Nausch B.,University of Vermont |
Rode F.,Neurosearch |
Rode F.,Novo Nordisk AS |
Jorgensen S.,Neurosearch |
And 18 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2014
Large-conductance Ca2+-activated K+ channels (BK, K Ca1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 μM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 μM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 μM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 μM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K+ concentration consistent with a K+ channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.
David-Bosne S.,University Paris - Sud |
Florent I.,CNRS Laboratory of Communication Molecules and Adaptation of Microorganisms |
Lund- Winther A.-M.,Pcovery Aps |
Lund- Winther A.-M.,University of Aarhus |
And 5 more authors.
FEBS Journal | Year: 2013
The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca 2+-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large-scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1-9 μm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 μm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6-specific inhibitors with good antimalarial activity. PfATP6, the P. falciparum SERCA, was investigated as an antimalarial drug target. It was expressed in yeast and large-scale purified, a chemical library of 1680 molecules was screened on its calcium-dependent ATPase activity and eight compounds were chosen for their effect on P. falciparum in vitro growth. This study describes a multidisciplinary approach allowing the selection of new potential antimalarials. © 2013 The Authors Journal compilation © 2013 FEBS.
Clausen J.D.,University of Aarhus |
Clausen J.D.,Pcovery ApS |
Bublitz M.,University of Aarhus |
Bublitz M.,University of Oxford |
And 5 more authors.
Structure | Year: 2016
Summary Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound at the catalytic site as a planar VO3 - in complex with water and Mg2+ in a dephosphorylation transition-state-like conformation. Validating bound VO3 - by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl- site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca2+-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3 - structure with that in E2·BeF3 - (E2P ground state analog) reveals multiple binding modes to the Ca2+-ATPase. © 2016 Elsevier Ltd. All rights reserved.
Mills T.A.,University of Manchester |
Greenwood S.L.,University of Manchester |
Devlin G.,University of Manchester |
Shweikh Y.,University of Manchester |
And 9 more authors.
Placenta | Year: 2015
Introduction Potassium (K+) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. Methods Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. Results Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K+ depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. Discussion Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance. © 2015 Elsevier Ltd.
PubMed | University of Manchester, University of Calabria and Pcovery Aps
Type: Journal Article | Journal: Placenta | Year: 2015
Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries.Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively.Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs.Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance.
Sorensen D.M.,Copenhagen University |
Moller A.B.,Copenhagen University |
Moller A.B.,Novo Nordisk AS |
Jakobsen M.K.,Copenhagen University |
And 6 more authors.
Journal of Biological Chemistry | Year: 2012
P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca2+ triggers dephosphorylation. Remarkably, Ca2+-induced dephosphorylation occurs at high apparent [Ca 2+] (Ki = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca2+ is a transported substrate but indicate the presence of a cytosolic low affinity Ca2+-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca2+ as a modifier of its function. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.