Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro

Aim:

To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.

Methods:

HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.

Results:

Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.

Conclusion:

p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.     

Discovery and characterization of novel small-molecule agonists of G protein-coupled receptor 119

Aim： GPR119 is a G protein-coupled receptor （GPCR） that is highly expressed in pancreatic β-cells and intestinal L-cells and facilitates glucose-stimulated insulin secretion （GSIS）. GPR119 may represent a novel target for the treatment of metabolic disorders. Here, we sought to identify novel small-molecule GPR119 agonists.
Methods： A cell-based high-throughput screening assay was established using HEK293 cells stably expressing GPR119 and pCRE-luc reporter plasmid （HEK293/GPR119/pCRE-luc）. A compound library composed of 1440 compounds was screened. Mouse β-cell line MIN-6 and isolated mouse islets were used to evaluate the effects of candidate compounds on GSIS in vitro.

Results： Three compounds with novel structures （ZSY-04, -06, and -13） were found to activate GPR119-mediated signaling and to induce GPR119 desensitization. The EC50 values of ZSY-04, -06, and -13 in stimulating intracellular cAMP accumulation in HEK293/GPR119 cells were 2.758, 3.046, and 0.778 μmol/L, respectively. Furthermore, all three compounds displayed high selectivity for GPR119, and did not activate other 9 GPCRs tested. Moreover, all three compounds significantly increased GSIS in both MIN-6 mouse β-cells and isolated mouse islets at concentration of 10 μmol/L.

Conclusion： Three novel small-molecule GPR119 agonists （ZSY-04, -06, and -13） with high receptor selectivity and capacity to induce GSIS in vitro were discovered. These compounds are potential candidates to be structurally optimized into drugs for the treatment of type 2 diabetes.     

Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects

BACKGROUND AND PURPOSE

Allyl isothiocyanate (AITC, mustard oil), a constituent of many common cruciferous vegetables (Brassicaceae), activates transient receptor potential of ankyrin type-1 (TRPA1) channels, claimed to regulate gastrointestinal contractility. In this study, we have investigated the effect of AITC on intestinal motility.

EXPERIMENTAL APPROACH

Effects of AITC were investigated in vivo on upper gastrointestinal transit in mice and in mouse isolated ileum [contractions induced by electrical field stimulation (EFS), acetylcholine and spontaneous contractility]. The contractor activity of AITC was studied in mouse isolated colon. The ability of TRPA1 channel antagonists to block AITC-induced elevation of intracellular Ca²⁺[Ca²⁺]_i was assessed in HEK293 cells transfected with rat TRPA1 channels.

KEY RESULTS

AITC increased [Ca²⁺]_i in HEK293 cells, reduced ileal contractility (acetylcholine-, EFS-induced contractions and spontaneous contractility), but contracted the isolated colon. Gentamicin and camphor (non-selective TRPA1 channel antagonists), HC-030031 and AP18 (selective TRPA1 channel agonists) inhibited AITC-induced effects in HEK293 cells but not in the ileum or colon. AITC-induced contractions were reduced by tetrodotoxin and strongly reduced by nifedipine, cyclopiazonic acid and ryanodine. In vivo, AITC reduced (following i.p. administration) or increased (following intragastric administration) upper gastrointestinal transit in mice These effects were not affected by HC-030031.

CONCLUSION AND IMPLICATIONS

AITC, depending, in vitro, on the regions of gut examined and, in vivo, on the route of administration, exerted both stimulatory and inhibitory effects on intestinal motility, which were not sensitive to TRPA1 channel antagonists. The proposition that TRPA1 channels are the primary targets for AITC to induce contraction should be revised.     

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE

Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I_f or I_h). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels.

EXPERIMENTAL APPROACH

HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings.

KEY RESULTS

In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I_f by 67% at −120 mV, while MEL57A (3 µM) reduced I_f by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I_h by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization.

CONCLUSIONS

Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.     

A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery

Aim:

To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity.

Methods:

Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry.

Results:

The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine^TM 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors.

Conclusion:

L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.     

Serum uric acid levels are associated with polymorphisms in the SLC2A9, SF1, and GCKR genes in a Chinese population

Aim:

Genome-wide association studies have identified several novel loci associated with serum uric acid concentrations in individuals of European descent. In the current study, we aimed to evaluate the associations between these loci and serum uric acid concentrations in a Chinese population.

Methods:

Fourteen single nucleotide polymorphisms (SNPs) mapped in or near 11 loci (PDZK1, GCKR, LRP2, SLC2A9, ABCG2, LRRC16A, SLC17A1, SLC17A3, SLC22A11, SLC22A12 and SF1) were genotyped in 2329 Chinese subjects in Shanghai. Serum biochemical parameters including uric acid concentrations were determined. All the variants were analyzed for gender differences since uric acid metabolism differed between genders.

Results

In males after adjustments for age and BMI, GCKR rs780094, SLC2A9 rs11722228 and SF1 rs606458 were associated with the uric acid concentrations, which were statistically significant (P=0.016, 0.001 and 0.03, respectively), whereas SLC2A9 rs3775948 was marginally associated with the uric acid concentrations (P=0.071). In females, SLC22A12 rs506338 was also marginally associated with the uric acid concentrations (P=0.057). The meta-analysis for combined data from both males and females revealed that rs3775948 and rs606458 were associated with the uric acid concentrations (P=0.036 and 0.043, respectively). Furthermore, the gender significantly affected the association of rs11722228 with serum uric acid levels (P=0.012).

Conclusion

The SLC2A9 rs11722228, SF1 rs606458 and GCKR rs780094 variants modulate uric acid concentrations in Chinese males, while SF1 rs606458 and SLC2A9 rs3775948 are associated with the uric acid concentrations in both Chinese males and females.     

A quantitative model of amphetamine action on the 5-HT transporter

Background and Purpose

Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), noradrenaline (NET) and 5-HT (SERT); influx of amphetamine leads to efflux of substrates. Various models have been proposed to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not likely in alternative access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis.

Experimental Approach

Xenopus laevis oocytes and HEK293 cells expressing human (h) SERT were voltage-clamped and exposed to 5-HT, p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA).

Key Results

In contrast to the currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 or 100 μM pCA decayed 10 or 100 times faster, respectively, after pCA removal.

Conclusions and Implications

This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multistate version of the alternative access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.     

Statins and fenofibrate affect skeletal muscle chloride conductance in rats by differently impairing ClC-1 channel regulation and expression

Background and purpose:

Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca²⁺-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl.

Experimental approach:

In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells.

Key results:

Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin-and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells.

Conclusions and implications:

Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca²⁺-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1.     

L655,240, acting as a competitive BACE1 inhibitor,efficiently decreases β-amyloid peptide production in HEK293-APPswe cells

Aim:

To identify a small molecule L655,240 as a novel β-secretase (BACE1) inhibitor and to investigate its effects on β-amyloid (Aβ) generation in vitro.

Methods:

Fluorescence resonance energy transfer (FRET) was used to characterize the inhibitory effect of L655,240 on BACE1. Surface plasmon resonance (SPR) technology-based assay was performed to study the binding affinity of L655,240 for BACE1. The selectivity of L655,240 toward BACE1 over other aspartic proteases was determined with enzymatic assay. The effects of L655,240 on Aβ40, Aβ42, and sAPPβ production were studied in HEK293 cells stably expressing APP695 Swedish mutant^K595N/M596L (HEK293-APPswe cells). The activities of BACE1, γ-secretase and α-secretase were assayed, and both the mRNA and protein levels of APP and BACE1 were evaluated using real-time PCR (RT-PCR) and Western blot analysis.

Results:

L655,240 was determined to be a competitive, selective BACE1 inhibitor (IC₅₀=4.47±1.37 μmol/L), which bound to BACE1 directly (K_D=17.9±0.72 μmol/L). L655,240 effectively reduced Aβ40, Aβ42, and sAPPβ production by inhibiting BACE1 without affecting the activities of γ-secretase and α-secretase in HEK293-APPswe cells. L655,240 has no effect on APP and BACE1 mRNA or protein levels in HEK293-APPswe cells.

Conclusion:

The small molecule L655,240 is a novel BACE1 inhibitor that can effectively decreases Aβ production in vitro, thereby highlighting its therapeutic potential for the treatment of Alzheimer''s disease.     

Modulation of A2a receptor antagonist on D2 receptor internalization and ERK phosphorylation

Aim:

To explore the effects of heterodimerization of D₂ receptor/A_2a receptor (D₂R/A_2aR) on D₂R internalization and D₂R downstream signaling in primary cultured striatal neurons and HEK293 cells co-expressing A_2aR and D₂R in vitro.

Methods:

Primary cultured rat striatal neurons and HEK293 cells co-expressing A_2aR and D₂R were treated with A_2aR- or D₂R-specific agonists. D₂R internalization was detected using a biotinylation assay and confocal microscopy. ERK, Src kinase and β-arrestin were measured using Western blotting. The interaction between A_2aR and D₂R was detected using bioluminescence resonance energy transfer (BRET) and immunoprecipitation.

Results:

D₂R and A_2aR were co-localized and formed complexes in striatal neurons, while both the receptors formed heterodimers in the HEK293 cells. In striatal neurons and the HEK293 cells, the D₂R agonist quinpirole (1 μmol/L) marked increased Src phosphorylation and β-arrestin recruitment, thereby D₂R internalization. Co-treatment with the A_2aR antagonist ZM241385 (100 nmol/L) significantly attenuated these D₂R-mediated changes. Furthermore, both ZM241385 (100 nmol/L) and the specific Src kinase inhibitor PP2 (5 μmol/L) blocked D₂R-mediated ERK phosphorylation. Moreover, expression of the mutant β-arrestin (319-418) significantly attenuated D₂R-mediated ERK phosphorylation in HEK293 cells expressing both D₂R and A_2aR, but not in those expressing D₂R alone.

Conclusion:

A_2aR antagonist ZM241385 significantly attenuates D₂R internalization and D₂R-mediated ERK phosphorylation in striatal neurons, involving Src kinase and β-arrestin. Thus, A_2aR/D₂R heterodimerization plays important roles in D₂R downstream signaling.     

Heterologous regulation of agonist-independent μ-opioid receptor phosphorylation by protein kinase C

BACKGROUND AND PURPOSE

Homologous agonist-induced phosphorylation of the μ-opioid receptor (MOR) is initiated at the carboxyl-terminal S375, followed by phosphorylation of T370, T376 and T379. In HEK293 cells, this sequential and hierarchical multi-site phosphorylation is specifically mediated by G-protein coupled receptor kinases 2 and 3. In the present study, we provide evidence for a selective and dose-dependent phosphorylation of T370 after activation of PKC by phorbol esters.

EXPERIMENTAL APPROACH

We used a combination of phospho site-specific antibodies, kinase inhibitors and siRNA knockdown screening to identify kinases that mediate agonist-independent phosphorylation of the MOR in HEK293 cells. In addition, we show with phospho site-specific antibodies were also used to study constitutive phosphorylation at S363 of MORs in mouse brain in vivo.

KEY RESULTS

Activation of PKC by phorbol esters or heterologous activation of substance P receptors co-expressed with MORs in the same cell induced a selective and dose-dependent phosphorylation of T370 that specifically requires the PKCα isoform. Inhibition of PKC activity did not compromise homologous agonist-driven T370 phosphorylation. In addition, S363 was constitutively phosphorylated in both HEK293 cells and mouse brain in vivo. Constitutive S363 phosphorylation required ongoing PKC activity. When basal PKC activity was decreased, S363 was also a substrate for homologous agonist-stimulated phosphorylation.

CONCLUSIONS AND IMPLICATIONS

Our results have disclosed novel mechanisms of heterologous regulation of MOR phosphorylation by PKC. These findings represent a useful starting point for definitive experiments elucidating the exact contribution of PKC-driven MOR phosphorylation to diminished MOR responsiveness in morphine tolerance and pathological pain.     

SLC22A2 gene 808 G/T variant is related to plasma lactate concentration in Chinese type 2 diabetics treated with metformin

Aim:

To investigate the potential relationship between the SLC22A2 gene polymorphism and blood lactate concentration in Shanghai Hans suffering from type 2 diabetes mellitus (T2DM).

Methods:

The SLC22A2 single nucleotide polymorphism (SNP) 808G/T was genotyped in 400 T2DM patients, including a metformin-treated group (n=200) and a non-metformin-treated group (n=200). Fasting plasma lactic acid levels were measured with an enzyme-electrode assay. Biochemical indexes, including plasma alanine aminotransferase (ALT), creatinine (Cr), and glycolated hemoglobin (HbA1c), were also measured.

Results:

The fasting plasma lactate concentration in the metformin-treated group was significantly higher than that in the non-metformin-treated group (1.29±0.45 mmol/L vs 1.18±0.44 mmol/L, P=0.015). Additionally, the ratio of patients with hyperlactacidemia was 8% (16/200) for the metformin-treated group and 5.5% (11/200) for the non-metformin-treated group, with no lactic acidosis found in either group. The frequency of the SLC22A2 808G/T T allele was 12.9%. Patients with the mutant genotype (TT) had a higher blood lactate concentration in the metformin-treated group than those in the non-metformin-treated group (t=2.492, P=0.013). This trend was not observed in the GG and GT genotypes when compared with metformin-treated and non-metformin-treated groups. Patients with the mutant genotype (TT) in the metformin-treated group also had a higher incidence of hyperlactacidemia compared with the GG genotype (40.0% vs 6.9%, P=0.050) in the metformin-treated group and the GG (6.0%, P=0.042) or GT (4.3%, P=0.043) genotypes in the non-metformin-treated group. In the metformin-treated group, there were significant gender differences in lactate concentrations in the TT (2.18±0.15 vs 1.04±0.27 mmol/L, P=0.008) and GG genotypes (1.40±0.51 vs 1.19±0.35 mmol/L, P=0.004). The lactate levels of women with the TT genotype were the highest in the metformin-treated group, but differences in lactate levels among the genotypes were not observed in the non-metformin-treated group.

Conclusion:

There is an 808G/T polymorphism in the SLC22A2 gene in Chinese Hans with T2DM. The 808G>T variance in the SLC22A2 gene can affect the plasma lactate level and the incidence of hyperlactacidemia in T2DM patients undergoing metformin therapy. Additionally, the female patients carrying the TT genotype are prone to lactatemia.     

Highly lipophilic 3-epi-betulinic acid derivatives as potent and selective TGR5 agonists with improved cellular efficacy

Aim:

TGR5 is a G protein-coupled receptor that is expressed in intestinal L-cells and stimulates glucagon-like peptide 1 (GLP-1) secretion. TGR5 may represent a novel target for the treatment of metabolic disorder. Here, we sought to design and synthesize a series of TGR5 agonists derived from the natural product betulinic acid.

Methods:

A series of betulinic acid derivatives were designed and synthesized. A cAMP assay was established using a HEK293 cell line expressing human TGR5. Luciferase reporter assay was established using HEK293 cells transfected with plasmids encoding human FXR and luciferase reporter. A human intestinal L-cell line NCI-H716 was used to evaluate the effects of the betulinic acid derivatives on GLP-1 secretion in vitro.

Results:

Biological data revealed that the 3-α-OH triterpenoids consistently show increased potency for TGR5 compared to their 3-β-OH epimers. 3-OH esterification increased the lipophilicity and TGR5 activity of 3-α betulinic derivatives and enhanced the activity differences between 3-α and 3-β derivatives. The 3-α-acyloxy betulinic acids also exhibited a significant dose-dependent GLP-1 secretion effect.

Conclusion:

This study demonstrates that highly lipophilic 3-epi-betulinic acid derivatives can be potent and selective TGR5 agonists with improved cellular efficacy, and our research here provides a new strategy for the design and development of potent TGR5 agonists.     

Mechanisms of zolpidem-induced long QT syndrome: acute inhibition of recombinant hERG K+ channels and action potential prolongation in human cardiomyocytes derived from induced pluripotent stem cells

Background and Purpose

Zolpidem, a short-acting hypnotic drug prescribed to treat insomnia, has been clinically associated with acquired long QT syndrome (LQTS) and torsade de pointes (TdP) tachyarrhythmia. LQTS is primarily attributed to reduction of cardiac human ether-a-go-go-related gene (hERG)/I_Kr currents. We hypothesized that zolpidem prolongs the cardiac action potential through inhibition of hERG K⁺ channels.

Experimental Approach

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hERG currents from Xenopus oocytes and from HEK 293 cells. In addition, hERG protein trafficking was evaluated in HEK 293 cells by Western blot analysis, and action potential duration (APD) was assessed in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

Key Results

Zolpidem caused acute hERG channel blockade in oocytes (IC₅₀ = 61.5 μM) and in HEK 293 cells (IC₅₀ = 65.5 μM). Mutation of residues Y652 and F656 attenuated hERG inhibition, suggesting drug binding to a receptor site inside the channel pore. Channels were blocked in open and inactivated states in a voltage- and frequency-independent manner. Zolpidem accelerated hERG channel inactivation but did not affect I–V relationships of steady-state activation and inactivation. In contrast to the majority of hERG inhibitors, hERG cell surface trafficking was not impaired by zolpidem. Finally, acute zolpidem exposure resulted in APD prolongation in hiPSC-derived cardiomyocytes.

Conclusions and Implications

Zolpidem inhibits cardiac hERG K⁺ channels. Despite a relatively low affinity of zolpidem to hERG channels, APD prolongation may lead to acquired LQTS and TdP in cases of reduced repolarization reserve or zolpidem overdose.     

A high-throughput screening system for G-protein-coupled receptors using β-lactamase enzyme complementation technology

Aim:

To establish a system for monitoring the activation of G-protein-coupled receptors (GPCRs) using β-lactamase enzyme fragment complementation (EFC) technology.

Methods:

Two inactive β-lactamase deletion fragments, bla(a) and bla(b), were fused to β-arrestin and GPCR, respectively. A stable cell line named HEK/293-β2a2, which expressed two fusion proteins, GPCR/bla(b) and β-arrestin2/bla(a), was generated under antibiotic selection. A natural compound library of high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs was used for high-throughput screening (HTS) of β2-adrenoceptor (β2AR) agonists against the cell line HEK/293-β2a2. The interested hits were validated by the measurement of second-messenger cyclic adenosine monophosphate (cAMP) production.

Results:

The stable cell line HEK/293-β2a2 responded to β2AR agonist/antagonist in a dose-dependent manner. The EC₅₀ value obtained for isoproterenol was 15.5 nmol/L, and the IC₅₀ value obtained for propranolol was 51.9 nmol/L. Furthermore, HTS was performed to identify β2AR agonists from the natural compound library we established. The Z′ factor value was determined to be 0.68. Three hits were identified from primary screening and found to be as potent as isoproterenol in a cAMP assay.

Conclusion:

A cell-based high-throughput functional assay was established to directly monitor the activation of GPCRs based on the interaction between agonist-activated GPCR and β-arrestin using β-lactamase EFC technology, which can be used to search for leads in the natural compound library.     

Paeoniflorin ameliorates ischemic neuronal damage in vitro via adenosine A1 receptor-mediated transactivation of epidermal growth factor receptor

Aim:

Paeoniflorin from Chinese herb Paeoniae Radix has been shown to ameliorate middle cerebral artery occlusion-induced ischemia in rats. The aim of this study was to investigate the mechanisms underlying the neuroprotective action of PF in cultured rat cortical neurons.

Methods:

Primary cultured cortical neurons of rats were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) insult. Cell survival was determined using MTT assay. HEK293 cells stably transfected with A₁R (HEK293/A₁R) were used for detailed analysis. Phosphorylation of the signaling proteins was evaluated by Western blot or immunoprecipitation. Receptor interactions were identified using co-immunoprecipitation and immunofluorescence staining.

Results:

Paeoniflorin (10 nmol/L to 1 μmol/L) increased the survival of neurons subjected to OGD/R. Furthermore, paeoniflorin increased the phosphorylation of Akt and ERK1/2 in these neurons. These effects were blocked by PI3K inhibitor wortmannin or MEK inhibitor U0126. Paeoniflorin also increased the phosphorylation of Akt and ERK1/2 in HEK293/A₁R cells. Both A₁R antagonist DPCPX and EGFR inhibitor AG1478 not only blocked paeoniflorin-induced phosphorylation of ERK1/2 and Akt in HEK293/A₁R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. In addition, paeoniflorin increased the phosphorylation of Src kinase and activation of MMP-2 in HEK293/A₁R cells. Both Src inhibitor PP2 and MMP-2/MMP-9 inhibitor BiPs not only blocked paeoniflorin-induced phosphorylation of ERK1/2 (and Akt) in HEK293/A₁R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R.

Conclusion:

Paeoniflorin promotes the survival of cultured cortical neurons by increasing Akt and ERK1/2 phosphorylation via A₁R-mediated transactivation of EGFR.     

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

Aim:

Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists.

Methods:

HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z′-factor.

Results:

RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC₅₀ value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z′-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC₅₀=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells.

Conclusion:

A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.     

Role of mixed ion channel effects in the cardiovascular safety assessment of the novel anti-MRSA fluoroquinolone JNJ-Q2

BACKGROUND AND PURPOSE

JNJ-Q2, a novel broad-spectrum fluoroquinolone with anti-methicillin-resistant Staphylococcus aureus activity, was evaluated in a comprehensive set of non-clinical and clinical cardiovascular safety studies. The effect of JNJ-Q2 on different cardiovascular parameters was compared with that of moxifloxacin, sparfloxacin and ofloxacin. Through comparisons with these well-known fluoroquinolones, the importance of effects on compensatory ion channels to the cardiovascular safety of JNJ-Q2 was investigated.

EXPERIMENTAL APPROACH

JNJ-Q2 and comparator fluoroquinolones were evaluated in the following models/test systems: hERG-transfected HEK293 cells sodium channel-transfected CHO cells, guinea pig right atria, arterially perfused rabbit left ventricular wedge preparations and in vivo studies in anaesthetized guinea pigs, anaesthetized and conscious telemetered dogs, and a thorough QT study in humans.

KEY RESULTS

The trend for effects of JNJ-Q2 on Tp–Te, QT, QRS and PR intervals in the non-clinical models and the plateau in QTc with increasing plasma concentration in humans are consistent with offsetting sodium and calcium channel activities that were observed in the non-clinical studies. These mixed ion channel activities result in the less pronounced or comparable increase in QTc interval for JNJ-Q2 compared with moxifloxacin and sparfloxacin despite its greater in vitro inhibition of I_Kr.

CONCLUSIONS AND IMPLICATIONS

Based on the non-clinical and clinical cardiovascular safety assessment, JNJ-Q2 has a safe cardiovascular profile for administration in humans with comparable or reduced potential to prolong QT intervals, compared with moxifloxacin. The results demonstrate the importance of compensatory sodium and calcium channel activity in offsetting potassium channel activity for compounds with a fluoroquinolone core.