1,4-Disubstituted Piperazin-2-Ones as Selective Late Sodium Current Inhibitors with QT Interval Shortening Properties in Isolated Rabbit Hearts.

Late sodium current (INa) inhibitors are a new subclass of antiarrhythmic agents. To overcome the drawbacks, e.g., low efficacy and inhibition effect on K+ current, of the FDA-approved late INa inhibitor ranolazine, chain amide 6a-6q, 1,4-disubstituted piperazin-2-ones 7a-7s, and their derivatives 8a-8n were successively designed, synthesized, and evaluated in vitro on the NaV1.5-transfected HEK293T cells by the whole-cell patch clamp recording assay at the concentration of 40 µM. Among the new skeleton compounds, 7d showed the highest efficacy (IC50 = 2.7 µM) and good selectivity (peak/late ratio >30 folds), as well as excellent pharmacokinetics properties in mice (T1/2 of 3.5 h, F = 90%, 3 mg/kg, po). It exhibited low hERG inhibition and was able to reverse the ATX-II-induced augmentation of late INa phenotype of LQT3 model in isolated rabbit hearts. These results suggest the application potentials of 7d in the treatments of arrhythmias related to the enhancement of late INa.

Coupling of Slack and NaV1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development.

Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidine's blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel NaV1.6 and Slack. NaV1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of NaV1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. NaV1.6-mediated sensitization requires the involvement of NaV1.6's N- and C-termini binding to Slack's C-terminus and is enhanced by transient sodium influx through NaV1.6. Moreover, disrupting the Slack-NaV1.6 interaction by viral expression of Slack's C-terminus can protect against SlackG269S-induced seizures in mice. These insights about a Slack-NaV1.6 complex challenge the traditional view of 'Slack as an isolated target' for anti-epileptic drug discovery efforts and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy.

A role of GABAA receptor α1 subunit in the hippocampus for rapid-acting antidepressant-like effects of ketamine.

Ketamine can produce rapid-acting antidepressant effects in treatment-resistant patients with depression. Although alterations in glutamatergic and GABAergic neurotransmission in the brain play a role in depression, the precise molecular mechanisms in these neurotransmission underlying ketamine's antidepressant actions remain largely unknown. Mice exposed to FSS (forced swimming stress) showed depression-like behavior and decreased levels of GABA (γ-aminobutyric acid), but not glutamate, in the hippocampus. Ketamine increased GABA levels and decreased glutamate levels in the hippocampus of mice exposed to FSS. There was a correlation between GABA levels and depression-like behavior. Furthermore, ketamine increased the levels of enzymes and transporters on the GABAergic neurons (SAT1, GAD67, GAD65, VGAT and GAT1) and astrocytes (EAAT2 and GAT3), without affecting the levels of enzymes and transporters (SAT2, VGluT1 and GABAAR γ2) on glutamatergic neurons. Moreover, ketamine caused a decreased expression of GABAAR α1 subunit, which was specifically expressed on GABAergic neurons and astrocytes, an increased GABA synthesis and metabolism in GABAergic neurons, a plasticity change in astrocytes, and an increase in ATP (adenosine triphosphate) contents. Finally, GABAAR antagonist bicuculline or ATP exerted a rapid antidepressant-like effect whereas pretreatment with GABAAR agonist muscimol blocked the antidepressant-like effects of ketamine. In addition, pharmacological activation and inhibition of GABAAR modulated the synthesis and metabolism of GABA, and the plasticity of astrocytes in the hippocampus. The present data suggest that ketamine could increase GABA synthesis and astrocyte plasticity through downregulation of GABAAR α1, increases in GABA, and conversion of GABA into ATP, resulting in a rapid-acting antidepressant-like action. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Multiple machine learning methods aided virtual screening of NaV 1.5 inhibitors.

Nav 1.5 sodium channels contribute to the generation of the rapid upstroke of the myocardial action potential and thereby play a central role in the excitability of myocardial cells. At present, the patch clamp method is the gold standard for ion channel inhibitor screening. However, this method has disadvantages such as high technical difficulty, high cost and low speed. In this study, novel machine learning models to screen chemical blockers were developed to overcome the above shortage. The data from the ChEMBL Database were employed to establish the machine learning models. Firstly, six molecular fingerprints together with five machine learning algorithms were used to develop 30 classification models to predict effective inhibitors. A validation and a test set were used to evaluate the performance of the models. Subsequently, the privileged substructures tightly associated with the inhibition of the Nav 1.5 ion channel were extracted using the bioalerts Python package. In the validation set, the RF-Graph model performed best. Similarly, RF-Graph produced the best result in the test set in which the Prediction Accuracy (Q) was 0.9309 and Matthew's correlation coefficient was 0.8627, further indicating the model had high classification ability. The results of the privileged substructures indicated Sulfa structures and fragments with large Steric hindrance tend to block Nav 1.5. In the unsupervised learning task of identifying sulfa drugs, MACCS and Graph fingerprints had good results. In summary, effective machine learning models have been constructed which help to screen potential inhibitors of the Nav 1.5 ion channel and key privileged substructures with high affinity were also extracted.

Kainate receptors GluK1 and GluK2 differentially regulate synapse morphology.

The regulation of dendritic spine morphology is a critical aspect of neuronal network refinement during development and modulation of neurotransmission. Previous studies revealed that glutamatergic transmission plays a central role in synapse development. AMPA receptors and NMDA receptors regulate spine morphology in an activity dependent manner. However, whether and how Kainate receptors (KARs) regulate synapse development remains poorly understood. In this study, we found that GluK1 and GluK2 may play distinct roles in synapse development. In primary cultured hippocampal neurons, we found overexpression of the calcium-permeable GluK2(Q) receptor variant increased spine length and spine head area compared to overexpression of the calcium-impermeable GluK2(R) variant or EGFP transfected, control neurons, indicating that Q/R editing may play a role in GluK2 regulation of synapse development. Intriguingly, neurons transfected with GluK1(Q) showed decreased spine length and spine head area, while the density of dendritic spines was increased, suggesting that GluK1(Q) and GluK2(Q) have different effects on synaptic development. Swapping the critical domains between GluK2 and GluK1 demonstrated the N-terminal domain (NTD) is responsible for the different effects of GluK1 and GluK2. In conclusion, Kainate receptors GluK1 and GluK2 have distinct roles in regulating spine morphology and development, a process likely relying on the NTD.

Improved Bioherbicidal Efficacy of Bipolaris eleusines through Herbicide Addition on Weed Control in Paddy Rice.

Bipolaris eleusines was mixed with herbicides to improve the control of barnyardgrass (Echinochloa crus-galli), a noxious weed in rice fields. The compatibility of B. eleusines with herbicides was evaluated for toxic effects on spore germination and mycelium growth in vitro tests, and varied effects were observed with different chemical products. Briefly, 25 g/L penoxsulam OD plus 10% bensulfuron-methyl WP were much more compatible with B. eleusines, and there was no inhibition of spore germination but the promotion of mycelium growth of B. eleusines at all treatment rates. Under greenhouse conditions, the coefficient of the specificity of B. eleusines conidial agent was determined as 3.91, closer to the herbicidal control of 2.89, showing it is highly specific between rice and barnyardgrass. Field experiments in 2011 and 2012 showed that B. eleusines conidial agent displayed good activity on barnyardgrass, monochoria [Monochoria vaginalis (Burm.f.) Presl. Ex Kunth.], and small-flower umbrella sedge (Cyperus difformis L.) and had no negative impact on the rice plant. It also reduced the loss of rice yield when compared with the non-treated control and could make this pathogen a conidial agent for commercial bioherbicidal development in the future.

Identification of poly(ADP-ribose)polymerase 1 and 2 (PARP1/2) as targets of andrographolide using an integrated chemical biology approach.

Here, we describe the identification of PARP1/2 as direct binding proteins of andrographolide (Andro) using protein microarray, surface plasmon resonance (SPR), and enzyme activity assays. We then evaluated the proliferation inhibition, apoptosis, and cell migration effects of Andro on the MDA-MB-436 cell line in vitro. The final biological evaluation confirmed that Andro was a highly effective single agent in the MDA-MB-436 xenograft model and had a low hERG-mediated cardiac toxicity. Therefore, Andro represents the first natural product, non-amide member of a novel nanomolar-potency PARP1/2 inhibitor family.

Extrasynaptic CaMKIIα is involved in the antidepressant effects of ketamine by downregulating GluN2B receptors in an LPS-induced depression model.

BACKGROUND: A subanesthetic dose of ketamine provides rapid and effective antidepressant effects, but the molecular mechanism remains elusive. It has been reported that overactivation of extrasynaptic GluN2B receptors is associated with the antidepressant effects of ketamine and the interaction between GluN2B and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) is important for GluN2B localization and activity. Here, we tested whether changes of CaMKIIα and GluN2B are involved in the antidepressant effects of ketamine. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally (i.p.) into male C57BL/6 mice. For the interventional study, mice were administrated with ketamine (10 mg/kg, i.p.) or a CaMKIIα inhibitor KN93. Behavioral alterations were evaluated by open-field, novelty-suppressed feeding, and forced-swimming tests. Physiological functions were evaluated by the body weight and fur coat state of mice. The levels of p-CaMKIIα, CaMKIIα, p-GluN2B, GluN2B, p-CREB, CREB, BDNF, GluR1, and GluR2 in the hippocampus were detected by western blotting. The interaction between GluN2B and CaMKIIα was studied using immunoprecipitation assay and small interfering RNA (siRNA) assays. The colocalizations of GluN2B/PSD95 and p-GluN2B/PSD95 were detected by immunofluorescence. The long-term potentiation (LTP) in SC-CA1 of the hippocampus was detected by electrophysiology. RESULTS: LPS injection induced depression-like behaviors, which were accompanied by significant increases in extrasynaptic p-CaMKIIα expression, extrasynaptic GluN2B localization, and phosphorylation and decreases in p-CREB, BDNF, and GluR1 expressions and LTP impairment. These changes were prevented by ketamine administration. Immunoprecipitation assay revealed that LPS induced an increase in the p-CaMKIIα-GluN2B interaction, which was attenuated by ketamine administration. SiRNA assay revealed that CaMKIIα knockdown reduced the level and number of clusters of GluN2B in the cultured hippocampal neurons. KN93 administration also reduced extrasynaptic p-CaMKIIα expression, extrasynaptic GluN2B localization, and phosphorylation and exerted antidepressant effects. CONCLUSION: These results indicate that extrasynaptic CaMKIIα plays a key role in the cellular mechanism of ketamine's antidepressant effect and it is related to the downregulation of extrasynaptic GluN2B localization and phosphorylation.

Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites.

The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins (i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2_NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating.

Signal peptide represses GluK1 surface and synaptic trafficking through binding to amino-terminal domain.

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.

Precise and robust binocular camera calibration based on multiple constraints.

Precise calibration of a binocular vision system is the foundation of binocular vision measurement. In this paper, we propose a highly precise and robust binocular camera calibration method, which is devoted to minimize the error between the geometric relation of 3D reconstructed feature points and the ground truth, such as adjacent distance error, collinear error, and right-angle error. In addition, the reprojection error and epipolar are introduced to satisfy the homography relation and epipolar geometry theory better. We optimize all intrinsic parameters, extrinsic parameters, and distortion parameters to minimize the objective function, which is the sum of a series of nonlinear least squares terms. Levenberg-Marquardt iterative algorithm is used to find the optimal solution of the camera parameters. To test the precision and robustness of the proposed method, both actual measurement experiments and Gauss noise-adding experiments are carried out. The experimental results show that compared with the other two calibration methods in the contrast experiment, the distance measurement error, collinear error, and right-angle error are reduced dramatically. It is noticeable that in Gauss noise-adding experiments, the calibration parameters estimated by the proposed method are more stable.

AtWNK9 is regulated by ABA and dehydration and is involved in drought tolerance in Arabidopsis.

WNK (with no lysine [K]) kinases play important regulatory roles in flowering, as well as salt and osmotic stress tolerance in plants. Here, we report that AtWNK9, a member of the Arabidopsis WNK gene family, was induced by exogenous abscisic acid (ABA) treatment and dehydration stress. Overexpression of AtWNK9 from the cauliflower mosaic virus 35S promoter in Arabidopsis resulted in increased sensitivity to ABA, strong inhibition of primary root elongation, increased proline accumulation, reduced stomatal aperture, and a reduced rate of water loss. In addition, plant survival under drought stress was improved compared to wild type. In contrast, a mutant with a T-DNA insertion in AtWNK9 showed reduced ABA sensitivity and an increased rate of water loss; further, it showed increased susceptibility to drought stress. The transcription of a number of ABA signaling components, including ABI1, ERA1, ABI3, and ABF3, was up-regulated in AtWNK9 transgenic plants and down-regulated in the wnk9 mutant in response to ABA. Some ABA-responsive and biosynthetic genes, as well as other drought-related genes, were altered at various levels in AtWNK9 transgenic plants and wnk9 mutants under dehydration stress. Overall, these findings suggest that AtWNK9 plays a positive role in ABA signaling and improves drought tolerance in transgenic Arabidopsis.