Synthesis and PKCθ inhibitory activity of a series of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles

A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCθ were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC₅₀ value of 18 nM for the inhibition of PKCθ.     

Agrin Regulation of ��3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction

Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of α3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the α3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the α3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na⁺ in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-α3 Na,K-ATPase interaction and suggest that agrin modulation of the α3 Na,K-ATPase is important in regulating heart function.Na,K-ATPases, or sodium pumps, are integral membrane enzymes found in all animal cells. Using energy from the hydrolysis of ATP they transport three Na⁺ ions out of the cell for every two K⁺ ions into the cell, resulting in a transmembrane chemical gradient that is reflected in the resting membrane potential and used to drive a variety of secondary transport processes. Each Na,K-ATPase is a heterodimer consisting of an α- and β-subunit. The α-subunit is the catalytic subunit and contains the binding sites for Na⁺ and K⁺. The β-subunit is required for pump function and targeting of the α-subunit to the plasma membrane. Four α- and three β-subunit genes have been identified. All combinations of α- and β-subunits form functional pumps, but developmental, cellular, and subcellular differences in expression suggest functional adaptation of the different isoforms (1).Na,K-ATPases play a central role in regulating the contractile activity of cardiac muscle (2). They are directly responsible for the Na⁺ gradient required for propagation of action potentials that initiate myocyte contraction. Moreover, because of the dependence of the Na⁺/Ca²⁺ exchanger (NCX)³ on the Na⁺ gradient as the source of counterions for transport of Ca²⁺ out of the cell, they play a critical role in Ca²⁺ homeostasis and excitation-contraction coupling. For example, inhibition of Na,K-ATPases by digoxin, ouabain, or other cardiac glycoside results in a decline of the Na⁺ gradient, reducing NCX activity and Ca²⁺ efflux. The inotropic effects of cardiac glycosides result from uptake of this “excess” cytoplasmic Ca²⁺ into the sarcoplasmic reticulum, raising the level of Ca²⁺ in intracellular stores, which, when released during excitation, enhances muscle contraction (3).In light of the importance of Na,K-ATPases for cardiac muscle function, it is not surprising that mechanisms have evolved to regulate their activity. Na,K-ATPases are susceptible to phosphorylation by either cAMP-dependent protein kinase or protein kinase C, and neurotransmitter- and peptide hormone-dependent activation of these cytoplasmic kinases have been shown to regulate pump activity (4). Other molecules exert their effects through direct interaction with the Na,K-ATPase. For example, phospholemman, a member of the FXYD family of membrane proteins expressed in heart, is tightly associated with the Na,K-ATPase and inhibits its function (5–7). Phosphorylation of phospholemman by either protein kinase C or cAMP-dependent protein kinase, however, relieves inhibition thereby restoring the activity of the pump (8, 9). Endogenous ouabain-like compounds have also been implicated in regulating Na,K-ATPase activity (10). Ouabain, or closely related molecules, is synthesized by the adrenal gland and hypothalamus, and increased circulating levels of these compounds observed in patients with congestive heart failure has been suggested as an adaptive response to improve heart function (11). Recent studies in the central nervous system have identified the protein agrin as a new endogenous ligand that regulates Na,K-ATPase function through interaction with its extracellular domains (12).Agrin was first identified as an extracellular matrix protein at the neuromuscular junction where, by signaling through a muscle-specific receptor tyrosine kinase called MuSK, it mediates the motor neuron-induced accumulation of acetylcholine receptors in the postsynaptic muscle fiber membrane (13). Agrin is also expressed in other tissues (14–16), but its function outside of the neuromuscular junction has been less well understood. Recently, however, we showed that agrin plays a role in regulating excitability of central nervous system neurons by binding to and inhibiting the activity of the α3 subunit-containing isoform of the Na,K-ATPase (12). Although both agrin (14, 16) and the α3 Na,K-ATPase (17) are expressed in heart, their potential interaction has not been explored. Here we show that the frequency of cardiac myocyte contraction is modulated by agrin regulation of α3 Na,K-ATPase activity.     

Regulation of Mitochondrial Iron Import through Differential Turnover of Mitoferrin 1 and Mitoferrin 2

Mitoferrin 1 and mitoferrin 2 are homologous members of the mitochondrial solute carrier family. Mitoferrin 1 is required for mitochondrial iron delivery in developing erythrocytes. Here we show that mitoferrin 1 and mitoferrin 2 contribute to mitochondrial iron delivery in a variety of cells. Reductions in mitoferrin 1 and/or mitoferrin 2 levels by RNA interference result in decreased mitochondrial iron accumulation, heme synthesis, and iron-sulfur cluster synthesis. The ectopic expression of mitoferrin 1 in nonerythroid cells silenced for mitoferrin 2 or the expression of mitoferrin 2 in cells silenced for mitoferrin 1 restored heme synthesis to “baseline” levels. The ectopic expression of mitoferrin 2, however, did not support hemoglobinization in erythroid cells deficient in mitoferrin 1. Mitoferrin 2 could not restore heme synthesis in developing erythroid cells because of an inability of the protein to accumulate in mitochondria. The half-life of mitoferrin 1 was increased in developing erythroid cells, while the half-life of mitoferrin 2 did not change. These results suggest that mitochondrial iron accumulation is tightly regulated and that controlling mitoferrin levels within the mitochondrial membrane provides a mechanism to regulate mitochondrial iron levels.Iron is a required element for all eukaryotes, but iron can be toxic at high concentrations. Consequently, the cellular acquisition of iron is highly regulated, as is the concentration of free iron in biological fluids. The regulation of iron concentration is extended to cellular organelles that either store or utilize iron. Mitochondria utilize iron for the synthesis of heme and iron-sulfur (Fe-S) clusters. These prosthetic groups are used within the mitochondria and are exported for use by cytosolic and nuclear proteins. The mechanisms that regulate mitochondrial iron levels are not known, although it is clear that mitochondrial iron levels must be regulated. For example, the loss of function mutations in genes that encode enzymes required for Fe-S cluster synthesis or the Atm1 transporter that exports Fe-S clusters, results in excessive mitochondrial iron accumulation in yeast and humans (for a review, see reference 11).The mechanisms that regulate mitochondrial iron pools are not well defined. Mitochondrial iron pools might be regulated at the level of import. Mitoferrin 1 (Mfrn1) has been shown to be required for mitochondrial iron import in developing erythroid cells. A mutation in zebrafish Mfrn1 (frascati) or the deletion of mouse Mfrn1 leads to defects in hemoglobinization due to a deficit in mitochondrial iron uptake (17). The phenotype of frascati zebrafish is restricted to developing red blood cells; other cell types showed no evidence of a mitochondrial iron phenotype. Mfrn1 has a paralogue, Mfrn2, and both genes have homologues MRS3 and MRS4 in Saccharomyces cerevisiae. Yeast with deletions of MRS3 and MRS4 grows poorly under low iron conditions due to impaired mitochondrial iron acquisition (5, 10, 13, 23). In yeast, the expression of Mfrn1 or Mfrn2 in Δmrs3 Δmrs4 cells can correct the poor growth under low iron conditions. The expression of either mouse or zebrafish Mfrn1 as a transgene in frascati zebrafish corrected the hemoglobin deficiency in cells, but the expression of Mfrn2 did not (17). These observations raise three questions. (i) What is the role of Mfrn2 in mitochondrial iron metabolism? (ii) Is iron transport into mitochondria regulated? (iii) If Mfrn2 transports iron into the mitochondria of vertebrate cells, why doesn''t Mfrn2 rescue the mitochondrial defect in Mfrn1-deficient zebrafish?Here, we show that Mfrn1 and Mfrn2 can transport iron into the mammalian mitochondria of nonerythroid cells. The ectopic expression of either Mfrn1 or Mfrn2 can restore mitochondrial iron transport in cells silenced for Mfrn2 and -1, respectively, but ectopic expression has little effect on increasing mitochondrial iron levels above the baseline values. Mitochondrial iron levels do not increase over the baseline because the levels of Mfrns are regulated posttranslationally. Mfrn1 accumulates in the mitochondria of developing red blood cells as a result of an increased protein half-life. In contrast, Mfrn2 does not accumulate in developing red blood cells or other cells, as the half-life of Mfrn2 protein remains constant.     

Long-term outcomes of patients receiving drug-eluting stents

Background

We sought to establish the long-term safety of drug-eluting stents compared with bare-metal stents in a usual care setting.

Methods

Using data from a prospective multicentre registry, we compared rates of death and of death or repeat revascularization during 3 years of follow-up of 6440 consecutive patients who underwent angioplasty with either drug-eluting or bare-metal stents between Apr. 1, 2003, and Mar. 31, 2006.

Results

Drug-eluting stents were inserted in 1120 patients and bare-metal stents in 5320. The drug-eluting stents were selected for patients who had a greater burden of comorbid illness, including diabetes mellitus (32.8% v. 20.8% in the bare-metal group, p < 0.001) and renal disease (7.4% v. 5.0%, p = 0.001). At 1-year follow-up, the drug-eluting stents were associated with a mortality of 3.0%, as compared with 3.7% with the bare-metal stents (adjusted odds ratio [OR] 0.62, 95% confidence interval [CI] 0.46–0.83). The rate of the composite outcome of death or repeat revascularization was 12.0% for the drug-eluting stents and 15.8% for the bare-metal stents (adjusted OR 0.40, 95% CI 0.33–0.49). In the subgroup of patients who had acute coronary syndromes, the adjusted OR for this composite outcome was 0.46 (95% CI 0.35–0.61). During the 3 years of observation, the relative risks for death and repeat revascularization varied over time. In year 1, there was an initial period of lower risk in the group with drug-eluting stents than in the group with bare-metal stents; this was followed by a shift toward outcome rates favouring bare-metal stents in years 2 and 3. The adjusted relative risk of the composite outcome of death or repeat revascularization associated with drug-eluting stents relative to bare-metal stents was 0.73 early in the first year of follow-up; it then rose gradually over time, to a peak of 2.24 at 3 years.

Interpretation

Drug-eluting stents are safe and effective in the first year following insertion. Thereafter, the possibility of longer term adverse events cannot be ruled out.Drug-eluting stents now comprise at least 85% of stents used in the United States and up to 40% or more of stents elsewhere. The overwhelming worldwide use of drug-eluting stents has, however, been tempered by the cost differential to bare-metal stents, the lack of data on long-term outcomes in large patient populations and, more recently, emerging concerns about safety because of reports of late thrombosis.^1–8The use of stents has been shown to reduce the rates of repeat revascularization and restenosis after angioplasty compared with angioplasty alone.⁹ Despite this, the long-term efficacy of stent use has been limited by the need for repeat revascularization owing to restenosis.¹⁰ Drug-eluting stents were developed to address this problem. Both clinical trials^11–20 and registry data^21–25 have shown reduced rates of restenosis with drug-eluting stents up to 4 years after implantation. This advantage appears to extend to patients with acute coronary syndromes: a recent 2-year follow-up study involving 7217 patients with acute coronary syndromes suggested that rates of death were lower among patients with drug-eluting stents than among those with bare-metal stents.²⁶The possibility of late thrombosis associated with drug-eluting stents is, however, a concern. Rates of late thrombosis have been reported to be 3.6–5.9 events per 1000 patients receiving drug-eluting stents.²⁷ This adverse event has been the subject of a review by the US Food and Drug Administration and has captured the attention of authoritative bodies around the world.Because of concerns about the long-term safety of drug-eluting stents, we compared the rates of death and of death or repeat revascularization over 3 years among patients who received either bare-metal or drug-eluting stents during angioplasty.     

C-5 Substituted heteroaryl 3-pyridinecarbonitriles as PKCθ inhibitors: Part I

We earlier reported that 3-pyridinecarbonitriiles with a 4-methylindolyl-5-amino group at C-4 and a phenyl group at C-5 were inhibitors of PKCθ. Keeping the group at C-4 of the pyridine core constant, we varied the water solubilizing group on the phenyl ring at C-5 and then replaced the C-5 phenyl ring with several monocyclic heteroaryl rings, including furan, thiophene and pyridine. Analog 6e with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-2-furyl group C-5 had an IC₅₀ value of 4.5 nM for the inhibition of PKCθ.     

Glutathione Peroxidase-1 Regulates Mitochondrial Function to Modulate Redox-dependent Cellular Responses

Glutathione peroxidase-1 (GPx-1) is a selenocysteine-containing enzyme that plays a major role in the reductive detoxification of peroxides in cells. In permanently transfected cells with approximate 2-fold overexpression of GPx-1, we found that intracellular accumulation of oxidants in response to exogenous hydrogen peroxide was diminished, as was epidermal growth factor receptor (EGFR)-mediated Akt activation in response to hydrogen peroxide or EGF stimulation. Knockdown of GPx-1 augmented EGFR-mediated Akt activation, whereas overexpression of catalase decreased Akt activation, suggesting that EGFR signaling is regulated by redox mechanisms. To determine whether mitochondrial oxidants played a role in these processes, cells were pretreated with a mitochondrial uncoupler prior to EGF stimulation. Inhibition of mitochondrial function attenuated EGF-mediated activation of Akt in control cells but had no additional effect in GPx-1-overexpressing cells, suggesting that GPx-1 overexpression decreased EGFR signaling by decreasing mitochondrial oxidants. Consistent with this finding, GPx-1 overexpression decreased global protein disulfide bond formation, which is dependent on mitochondrially produced oxidants. GPx-1 overexpression, in permanently transfected or adenovirus-treated cells, also caused overall mitochondrial dysfunction with a decrease in mitochondrial potential and a decrease in ATP production. GPx-1 overexpression also decreased EGF- and serum-mediated [³H]thymidine incorporation, indicating that alterations in GPx-1 can attenuate cell proliferation. Taken together, these data suggest that GPx-1 can modulate redox-dependent cellular responses by regulating mitochondrial function.Accumulation of reactive oxygen species (ROS),² such as superoxide anion and hydrogen peroxide, is thought to contribute to cellular damage, apoptosis, and cell death (1–3); however, ROS production is part of normal cellular metabolism, and evidence is accumulating that hydrogen peroxide, in particular, may function as a signaling molecule necessary for cell growth and survival (4–8). Superoxide is generated as a byproduct of mitochondrial respiration and by cellular redox enzymes, such as NADPH oxidase, that are stimulated through receptor-mediated mechanisms (9). Hydrogen peroxide is formed from the dismutation of superoxide, which occurs spontaneously or can be catalyzed by superoxide dismutase (10) or, alternatively, is produced by the two-electron enzymatic reduction of molecular oxygen by various oxidases, such as xanthine oxidase (11). Recent studies also suggest that hydrogen peroxide may be directly generated by receptor-ligand interactions (12). One mechanism by which hydrogen peroxide may modulate signal transduction is through the reversible oxidation of proteins at redox-active cysteines, including, for example, thiols in tyrosine kinase phosphatases. Oxidation and inactivation of phosphatases, such as PTEN, have been shown to promote the activity of the pro-growth and -survival kinase, Akt (13).Antioxidant enzymes, such as glutathione peroxidase, catalase, and peroxiredoxins, serve to eliminate hydrogen peroxide, thereby regulating cellular responses to this endogenous oxidant. GPx-1 is a selenoprotein and one of a family of peroxidases that reductively inactivate peroxides using glutathione as a source of reducing equivalents (14, 15). GPx-1, in particular, is a major intracellular antioxidant enzyme that is found in the cytoplasm and mitochondria of all cell types. In cell culture models as well as in genetic mouse models, GPx-1 overexpression is associated with enhanced protection against oxidative stress (16–19); however, GPx-1-overexpressing mice can become obese and insulin-resistant, and have attenuated insulin-mediated activation of Akt (20). Thus, to study how GPx-1 modulates the effects of cellular oxidants on cell signaling and cell growth, we analyzed cellular responses to hydrogen peroxide and EGF in permanently transfected cells overexpressing GPx-1.     

Targeted genome-wide investigation identifies novel SNPs associated with diabetic nephropathy

Loci contributing to complex disease have been identified by focusing on genome-wide scans utilising non-synonymous single nucleotide polymorphisms (nsSNPs). We employed Illumina's HNS12 BeadChip (13,917 high-value SNPs) which was specifically designed to capture nsSNPs and ideally complements more dense genome-wide association studies that fail to consider many of these putatively functional variants. The HNS12 panel also includes 870 tag SNPs covering the major histocompatibility region. All individuals genotyped in this study were Caucasians with (cases) and without (controls) diabetic nephropathy. About 449 individuals with type 2 diabetes (203 cases, 246 controls) were genotyped in the initial study. 1,467 individuals with type 1 diabetes (718 cases, 749 controls) were genotyped in the follow up study. 11,152 SNPs were successfully analysed and ranked for association with diabetic nephropathy based on significance (P) values. The top ranked 32 SNPs were subsequently genotyped using MassARRAY iPLEX(?) and TaqMan technologies to investigate association of these polymorphisms with nephropathy in individuals with type 1 diabetes. The top ranked nsSNP, rs1543547 (P?=?10(-5)), is located in RAET1L, a major histocompatibility class I-related gene at 6q25.1. Of particular interest, multiple nsSNPs within the top ranked (0.2%) SNPs are within several plausible candidate genes for nephropathy on 3q21.3 and 6p21.3.