephrine reuptake currently being investigated in clinical studies for the treatment of attention-deficit/hyperactivity disorder (ADHD). Uniquely, relative to current ADHD medications, dasotraline has a slow absorption and long elimination half-life. Here we relate the pharmacokinetics and pharmacodynamics of dasotraline to reduction in ADHD symptoms based on simulated clinical trial outcomes., Methods: Dasotraline pharmacokinetics were analyzed by population pharmacokinetic methodologies using data collected from 395 subjects after single or multiple oral dose administrations ranging from 0.2 to 36 mg (three phase I studies and one phase II ADHD study). Population pharmacokinetic and pharmacodynamic models related individual dasotraline exposures to norepinephrine metabolite 3,4-dihydroxyphenylglycol (DHPG) concentrations, ADHD symptoms, and study discontinuation (probability of dropout)., Results: Dasotraline pharmacokinetics were described by a one-compartment model with dual (linear plus nonlinear) elimination. In an ADHD population treated with dasotraline 4 or 8 mg/day, dasotraline was characterized by a mean apparent half-life of 47 h and plasma concentrations reached steady-state by 10 days of dosing. A population pharmacokinetic and pharmacodynamic model of DHPG indicated clinically significant norepinephrine transporter inhibition was achieved as a function of time-matched dasotraline concentrations. Dasotraline exposure reduced ADHD symptoms in a sigmoid E max time-course model. Clinical trial simulations described the effects of dose, duration, and sample size on clinical outcomes., Conclusion: These results related dasotraline pharmacokinetics to pharmacological activity in ADHD, and support the novel concept that maintaining constant, steady-state dopamine and norepinephrine reuptake inhibition throughout a 24-h dosing interval is a novel pharmacological approach to the management of ADHD symptoms. Clinicaltrials.gov identifier: NCT01692782.

Aims: The aim of this study was to evaluate the abuse potential of dasotraline, a novel dopamine and norepinephrine reuptake inhibitor with slow absorption (tmax, 10-12h) and elimination (t1/2=47-77 h) that is in development for the treatment of attention deficit hyperactivity disorder (ADHD)., Methods: Recreational stimulant users (N=48) who had specific experience with cocaine, and who were able to distinguish methylphenidate (60 mg) versus placebo in a qualification session, were randomized, in a 6-period, double-blind, crossover design, to receive single doses of dasotraline 8 mg, 16 mg, and 36 mg, methylphenidate (MPH) 40 mg and 80 mg, and placebo. The primary endpoint was the Drug Liking Visual Analog Scale (VAS) score at the time of peak effect (Emax)., Results: There were no significant differences between the 3 doses of dasotraline and placebo on the drug liking VAS at Emax, and on most secondary endpoints. Both doses of MPH had significantly higher VAS-drug liking scores at Emax relative to both placebo (P<0.001 for all comparisons) and dasotraline 8 mg (P<0.001), 16 mg (P<0.001) and 36 mg (P<0.01). The increase in heart rate for MPH and dasotraline 36 mg showed a time-course that closely matched subject-rated measures such as Any Effects VAS., Conclusions: In this study, dasotraline was found to have low potential for abuse, which may be, in part, related to its established pharmacokinetics (PK) profile, which is characterized by slow absorption and gradual elimination., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by symptoms of inattention, hyperactivity, and impulsivity associated with clinically significant impairment in functioning. ADHD has an early onset, but frequently persists, with a prevalence estimate of 4% in adults. Dasotraline is a novel compound that is a potent inhibitor of dopamine and norepinephrine transporters that achieves stable plasma concentrations with once-daily dosing. In this study, adult outpatients meeting DSM-IV-TR criteria for ADHD were randomized to 4 weeks of double-blind, once-daily treatment with dasotraline 4 and 8 mg/day or placebo. The primary efficacy end point was change from baseline at week 4 in the ADHD Rating Scale, Version IV (ADHD RS-IV) total score. Secondary efficacy end points included the Clinical Global Impression, Severity (CGI-S) scale, modified for ADHD symptoms. Least squares (LS) mean improvements at week 4 in ADHD RS-IV total score were significantly greater for dasotraline 8 mg/day vs placebo (-13.9 vs -9.7; P=0.019), and nonsignificantly greater for 4 mg/day (-12.4; P=0.076). The LS mean improvements in modified CGI-S were significantly greater at week 4 for dasotraline 8 mg/day vs placebo (-1.1 vs -0.7; P=0.013), and for 4 mg/day vs placebo (-1.1 vs -0.7; P=0.021). The most frequent adverse events reported were insomnia, decreased appetite, nausea, and dry mouth. Discontinuations due to treatment-emergent adverse events were 10.3% and 27.8% of patients in 4 and 8 mg/day treatment groups, respectively. This study provides preliminary evidence that once-daily dosing with dasotraline, a long-acting, dual monoamine reuptake inhibitor, may be a safe and efficacious treatment for adult ADHD.

During recent years, small conductance Ca-activated K (SK) channels have been reported to play a role in cardiac electrophysiology. SK channels seem to be expressed in atria and ventricles, but from a functional perspective, atrial activity is predominant. A general notion seems to be that cardiac SK channels are predominantly coming into play during arrhythmogenic events where intracellular concentration of Ca is increased. During ventricular fibrillation (VF), a surge of [Ca]i has the potential to bind to and open SK channels. To obtain mechanistic insight into possible roles of SK channels during VF, we conducted experiments with an SK channel pore blocker (ICA) and a negatively allosteric modulator (NS8395) in a Langendorff-perfused heart model. Both compounds increased the action potential duration, effective refractory period, and Wenckebach cycle length to comparable extents. Despite these similarities, the SK channel modulator was found to revert and prevent VF more efficiently than the SK channel pore blocker. In conclusion, either negative allosteric modulation of the SK channel with NS8593 is more favorable than pure channel block with ICA or the 2 compounds have different selectivity profiles that makes NS8593 more antiarrhythmic than ICA in a setting of VF.

A series of N-aryl-naphthylamines, exemplified by the structures 11-16, were chosen for an in-house library screening to assay their ability to disrupt the interaction between the LEDGF cofactor and the HIV integrase. Structure modification led also to design and synthesize new compounds 17a-f. Compounds 11e,h,k,n, 13b, and 14 showed good activity in AlphaScreen assay. The most active compound 11e (IC50 = 2.5 μM) was selected for molecular modeling studies and showed a binding mode similar to the one of the known LEDGIN 8., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

The therapeutic efficacy of arsenic trioxide (ATO) for treatments of solid tumors is restricted by its drug resistance and chemotoxicity. In this study, we investigated ATO sensitization and detoxification effect of the Poly (ADP ribose) polymerase-1 (PARP-1) inhibitor 4-Amino-1,8-naphthalimide (4AN) in the hepatocellular carcinoma cell line HepG2. We firstly reported that ATO treatment induced the activation of Nuclear factor of κB (NF-κB) and its downstream anti-apoptosis and pro-inflammatory effectors in a PARP-1-dependent manner and thus conferred HepG2 cells with ATO resistance and toxicity. 4AN significantly suppressed the ATO-induced NF-κB activation, which promotes the apoptotic response and alleviates the inflammatory reaction induced by ATO, resulting in sensitization and detoxification against ATO. We also demonstrated that the ATO-induced activation of PARP-1 and NF-κB was closely associated with the oxidative DNA damage mediated by the generated reactive oxygen species (ROS). Furthermore, the attenuation of ATO-induced ROS and the resulting oxidative DNA damage by N-acetyl-L-cysteine (NAC), a potent antioxidant, significantly reduced the activation of PARP-1 and NF-κB in ATO-treated cells. Our study provides novel insights into the mechanism of the PARP-1-mediated NF-κB signaling pathway in ATO resistance and toxicity in anticancer treatments. This study also highlights the application potential of PARP-1 inhibitors in ATO-based anti-cancer treatments and in prevention of NF-κB-mediated therapeutic resistance and toxicity.

Background: Small-conductance calcium-activated potassium (SK) channels have been found to play an important role in atrial repolarization and atrial fibrillation (AF)., Objective: The purpose of this study was to investigate the existence and functional role of SK channels in the equine heart., Methods: Cardiac biopsies were analyzed to investigate the expression level of the most prominent cardiac ion channels, with special focus on SK channels, in the equine heart. Subcellular distribution of SK isoform 2 (SK2) was assessed by immunohistochemistry and confocal microscopy. The electrophysiologic and anti-AF effects of the relative selective SK channel inhibitor NS8593 (5 mg/kg IV) were evaluated in anesthetized horses, focusing on the potential of NS8593 to terminate acute pacing-induced AF, drug-induced changes in atrial effective refractory period, AF duration and vulnerability, and ventricular depolarization and repolarization times., Results: Analysis revealed equivalent mRNA transcript levels of the 3 SK channel isoforms in atria compared to ventricles. Immunohistochemistry and confocal microscopy displayed a widespread distribution of SK2 in both atrial and ventricular cardiomyocytes. NS8593 terminated all induced AF episodes (duration ≥15 minutes), caused pronounced prolongation of atrial effective refractory period, and reduced AF duration and vulnerability. QRS duration and QTc interval were not affected by treatment., Conclusion: SK channels are widely distributed in atrial and ventricular cardiomyocytes and contribute to atrial repolarization. Inhibition by NS8593 terminates pacing-induced AF of short duration and decreases AF duration and vulnerability without affecting ventricular conduction and repolarization. Thus, inhibition by NS8593 demonstrates clear atrial antiarrhythmic properties in healthy horses., (Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori infects approximately half of the world population and is a major cause of gastritis, peptic ulcer, and gastric cancer. Moreover, this bacterium has quickly developed resistance to all major antibiotics. Recently, we developed a novel liposomal linolenic acid (LipoLLA) formulation, which showed potent bactericidal activity against several clinical isolated antibiotic-resistant strains of H. pylori including both the spiral and coccoid form. In addition, LipoLLA had superior in vivo efficacy compared to the standard triple therapy. Our data showed that LipoLLA associated with H. pylori cell membrane. Therefore, in this study, we investigated the possible antibacterial mechanism of LipoLLA against H. pylori. The antibacterial activity of LipoLLA (C18:3) was compared to that of liposomal stearic acid (LipoSA, C18:0) and oleic acid (LipoOA, C18:1). LipoLLA showed the most potent bactericidal effect and completely killed H. pylori within 5 min. The permeability of the outer membrane of H. pylori increased when treated with LipoOA and LipoLLA. Moreover, by detecting released adenosine triphosphate (ATP) from bacteria, we found that bacterial plasma membrane of H. pylori treated with LipoLLA exhibited significantly higher permeability than those treated with LipoOA, resulting in bacteria cell death. Furthermore, LipoLLA caused structural changes in the bacterial membrane within 5 min affecting membrane integrity and leading to leakage of cytoplasmic contents, observed by both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Our findings showing rapid bactericidal effect of LipoLLA suggest it is a very promising new, effective anti-H. pylori agent.

Objective: To study the effect and mechanism of 4-amino-1, 8-naphthalimide (4-AN) on the sensitive effect of arsenic trioxide (ATO) in hepatocellular carcinoma cells., Methods: Hepatocellular carcinoma HepG2 cells were divided into two groups according to whether they were treated with 4-AN or not. Cell viability was evaluated by MTT assay, population doubling experiment and colony formation assay; genic mechanism was explored by 8-OH-dG assay, single cell gel electrophoresis (comet assay) and microriucleus test., Results: At 2-10 micromol/L concentration of ATO, the cell viability and colony formation efficiency of the combinatio group (4-AN+ATO) were significantly lower than that of the ATO group (P<0.05); moreover, the tail-length (L-Tail) and olive tail moment (OTM) in comet assay were notablely higher than that of the ATO group (P<0.05). At 2-20 micromol/L concentration of ATO, the population doubling time and 8-OH-dG in combination group were significantly higher than that of ATO group (P<0.05). Results from DNA damage repair assay showed that the efficiency of DNA damage repair in combination group was remarkably lower than that of ATO group (P<0.05). At 5-20 micromol/L concentration of ATO, the frequency of micronucleated cells in combination group was significantly higher than that of ATO group (P<0.05)., Conclusion: 4-AN can significantly increase the sensitivity of ATO in treatment with hepatocellular carcinoma cells and prevent DNA damage repair may be a primary mechanism for this effect.

Two Schiff base compounds, N,N'-bis(2-methoxy phenylidene)-1,5-diamino naphthalene (L(1)) and N,N'-bis(3,4,5-trimethoxy phenylidene)-1,5-diamino naphthalene (L(2)) were synthesized and characterized by the analytical and spectroscopic methods. The electrochemical and photoluminescence properties of the Schiff bases were investigated in the different conditions. The compounds L(1) and L(2) show the reversible redox processes at some potentials. The sensor properties of the Schiff bases were examined and color changes were observed upon addition of the metal cations, such as Hg(II), Cu(II), Co(II) and Al(III). The Schiff base compounds show the bathochromic shift from 545 to 585 nm. The single crystals of the compounds (L(1)) and (L(2)) were obtained from the methanol solution and characterized structurally by the X-ray crystallography technique. The molecule L(2) is centrosymmetric whereas the L(1) has no crystallographically imposed molecular symmetry. However, the molecular structures for these compounds are quite similar, differing principally in the conformation about methoxy groups and the dihedral angle between the two aromatic rings and diamine naphthalene., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Arsenic trioxide (ATO) is successfully used to treat hematological malignancies. However, the clinical application of the agent in solid tumors is largely limited by its dose-dependent toxicity which results from the high intrinsic resistance of the cancer cells. In this study, we firstly identified a series of sensitization effects of 4AN, a PARP-1 inhibitor, on human hepatocellular carcinoma cell line HepG2 to ATO treatment. We showed that treatment of HepG2 cells with 4AN promoted ATO-induced cell death in a synergistic manner. The ATO-sensitization by 4AN was associated with its effect on abrogation of ATO-induced G2/M checkpoint which impairs DNA damage repair and promotes cell apoptosis. Further analysis demonstrated that the ATO-induced G2/M checkpoint was closely related to a decrease in cyclin B1, a key G2/M mediator; whereas 4AN up-regulated the expression of cyclin B1 in ATO-treated cells, which may be at least partly responsible for its effect on abrogation of ATO-induced G2/M checkpoint. This was further supported by the result showing that down-regulation of cyclin B1 using siRNA could restore the G2/M checkpoint in cells co-treated with ATO and 4AN, thereby improving DNA damage repair and decreasing apoptosis. Our study indicates that the abrogation of G2/M checkpoint and the suppression of DNA damage repair contribute to ATO-sensitization by PARP-1 inhibitor in HepG2 cells, which provides a novel insight into the chemo-sensitization mechanism of PARP-1 inhibitor., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Cytoplasmic free Mg(2+) concentration ([Mg(2+)]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K(+) (Ca(2+)- and Mg(2+)-free) solution, [Mg(2+)]i decreased from ? 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg(2+)]i was recovered by perfusion with Ca(2+)-free Tyrode's solution containing 1 mM Mg(2+). The time course of the [Mg(2+)]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg(2+) influx rate. The Mg(2+) influx rate was inversely related to [Mg(2+)]i, being higher at low [Mg(2+)]i. The Mg(2+) influx rate was augmented by the high extracellular Mg(2+) concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K(+). Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg(2+) influx rate with half inhibitory concentrations (IC50) of, respectively, 17 ?M, 2.0 ?M, and 22 ?M. We also studied Ni(2+) influx by fluorescence quenching of intracellular furaptra by Ni(2+). The Ni(2+) influx was activated by lowering intra- and extracellular Mg(2+) concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg(2+) influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg(2+) influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ? 50% by 100 ?M 2-APB or 10 ?M NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg(2+) influx in rat ventricular myocytes.

Ion channels play pivotal roles in regulating important functions of macrophages, such as cytokine and chemokine production, migration, proliferation, phagocytosis and others. In this study, we have identified the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) for the first time in macrophages. TRPM7 activity is differentially regulated in macrophages, i.e. current density in TRPM7 is significantly larger in anti-inflammatory M2-type macrophages than in untreated and in pro-inflammatory M1-type macrophages, whereas mRNA levels of TRPM7 remain unchanged upon cell polarisation. The specific TRPM7 inhibitors NS8593 and FTY720 abolish proliferation of macrophages induced by interleukin-4 (IL-4) and macrophage colony-stimulating factor (M-CSF), respectively, whereas proliferation arrest was not accompanied by induction of apoptosis or necrosis in macrophages. Furthermore, NS8593 and FTY720 prevented polarisation of macrophages towards the anti-inflammatory M2 phenotype. Inhibition of TRPM7 reduced IL-4-induced upregulation of arginase-1 (Arg1) mRNA levels and Arg1 activity, and abolished the inhibitory effects of IL-4 or M-CSF on LPS-induced TNF-α production by macrophages. In summary, our data suggest a main role of TRPM7 in the regulation of macrophage proliferation and polarisation., (© 2014. Published by The Company of Biologists Ltd.)

Microglia rapidly respond to CNS injury and disease and can assume a spectrum of activation states. While changes in gene expression and production of inflammatory mediators have been extensively described after classical (LPS-induced) and alternative (IL4-induced) microglial activation, less is known about acquired de-activation in response to IL10. It is important to understand how microglial activation states affect their migration and invasion; crucial functions after injury and in the developing CNS. We reported that LPS-treated rat microglia migrate very poorly, while IL4-treated cells migrate and invade much better. Having discovered that the lamellum of migrating microglia contains a large ring of podosomes--microscopic structures that are thought to mediate adhesion, migration and invasion--we hypothesized that IL4 and IL10 would differentially affect podosome expression, gene induction, migration and invasion. Further, based on the enrichment of the KCa2.3/SK3 Ca2+-activated potassium channel in microglial podosomes, we predicted that it regulates migration and invasion. We found both similarities and differences in gene induction by IL4 and IL10 and, while both cytokines increased migration and invasion, only IL10 affected podosome expression. KCa2.3 currents were recorded in microglia under all three activation conditions and KCNN3 (KCa2.3) expression was similar. Surprisingly then, of three KCa2.3 inhibitors (apamin, tamapin, NS8593), only NS8593 abrogated the increased migration and invasion of IL4 and IL10-treated microglia (and invasion of unstimulated microglia). This discrepancy was explained by the observed block of TRPM7 currents in microglia by NS8593, which occurred under all three activation conditions. A similar inhibition of both migration and invasion was seen with a TRPM7 inhibitor (AA-861) that does not block KCa2.3 channels. Thus, we conclude that TRPM7 (not KCa2.3) contributes to the enhanced ability of microglia to migrate and invade when in anti-inflammatory states. This will be an important consideration in developing TRPM7 inhibitors for treating CNS injury.

A series of aromatic and aliphatic bis[aminomethylidene(bisphosphonic)] acids was synthesized in the reaction of triethylphosphite with isonitriles followed by hydrolysis or dealkylation. The in vitro anti-proliferative effect of all synthesized tetraphosphonic acids against MCF-7 breast cancer cells, J774E macrophages and HL-60 promyelocytic leukemia cells was determined. Three aromatic derivatives (5a, 5f and 5j) showed a similar or higher anti-proliferative activity than zoledronic acid., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: Small-conductance calcium-activated potassium (SK) channels are expressed in the heart of various species, including humans. The aim of the present study was to address whether SK channels play a functional role in human atria., Methods and Results: Quantitative real-time PCR analyses showed higher transcript levels of SK2 and SK3 than that of the SK1 subtype in human atrial tissue. SK2 and SK3 were reduced in chronic atrial fibrillation (AF) compared with sinus rhythm (SR) patients. Immunohistochemistry using confocal microscopy revealed widespread expression of SK2 in atrial myocytes. Two SK channel inhibitors (NS8593 and ICAGEN) were tested in heterologous expression systems revealing ICAGEN as being highly selective for SK channels, while NS8593 showed less selectivity for these channels. In isolated atrial myocytes from SR patients, both inhibitors decreased inwardly rectifying K(+) currents by ∼15% and prolonged action potential duration (APD), but no effect was observed in myocytes from AF patients. In trabeculae muscle strips from right atrial appendages of SR patients, both compounds increased APD and effective refractory period, and depolarized the resting membrane potential, while only NS8593 induced these effects in tissue from AF patients. SK channel inhibition did not alter any electrophysiological parameter in human interventricular septum tissue., Conclusions: SK channels are present in human atria where they participate in repolarization. SK2 and SK3 were down-regulated and had reduced functional importance in chronic AF. As SK current was not found to contribute substantially to the ventricular AP, pharmacological inhibition of SK channels may be a putative atrial-selective target for future antiarrhythmic drug therapy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.

Background: Recent evidence points to functional Ca²⁺-dependent K⁺ (SK) channels in the heart that may govern atrial fibrillation (AF) risk, but the underlying mechanisms are unclear. This study addressed the role of SK channels in atrial repolarization and AF persistence in a canine AF model., Methods and Results: Electrophysiological variables were assessed in dogs subjected to atrial remodeling by 7-day atrial tachypacing (AT-P), as well as controls. Ionic currents and single-channel properties were measured in isolated canine atrial cardiomyocytes by patch clamp. NS8593, a putative selective SK blocker, suppressed SK current with an IC₅₀ of ≈5 μmol/L, without affecting Na⁺, Ca²⁺, or other K⁺ currents. Whole-cell SK current sensitive to NS8593 was significantly larger in pulmonary vein (PV) versus left atrial (LA) cells, without a difference in SK single-channel open probability (P(o)), whereas AT-P enhanced both whole-cell SK currents and single-channel P(o). SK-current block increased action potential duration in both PV and LA cells after AT-P; but only in PV cells in absence of AT-P. SK2 expression was more abundant at both mRNA and protein levels for PV versus LA in control dogs, in both control and AT-P; AT-P upregulated only SK1 at the protein level. Intravenous administration of NS8593 (5 mg/kg) significantly prolonged atrial refractoriness and reduced AF duration without affecting the Wenckebach cycle length, left ventricular refractoriness, or blood pressure., Conclusions: SK currents play a role in canine atrial repolarization, are larger in PVs than LA, are enhanced by atrial-tachycardia remodeling, and appear to participate in promoting AF maintenance. These results are relevant to the potential mechanisms underlying the association between SK single-nucleotide polymorphisms and AF and suggest SK blockers as potentially interesting anti-AF drugs.