Pharmacophore‐based discovery of inhibitors of a novel drug/proton antiporter in human brain endothelial hCMEC/D3 cell line

Background and Purpose

An influx drug/proton antiporter of unknown structure has been functionally demonstrated at the blood–brain barrier. This transporter, which handles some psychoactive drugs like diphenhydramine, clonidine, oxycodone, nicotine and cocaine, could represent a new pharmacological target in drug addiction therapy. However, at present there are no known drugs/inhibitors that effectively inhibit/modulate this transporter in vivo.

Experimental Approach

The FLAPpharm approach was used to establish a pharmacophore model for inhibitors of this transporter. The inhibitory potency of 44 selected compounds was determined against the specific substrate, [³H]‐clonidine, in the human cerebral endothelial cell line hCMEC/D3 and ranked as good, medium, weak or non‐inhibitor.

Key Results

The pharmacophore model obtained was used as a template to screen xenobiotic and endogenous compounds from databases [Specs, Recon2, Human Metabolome Database (HMDB), human intestinal transporter database], and hypothetical candidates were tested in vitro to determine their inhibitory capacity with [³H]‐clonidine. According to the transporter database, 80% of the proton antiporter inhibitor candidates could inhibit P‐glycoprotein/MDR1/ABCB1 and specificity is improved by reducing inhibitor size/shape and increasing water solubility. Virtual screening results using HMDB and Recon2 for endogenous compounds appropriately scored tryptamine as an inhibitor.

Conclusions and Implications

The pharmacophore model for the proton‐antiporter inhibitors was a good predictor of known inhibitors and allowed us to identify new good inhibitors. This model marks a new step towards the discovery of this drug/proton antiporter and will be of great use for the discovery and design of potent inhibitors that could potentially help to assess and validate its pharmacological role in drug addiction in vivo.

Abbreviations

ADE

absorption, distribution and elimination

BBB

blood–brain barrier

DPBS

Dulbecco''s PBS

DPH

diphenhydramine

G‐I

good inhibitor

Glob‐Prod flap

global product similarity score

Glob‐Sum flap

global sum similarity score

KH

Krebs–HEPES buffer

M‐I

medium inhibitor

N‐I

non‐inhibitor

PCA

principal component analysis

P‐gp

P‐glycoprotein

PK

pharmacokinetics

W‐I

weak inhibitor

     

Salvage of nicotinamide adenine dinucleotide plays a critical role in the bioenergetic recovery of post‐hypoxic cardiomyocytes

Background and Purpose

Ischaemic heart disease can lead to serious, life‐threatening complications. Traditional therapies for ischaemia aim to increase oxygen delivery and reduce the myocardial ATP consumption by increasing the coronary perfusion and by suppressing cardiac contractility, heart rate or blood pressure. An adjunctive treatment option for ischaemia is to improve or optimize myocardial metabolism.

Experimental Approach

Metabolic suppression in the ischaemic heart is characterized by reduced levels of high‐energy molecules: ATP and NAD⁺. Because NAD⁺ is required for most metabolic processes that generate ATP, we hypothesized that restoration of NAD⁺ would be a prerequisite for ATP regeneration and examined the role of the major NAD⁺ anabolic and catabolic pathways in the bioenergetic restoration process following oxygen–glucose deprivation injury in a cardiomyocyte cell line (H9c2 cells).

Key Results

Salvage of NAD⁺ via nicotinamide phosphoribosyl transferase was essential for bioenergetic recovery in cardiomyocytes. Blockade of nicotinamide phosphoribosyl transferase prevented the restoration of the cellular ATP pool following oxygen–glucose deprivation injury by inhibiting both the aerobic and anaerobic metabolism in the cardiomyocytes. NAD⁺ consumption by PARP‐1 also undermined the recovery processes, and PARP inhibition significantly improved the metabolism and increased cellular ATP levels in cardiomyocytes.

Conclusions and Implications

We conclude that the NAD⁺ salvage pathway is essential for bioenergetic recovery in post‐hypoxic cardiomyocytes and PARP inhibition may represent a potential future therapeutic intervention in ischaemic heart disease.

Abbreviations

CVD

cardiovascular disease

FK866

(E)‐N‐[4‐(1‐benzoylpiperidin‐4‐yl)butyl]‐3‐(pyridin‐3‐yl)acrylamide

JC‐1

5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide

MitoSOX Red

MitoSOX™ Red mitochondrial superoxide indicator

MTT

3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide

NamPRT

nicotinamide phosphoribosyltransferase

NMNAT

nicotinamide mononucleotide adenylyl transferase

OGD

oxygen–glucose deprivation

PJ34

N‐(6‐oxo‐5,6‐dihydrophenanthridin‐2‐yl)‐(N,N‐dimethylamino) acetamide hydrochloride

RNF146

ring finger protein 146

     

Blood–brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P‐glycoprotein on a novel verapamil influx carrier

Background and Purpose

The respective impact and interplay between ABC (P‐glycoprotein/P‐gp/Abcb1a, BCRP/ABCG2, MRP/ABCC) and SLC transporter functions at the blood–brain barrier (BBB) and blood–retinal barriers (BRB) are incompletely understood.

Experimental Approach

We measured the initial cerebral and retinal distribution of selected ABC substrates by in situ carotid perfusion using P‐gp/Bcrp knockout mice and chemical ABC/SLC modulation strategies. P‐gp, Bcrp, Mrp1 and Mrp4 were studied by confocal retina imaging.

Key Results

Chemical or physical disruption of P‐gp increased [³H]‐verapamil transport by ~10‐fold at the BBB and ~1.5‐fold at the BRB. [³H]‐Verapamil transport involved influx‐mediated by an organic cation clonidine‐sensitive/diphenhydramine‐sensitive proton antiporter at both barriers; this effect was unmasked when P‐gp was partially or fully inhibited/disrupted at the BBB. Studies of [³H]‐mitoxantrone and [³H]‐zidovudine transport suggested, respectively, that Bcrp efflux was less involved at the BRB than BBB, whereas Mrps were significantly and similarly involved at both barriers. Confocal imaging showed that P‐gp and Bcrp were expressed in intra‐retinal vessels (inner BRB/iBRB) but absent from the blood/basal membrane of cells of the retinal pigment epithelium (outer BRB/oBRB/RPE) where, in contrast, Mrp1 and Mrp4 were localized.

Conclusions and Implications

P‐gp, Bcrp, Mrp1 and Mrp4 are differentially expressed at the outer and inner BRB, resulting in an altered ability to limit substrate distribution at the retina as compared with the BBB. [³H]‐Verapamil distribution is not P‐gp‐specific and involves a proton antiporter at both the BBB and BRB. However, this transport is concealed by P‐gp at the BBB, but not at the BRB, where P‐gp activity is reduced.

Abbreviations

ABC

ATP‐binding cassette

BBB

blood–brain barrier

BRB

blood–retina barrier

CV

choroidal vessels

DPH

diphenhydramine; Elac, elacridar

iBRB

inner blood–retina barrier

KO

knockout

oBRB

outer blood–retina barrier

PD

pharmacodynamics

P‐gp

P‐glycoprotein

pH_e

extracellular/vascular pH

pH_i

intracellular pH

PK

pharmacokinetics

RPE

retinal pigment epithelial cells

SLC

solute carrier

TKO

triple knockout [Abcb1a^−/−, Abcb1b^−/−, Abcg2^−/−] mice

WT

wild type

     

Hepatitis C: recent successes and continuing challenges in the development of improved treatment modalities

Despite the progress being made toward development of less-toxic and simpler alternatives to the current peg-IFN standard-of-care therapy for chronic hepatitis C, the highly replicative nature of HCV infection and the error-prone nature of its viral RNA synthesis pose extraordinary challenges to drug development. Peg-IFN is likely to remain a mainstay of therapy for the foreseeable future, or until such time that multiple direct-acting STAT-C inhibitors are available and shown to provide a sufficiently high barrier to resistance when used in combination.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

Background and Purpose

In the phase III clinical trial, RELAX‐AHF, serelaxin caused rapid and long‐lasting haemodynamic changes. However, the cellular mechanisms involved are unclear in humans.

Experimental Approach

This study examined the effects of serelaxin in co‐cultures of human primary endothelial cells (ECs) and smooth muscle cells (SMCs) on cAMP and cGMP signalling.

Key Results

Stimulation of HUVECs or human coronary artery endothelial cells (HCAECs) with serelaxin, concentration‐dependently increased cGMP accumulation in co‐cultured SMCs to a greater extent than in monocultures of either cell type. This was not observed in human umbilical artery endothelial cells (HUAECs) that do not express the relaxin receptor, RXFP1. Treatment of ECs with l‐N^G‐nitro arginine (NOARG; 30 μM, 30 min) inhibited serelaxin‐mediated (30 nM) cGMP accumulation in HUVECs, HCAECs and co‐cultured SMCs. In HCAECs, but not HUVECs, pre‐incubation with indomethacin (30 μM, 30 min) also inhibited cGMP accumulation in SMCs. Pre‐incubation of SMCs with the guanylate cyclase inhibitor ODQ (1 μM, 30 min) had no effect on serelaxin‐mediated (30 nM) cGMP accumulation in HUVECs and HCAECs but inhibited cGMP accumulation in SMCs. Serelaxin stimulation of HCAECs, but not HUVECs, increased cAMP accumulation concentration‐dependently in SMCs. Pre‐incubation of HCAECs with indomethacin, but not l‐NOARG, abolished cAMP accumulation in co‐cultured SMCs, suggesting involvement of prostanoids.

Conclusions and Implications

In co‐cultures, treatment of ECs with serelaxin caused marked cGMP accumulation in SMCs and with HCAEC also cAMP accumulation. Responses involved EC‐derived NO and with HCAEC prostanoid production. Thus, serelaxin differentially modulates vascular tone in different vascular beds.

Abbreviations

AHF

acute heart failure

DEA

diethylamine NONOate

ECs

endothelial cells

HCAEC

human coronary artery endothelial cell

HUAEC

human umbilical artery endothelial cell

HUASMC

human umbilical artery smooth muscle cell

HUVSMC

human umbilical vein smooth muscle cell

l‐NOARG

l‐N^G‐nitro arginine

SMCs

smooth muscle cells

     

Meningeal blood flow is controlled by H2S‐NO crosstalk activating a HNO‐TRPA1‐CGRP signalling pathway

Background and Purpose

Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO–derived compound, nitroxyl (HNO), generated through reaction with endogenous H₂S. We investigated the involvement of HNO in CGRP release and meningeal blood flow.

Experimental Approach

Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H₂S were localized histochemically with specific sensors.

Key Results

Topical administration of the NO donor diethylamine‐NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H₂S synthesis, reduced this effect. Administration of Na₂S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP _8‐37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na₂S dose‐dependently increased CGRP release two‐ to threefold. Co‐administration of diethylamine‐NONOate facilitated CGRP release, while inhibition of endogenous NO or H₂S synthesis lowered basal CGRP release. NO and H₂S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L‐NMMA and oxamic acid.

Conclusions and Implications

NO and H₂S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO‐TRPA1‐CGRP signalling pathway may be relevant to the pathophysiology of headaches.

Abbreviations

CBS

cystathionine β‐synthase

CSE

cystathionine γ‐lyase

Cy3

cyanine dye 3

DAF

4‐amino‐5‐methylamino‐2′,7′‐difluoresceine diacetate

HNO

nitroxyl

iNOS

inducible NOS

L‐NMMA

L‐NG‐monomethylarginine acetate

MMA

middle meningeal artery

MST

mercaptopyruvate sulfurtransferase

nNOS

neuronal NOS

NONOate

diethylamine‐NONOate, DEANONOate

ODQ

1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one

sGC

soluble GC

SIF

synthetic interstitial fluid

TRPA1

transient receptor potential ankyrin 1 channel

Tables of Links

Priorities for mixtures health effects research

In order to better inform scientific decision making in the occupational environment, we need a better understanding of the toxicology of mixed exposures. In particular, we need an understanding of the dose–response relationship from the level of individual or population exposure down to the molecular level (and then back up again from the molecular level to the specific health-related response of the organism as a whole). Mixtures toxicology is proving to be different from single-chemical toxicology in several fundamental but barely recognized ways:

• complex chemical mixtures may consist of thousands of (often unidentified) components, each often at very low doses, but together constituting significant exposure levels;

• exposure is nearly always via multiple routes, pathways;

• other stressors such as noise, heat, infection, etc., may play a significant role in the overall environmental health response;

• interactions are potentially many and varied: pharmacokinetic and pharmacodynamic interactions may occur at the same site, or at different sites via complex physiological processes (including defense mechanisms);

• cumulative effects of different exposures/stresses over time need to be considered (altering the “baseline” susceptibility of the individual).

Knowledge gained in mixtures research should be able to improve current risk assessment and mitigation or intervention methods. In NIOSH's National Occupational Research Agenda (NORA) three priority areas have been identified:

• Epidemiology: Improvement of statistical tools to identify mixed effects from available epidemiological data (“confounding factors are mixtures effects”). Awareness of disease states with significant environmental components (Gulf War Syndrome (GWS), Chronic Fatigue Syndrome (CFS), Multiple Chemical Sensitivity (MCS), etc.). Recognition and investigation of the complexity of disease related responses to multiple (simultaneous and serial) stressors (immune system, endocrine system, nervous system, etc.). Wider emphasis on relatively new concepts such as susceptibility, which links the genetic and environmental components of a disease. Disease itself is of course also a stressor.

• Laboratory approaches: Methods need to be developed to understand and integrate experimental data from the molecular to the whole organism level for understanding multiple data (proteomics, genomics, and metabolomics/metabonomics) from mixed exposures. Understanding and prediction of precursors to adverse health effects will inevitably lead to identification of useful biomarkers of effect, and to earlier and more effective intervention strategies. We also need to improve our ability to forecast interaction effects from mixed exposures using less costly cellular-based screening tools and computer modeling (e.g. QSAR), and develop improved models for large-scale studies of the nature of chemical interactions that lump responses by chemical classes.

• Modeling as integrator of data: Development and validation of mechanism-based models and predictive tools are essential for improving current risk assessment processes for mixtures. For example, current linked physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) models for multiple simultaneous exposures to chemicals (such as BTEX) need to be extended to more complex mixtures, and new statistical methods of dealing with possibly thousands of components need to be developed. Ultimately, such models may become an integral part of a model of the “virtual human” via computer simulation.

Pro‐cognitive activity in rats of 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide,a positive allosteric modulator of the α7 nicotinic acetylcholine receptor

Background and Purpose

α7 nicotinic acetylcholine receptors (α7 nAChRs) may represent useful targets for cognitive improvement. The aim of this study is to compare the pro‐cognitive activity of selective α7‐nAChR ligands, including the partial agonists, DMXBA and A‐582941, as well as the positive allosteric modulator, 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide (PAM‐2).

Experimental Approach

The attentional set‐shifting task (ASST) and the novel object recognition task (NORT) in rats, were used to evaluate the pro‐cognitive activity of each ligand [i.e., PAM‐2 (0.5, 1.0, and 2.0 mg·kg⁻¹), DMXBA and A‐582941 (0.3 and 1.0 mg·kg⁻¹)], in the absence and presence of methyllycaconitine (MLA), a selective competitive antagonist. To determine potential drug interactions, an inactive dose of PAM‐2 (0.5 mg·kg⁻¹) was co‐injected with inactive doses of either agonist ‐ DMXBA: 0.1 (NORT); 0.3 mg·kg⁻¹ (ASST) or A‐582941: 0.1 mg·kg⁻¹.

Key Results

PAM‐2, DMXBA, and A‐582941 improved cognition in a MLA‐dependent manner, indicating that the observed activities are mediated by α7 nAChRs. Interestingly, the co‐injection of inactive doses of PAM‐2 and DMXBA or A‐582941 also improved cognition, suggesting drug interactions. Moreover, PAM‐2 reversed the scopolamine‐induced NORT deficit. The electrophysiological results also support the view that PAM‐2 potentiates the α7 nAChR currents elicited by a fixed concentration (3 μM) of DMXBA with apparent EC₅₀ = 34 ± 3 μM and E_max = 225 ± 5 %.

Conclusions and Implications

Our results support the view that α7 nAChRs are involved in cognition processes and that PAM‐2 is a novel promising candidate for the treatment of cognitive disorders.

Abbreviations

α7 nAChR

nicotinic acetylcholine receptor with α7 subunit

AD

Alzheimer''s disease

apparent EC₅₀

enhancement potency

ASST

attentional set‐shifting task

CD

compound discrimination

DI

discrimination index

E

exploration time

ED

extra‐dimensional

E_max

ligand efficacy

ID

intra‐dimensional

ITI

inter‐trial interval

MLA

methyllycaconitine

NORT

novel object recognition task

n_H

Hill coefficient

PAM

positive allosteric modulator

PAM‐2

3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide

Rev

reversal of discrimination

SD

simple discrimination

T1

familiarisation trial

T2

retention trial

     

The role of hexokinase in cardioprotection – mechanism and potential for translation

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose‐6‐phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre‐ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B‐cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target.

Linked Articles

This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

Abbreviations

Akt

also known as PKB

AMPK

AMP‐activated PK

ANT

adenine nucleotide translocase

Bad

Bcl‐2‐associated death promoter

Bak

Bcl‐2 homologous antagonist/killer

Bax

Bcl‐2‐like protein 4

Bcl‐2

B‐cell lymphoma 2

Bcl‐xL

Bcl‐2‐extra large

CK

creatine kinase

CsA

cyclosporine A

CyP‐D

cyclophilin D

Drp1

dynamin‐related protein 1

G‐6‐P

glucose‐6‐phosphate

GSK3β

glycogen synthase kinase 3β

HK

hexokinase

I/R

ischaemia/reperfusion

IF1

ATP synthase inhibitor factor 1

IMM

inner mitochondrial membrane

IP

ischaemic preconditioning

mPTP

mitochondrial permeability transition pore

OMM

outer mitochondrial membrane

Opa‐1

optic athrophy 1

PCr

phosphocreatine

PPIase

peptidylprolyl isomerase

ROS

reactive oxygen species

SR

sarcoplasmic reticulum

T₀

time of ischaemic rigor start

TAT‐HK2

cell‐permeable peptide of HK2 binding domain

TP

temperature preconditioning

TSPO

translocator protein of the outer membrane

VDAC

voltage‐dependent anion channel

Tables of Links

Activation of M 3 cholinoceptors attenuates vascular injury after ischaemia/reperfusion by inhibiting the Ca 2+/calmodulin‐dependent protein kinase II pathway

Background and Purpose

The activation of M ₃ cholinoceptors (M ₃ receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)‐induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) regulation.

Experimental Approach

Rats were given choline (10 mg·kg⁻¹, i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M ₃ receptor antagonist, 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), was injected (0.12 μg·kg⁻¹, i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca²⁺‐cycling proteins were also assessed.

Key Results

Choline treatment attenuated I/R‐induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up‐regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca²⁺‐cycling proteins, including Na⁺/Ca²⁺ exchanger, inositol 1,4,5‐trisphosphate receptor, sarcoplasmic reticulum Ca²⁺‐ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4‐DAMP.

Conclusions and Implications

Our data suggest that inhibition of the ROS‐mediated CaMKII pathway and modulation of Ca²⁺‐cycling proteins may be novel mechanisms underlying choline‐induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M ₃ receptors in the vasculature, providing a new therapeutic strategy for I/R‐induced vascular injury.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

4‐DAMP

4‐diphenylacetoxy‐N‐methylpiperidine methiodide

CaMKII

Ca²⁺/calmodulin‐dependent protein kinase II

DHE

dihydroethidium

I/R

ischaemia/reperfusion

IP₃R

inositol 1,4,5‐trisphosphate receptor

NAC

N‐acetyl‐L‐cysteine

NCX

Na⁺/Ca²⁺ exchanger

PLB

phospholamban

ROS

reactive oxygen species

SERCA

sarcoplasmic reticulum Ca²⁺‐ATPase

SNP

sodium nitroprusside

Tables of Links

The virtual heart as a platform for screening drug cardiotoxicity

To predict the safety of a drug at an early stage in its development is a major challenge as there is a lack of in vitro heart models that correlate data from preclinical toxicity screening assays with clinical results. A biophysically detailed computer model of the heart, the virtual heart, provides a powerful tool for simulating drug–ion channel interactions and cardiac functions during normal and disease conditions and, therefore, provides a powerful platform for drug cardiotoxicity screening. In this article, we first review recent progress in the development of theory on drug–ion channel interactions and mathematical modelling. Then we propose a family of biomarkers that can quantitatively characterize the actions of a drug on the electrical activity of the heart at multi‐physical scales including cellular and tissue levels. We also conducted some simulations to demonstrate the application of the virtual heart to assess the pro‐arrhythmic effects of cisapride and amiodarone. Using the model we investigated the mechanisms responsible for the differences between the two drugs on pro‐arrhythmogenesis, even though both prolong the QT interval of ECGs. Several challenges for further development of a virtual heart as a platform for screening drug cardiotoxicity are discussed.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

AE

allosteric effector

APD

action potential duration

APD₉₀

APD at 90% repolarization

APs

action potentials

BCL

basic cycle length

CV

conduction velocity

CVR

conduction velocity restitution

ERP

effective refractory period

GR

guarded receptor

HH

Hodgkin–Huxley

I_CaL

L‐type Ca²⁺ current

I_Kr

delayed rectifier K⁺ channel current

I_Ktof

fast component of the cardiac transient outward current

I_Na

Na⁺ channel current

LQTs

long QT syndrome

MR

modulated receptor

QTc

corrected QT interval

VW

vulnerable window

WL

wavelength

Tables of Links

Continuous inhibition of 11β‐hydroxysteroid dehydrogenase type I in adipose tissue leads to tachyphylaxis in humans and rats but not in mice

Background and Purpose

11β‐hydroxysteroid dehydrogenase type I (11β‐HSD1), a target for Type 2 diabetes mellitus, converts inactive glucocorticoids into bioactive forms, increasing tissue concentrations. We have compared the pharmacokinetic‐pharmacodynamic (PK/PD) relationship of target inhibition after acute and repeat administration of inhibitors of 11β‐HSD1 activity in human, rat and mouse adipose tissue (AT).

Experimental Approach

Studies included abdominally obese human volunteers, rats and mice. Two specific 11β‐HSD1 inhibitors (AZD8329 and COMPOUND‐20) were administered as single oral doses or repeat daily doses for 7–9 days. 11β‐HSD1 activity in AT was measured ex vivo by conversion of ³H‐cortisone to ³H‐cortisol.

Key Results

In human and rat AT, inhibition of 11β‐HSD1 activity was lost after repeat dosing of AZD8329, compared with acute administration. Similarly, in rat AT, there was loss of inhibition of 11β‐HSD1 activity after repeat dosing with COMPOUND‐20 with continuous drug cover, but effects were substantially reduced if a ‘drug holiday’ period was maintained daily. Inhibition of 11β‐HSD1 activity was not lost in mouse AT after continuous cover with COMPOUND‐20 for 7 days.

Conclusions and Implications

Human and rat AT, but not mouse AT, exhibited tachyphylaxis for inhibition of 11β‐HSD1 activity after repeat dosing. Translation of observed efficacy in murine disease models to human for 11β‐HSD1 inhibitors may be misleading. Investigators of the effects of 11β‐HSD1 inhibitors should confirm that desired levels of enzyme inhibition in AT can be maintained over time after repeat dosing and not rely on results following a single dose.

Abbreviations

11β‐HSD1

11β‐hydroxysteroid dehydrogenase type I

PK/PD

pharmacokinetic‐pharmacodynamic

AT

adipose tissue

DIO

diet induced obese

IHC

International Conference on Harmonisation

GCP

Good Clinical Practice

b.i.d.

twice daily

u.i.d.

once daily

HPMC

hydroxypropylmethylcellulose

IC₇₀

concentration that delivers 70% of the maximum effect

IC₉₀

concentration that delivers 90% of the maximum effect

fu

fraction unbound

C_max

maximum achieved concentration

C_min

minimum or trough concentration

E₀

baseline

E_max

maximum effect

ANCOVA

analysis of covariance

     

Molecular mechanism of positive allosteric modulation of the metabotropic glutamate receptor 2 by JNJ‐46281222

Background and Purpose

Allosteric modulation of the mGlu₂ receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu₂ positive allosteric modulator (PAM) JNJ‐46281222 and its radiolabelled counterpart [³H]‐JNJ‐46281222. Using this novel tool, we also describe the allosteric effect of orthosteric glutamate binding and the presence of a bound G protein on PAM binding and use computational approaches to further investigate the binding mode.

Experimental Approach

We have used radioligand binding studies, functional assays, site‐directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ‐46281222.

Key Results

JNJ‐46281222 is an mGlu₂‐selective, highly potent PAM with nanomolar affinity (K _D = 1.7 nM). Binding of [³H]‐JNJ‐46281222 was increased by the presence of glutamate and greatly reduced by the presence of GTP, indicating the preference for a G protein bound state of the receptor for PAM binding. Its allosteric binding site was visualized and analysed by a computational docking and molecular dynamics study. The simulations revealed amino acid movements in regions expected to be important for activation. The binding mode was supported by [³H]‐JNJ‐46281222 binding experiments on mutant receptors.

Conclusion and Implications

Our results obtained with JNJ‐46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu₂ allosteric modulation. The computational simulations and mutagenesis provide a plausible binding mode with indications of how the ligand permits allosteric activation. This study is therefore of interest for mGlu₂ and class C receptor drug discovery.

Abbreviations

JNJ‐46281222

3‐(Cyclopropylmethyl)‐7‐[(4‐phenyl‐1‐piperidinyl)methyl]‐8‐(trifluoromethyl)‐1,2,4‐triazolo[4,3‐a]pyridine

NAM

negative allosteric modulator

PAM

positive allosteric modulator

VFT

Venus Flytrap domain

     

Threonine532 phosphorylation in ClC‐3 channels is required for angiotensin II‐induced Cl? current and migration in cultured vascular smooth muscle cells

Background and Purpose

Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl⁻ current, but the underlying mechanism is not clear.

Experimental Approach

The A10 cell line and primary cultures of VSMC from control, ClC‐3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole‐cell patch clamp, co‐immunoprecipitation, site‐specific mutagenesis and Western blotting,

Key Results

In VSMC, AngII induced Cl⁻ currents was carried by the chloride ion channel ClC‐3. This current was absent in VSMC from ClC‐3 channel null mice. The AngII‐induced Cl⁻ current involved interactions between ClC‐3 channels and Rho‐kinase 2 (ROCK2), shown by N‐ or C‐terminal truncation of ClC‐3 protein, ROCK2 siRNA and co‐immunoprecipitation assays. Phosphorylation of ClC‐3 channels at Thr⁵³² by ROCK2 was critical for AngII‐induced Cl⁻ current and VSMC migration. The ClC‐3 T532D mutant (mutation of Thr⁵³² to aspartate), mimicking phosphorylated ClC‐3 protein, significantly potentiated AngII‐induced Cl⁻ current and VSMC migration, while ClC‐3 T532A (mutation of Thr⁵³² to alanine) had the opposite effects. AngII‐induced cell migration was markedly decreased in VSMC from ClC‐3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII‐induced cerebrovascular remodelling was decreased in ClC‐3 null mice, possibly by the ROCK2 pathway.

Conclusions And Implications

ClC‐3 protein phosphorylation at Thr⁵³² by ROCK2 is required for AngII‐induced Cl⁻ current and VSMC migration that are involved in AngII‐induced vascular remodelling in hypertension.

Abbreviations

ΔCT

C‐terminal truncated ClC‐3

ΔNT

N‐terminal truncated ClC‐3

BASMC

basilar artery smooth muscle cells

caROCK2

constitutively active ROCK2

dnROCK2

dominant negative ROCK2

N1

EGFP‐N1 plasmid

VRCC

volume‐regulated chloride channel

VSMC

vascular smooth muscle cells

     

Nuciferine relaxes rat mesenteric arteries through endothelium‐dependent and ‐independent mechanisms

Background and Purpose

Nuciferine, a constituent of lotus leaf, is an aromatic ring‐containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanism

Experimental Approach

Nuciferine‐induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca²⁺ level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging.

Key Results

Nuciferine induced relaxations in arterial segments pre‐contracted by KCl or phenylephrine. Nuciferine‐elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L‐NAME or the NO‐sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser¹¹⁷⁷ and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca²⁺ influx. Under endothelium‐free conditions, nuciferine attenuated CaCl₂‐induced contraction in Ca²⁺‐free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl₂. Nuciferine also suppressed Ca²⁺ influx in Ca²⁺‐free K⁺‐containing solution in VSMCs.

Conclusions and Implications

Nuciferine has a vasorelaxant effect via both endothelium‐dependent and ‐independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

[Ca²⁺]_i

intracellular calcium

[NO]_i

intracellular NO

DAF‐FM DA

4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate

EDHF

endothelium‐derived hyperpolarizing factor

eNOS

endothelial NOS

Fluo‐4 AM

fluo‐4‐acetoxymethylester

iNOS

inducible NOS

L‐NAME

Nω‐nitro‐L‐arginine methyl ester

ODQ

1H‐[1,2,4]oxadizolo[4,3‐a]quinoxalin‐1‐one

VDCCs

voltage‐dependent Ca²⁺ channels

VSMCs

vascular smooth muscle cells

Tables of Links

Advances in exploring the role of microRNAs in the pathogenesis,diagnosis and therapy of cardiac diseases in China

Cardiovascular disease has become the most serious health threat and represents the major cause of morbidity and mortality in China, as in other industrialized nations. During the past few decades, China''s economic boom has tremendously improved people''s standard of living but has also changed their lifestyle, increasing the prevalence of cardiovascular disease, the so‐called ‘disease of modern civilization’. This new trend has attracted a significant amount of research. Many of the studies conducted by Chinese investigators are orientated towards understanding the molecular mechanisms of cardiovascular disease. At the molecular level, the long‐standing consensus is that cardiovascular disease is associated with a sequence mutation (genetic anomaly) and expression deregulation (epigenetic disorder) of protein‐coding genes. However, new research data have established the non‐protein‐coding genes microRNAs (miRNAs) as a central regulator of the pathogenesis of cardiac disease and a potential new therapeutic target for cardiovascular disease. These small non‐coding RNAs have also been subjected to extensive, rigorous investigations by Chinese researchers. Over the years, a large body of studies on miRNAs in cardiovascular disease has been conducted by Chinese investigators, yielding fruitful research results and a better understanding of miRNAs as a new level of molecular mechanisms for the pathogenesis of cardiac disease. In this review, we briefly summarize the current status of research in the field of miRNAs and cardiovascular disease in China, highlighting the advances made in elucidating the role of miRNAs in various cardiac conditions, including cardiac arrhythmia, myocardial ischaemia, cardiac hypertrophy and heart failure. We have also examined the potential of miRNAs as novel diagnostic biomarkers and therapeutic targets.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

AF

atrial fibrillation

AMI

acute myocardial infarction

APD

action potential duration

Cx43

connexin 43 (also known as GJA1, gap junction protein, α1)

Drp1

dynamin‐related protein‐1

E‐C

excitation‐contraction

HSP60

heat shock protein 60

I_CaL

L type calcium current

JP2

junctophilin‐2

KCNJ2

gene for potassium inwardly rectifying channel, subfamily J, member 2 also known as K_ir2.1, inwardly rectifying potassium channel 2.1

LNA

locked nucleic acid

MI

myocardial infarct

miRNAs

microRNAs

NFAT

nuclear factor of activated T‐cells

SERCA2a

sarcoplasmic reticulum calcium ATPase

K_Ca2.3 (also known as SK3)

small conductance calcium‐activated potassium channels 3

TGF‐β1

transforming growth factor‐β1

TGFBR2

transforming growth factor‐β receptor II

Tables of Links

Actions of KMUP‐1, a xanthine and piperazine derivative,on voltage‐gated Na+ and Ca2+‐activated K+ currents in GH3 pituitary tumour cells

Background and Purpose

7‐[2‐[4‐(2‐Chlorophenyl)piperazinyl]ethyl]‐1,3‐dimethylxanthine (KMUP‐1) is a xanthine‐based derivative. It has soluble GC activation and K⁺‐channel opening activity. Effects of this compound on ion currents in pituitary GH₃ cells were investigated in this study.

Experimental Approach

The aim of this study was to evaluate effects of KMUP‐1 on the amplitude and gating of voltage‐gated Na⁺ current (I _Na) in pituitary GH₃ cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca²⁺‐activated K⁺ current and the activity of large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels were also studied.

Key Results

KMUP‐1 depressed the transient and late components of I _Na with different potencies. The IC₅₀ values required for its inhibitory effect on transient and late I _Na were 22.5 and 1.8 μM respectively. KMUP‐1 (3 μM) shifted the steady‐state inactivation of I _Na to a hyperpolarized potential by −10 mV, despite inability to alter the recovery of I _Na from inactivation. In cell‐attached configuration, KMUP‐1 applied to bath increased BK_Ca‐channel activity; however, in inside‐out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP‐1 decreased the peak I _Na with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A.

Conclusions and Implications

Apart from activating BK_Ca channels, KMUP‐1 preferentially suppresses late I _Na. The effects of KUMP‐1 on ion currents presented here constitute an underlying ionic mechanism of its actions.

Abbreviations

AC

action current

AP

action potential

BK_Ca

channel large‐conductance Ca²⁺‐activated K⁺ channel

I_K(Ca)

Ca²⁺‐activated K⁺ current

I_Na

voltage‐gated Na⁺ current

I–V

current versus voltage

K_ATP

channel ATP‐sensitive K⁺ channel

ODQ

1H‐[1,2,4]oxadiazolo‐[4,3‐a] quinoxalin‐1‐one

TEA

tetraethylammonium chloride

τ_inact(S)

slow component of inactivation time constant for I _Na

YC‐1

3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole