Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels.Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships.Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27–37 μM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 μM) and Q52R (IC50 55 μM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2–3 μM), while Q52R inhibition was 15–20% at 10 μM.Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

Inhibition of the Nav1.7 Channel in the Trigeminal Ganglion Relieves Pulpitis Inflammatory Pain

Pulpitis causes significant changes in the peripheral nervous system, which induce hyperalgesia. However, the relationship between neuronal activity and Nav1.7 expression following pulpal noxious pain has not yet been investigated in the trigeminal ganglion (TG). The aim of our study was to verify whether experimentally induced pulpitis activates the expression of Nav1.7 peripherally and the neuronal activities of the TGs can be affected by Nav1.7 channel inhibition. Acute pulpitis was induced through allyl isothiocyanate (AITC) application to the rat maxillary molar tooth pulp. Three days after AITC application, abnormal pain behaviors were recorded, and the rats were euthanized to allow for immunohistochemical, optical imaging, and western blot analyses of the Nav1.7 expression in the TG. A significant increase in AITC-induced pain-like behaviors and histological evidence of pulpitis were observed. In addition, histological and western blot data showed that Nav1.7 expressions in the TGs were significantly higher in the AITC group than in the naive and saline group rats. Optical imaging showed that the AITC group showed higher neuronal activity after electrical stimulation of the TGs. Additionally, treatment of ProTxII, selective Nav1.7 blocker, on to the TGs in the AITC group effectively suppressed the hyperpolarized activity after electrical stimulation. These findings indicate that the inhibition of the Nav1.7 channel could modulate nociceptive signal processing in the TG following pulp inflammation.

Absolute Structure Determination and Kv1.5 Ion Channel Inhibition Activities of New Debromoaplysiatoxin Analogues

Potassium channel Kv1.5 has been considered a key target for new treatments of atrial tachyarrhythmias, with few side effects. Four new debromoaplysiatoxin analogues with a 6/6/12 fused ring system were isolated from marine cyanobacterium Lyngbya sp. Their planar structures were elucidated by HRESIMS, 1D and 2D NMR. The absolute configuration of oscillatoxin J (1) was determined by single-crystal X-ray diffraction, and the absolute configurations of oscillatoxin K (2), oscillatoxin L (3) and oscillatoxin M (4) were confirmed on the basis of GIAO NMR shift calculation followed by DP4 analysis. The current study confirmed the absolute configuration of the pivotal chiral positions (7S, 9S, 10S, 11R, 12S, 15S, 29R and 30R) at traditional ATXs with 6/12/6 tricyclic ring system. Compound 1, 2 and 4 exhibited blocking activities against Kv1.5 with IC50 values of 2.61 ± 0.91 µM, 3.86 ± 1.03 µM and 3.79 ± 1.01 µM, respectively. However, compound 3 exhibited a minimum effect on Kv1.5 at 10 µM. Furthermore, all of these new debromoaplysiatoxin analogs displayed no apparent activity in a brine shrimp toxicity assay.

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

Metabolic demand regulates selective cell death in Parkinson′s Disease

Parkinson′s Disease (PD) is a debilitating neurodegenerative condition that affects over 10 million people across the world, causing tremors and muscle weakness. Its mechanisms are unknown, but one key feature is selective cell death: neurons in the Substantia Nigra Pars Compacta (SNc) die, but their neighbors, the cells in the Ventral Tegmental Area (VTA), remain healthy. To study this phenomenon, we used an established single neuron model of the SNc, adapting its biophysical and bioenergetic properties to match that of the VTA. We discovered that reducing calcium influx correlates with higher ATP and lower ROS concentrations in the cell, suggesting in silico the importance of calcium influx in metabolic stress and selective vulnerability for Parkinson′s Disease. Future efforts may target calcium channel inhibition as a therapeutic strategy, although caution is needed with potential metabolic side effects.

CRAC Channel Controls the Differentiation of Pathogenic B Cells in Lupus Nephritis

Store-operated Ca2+ release-activated Ca2+ (CRAC) channel is the main Ca2+ influx pathway in lymphocytes and is essential for immune response. Lupus nephritis (LN) is an autoimmune disease characterized by the production of autoantibodies due to widespread loss of immune tolerance. In this study, RNA-seq analysis revealed that calcium transmembrane transport and calcium channel activity were enhanced in naive B cells from patients with LN. The increased expression of ORAI1, ORAI2, and STIM2 in naive B cells from patients with LN was confirmed by flow cytometry and Western blot, implying a role of CRAC channel in B-cell dysregulation in LN. For in vitro study, CRAC channel inhibition by YM-58483 or downregulation by ORAI1-specific small-interfering RNA (siRNA) decreased the phosphorylation of Ca2+/calmodulin-dependent protein kinase2 (CaMK2) and suppressed Blimp-1 expression in primary human B cells, resulting in decreased B-cell differentiation and immunoglobulin G (IgG) production. B cells treated with CaMK2-specific siRNA showed defects in plasma cell differentiation and IgG production. For in vivo study, YM-58483 not only ameliorated the progression of LN but also prevented the development of LN. MRL/lpr lupus mice treated with YM-58483 showed lower percentage of plasma cells in the spleen and reduced concentration of anti-double-stranded DNA antibodies in the sera significantly. Importantly, mice treated with YM-58483 showed decreased immune deposition in the glomeruli and alleviated kidney damage, which was further confirmed in NZM2328 lupus mice. Collectively, CRAC channel controlled the differentiation of pathogenic B cells and promoted the progression of LN. This study provides insights into the pathogenic mechanisms of LN and that CRAC channel could serve as a potential therapeutic target for LN.

Modelling altered signalling of G-protein coupled receptors in inflamed environment to advance drug design

We previously reported the successful design, synthesis and testing of the prototype opioid painkiller NFEPP that does not elicit adverse side effects. Uniquely, this design was based on mathematical modelling of extracellular interactions between G-protein coupled receptors (GPCRs) and ligands, recognizing that GPCRs function differently under pathological versus healthy conditions. We now present a novel stochastic model of GPCR function that includes intracellular dissociation of G-protein subunits and modulation of plasma membrane calcium channels associated with parameters of inflamed and healthy tissue (pH, radicals). The model is validated against in vitro experimental data for NFEPP and fentanyl ligands at different pH values. We found markedly reduced calcium channel inhibition induced by NFEPP at normal pH compared to lower pH, in contrast to the effect of fentanyl, and enhanced constitutive G-protein activation but lower probability of ligand binding with increasing radical concentrations. By means of molecular dynamics simulations, we also assessed qualitative changes of reaction rates due to additional disulfide bridges inside the GPCR binding pocket. The results suggest that, compared to radicals, low pH is a more important determinant of overall GPCR function in an inflamed environment. Future drug design efforts should take this into account.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-Like Toxin Binding Properties

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.