Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

Design, synthesis, and biological activity of potent and selective inhibitors of blood coagulation factor Xa.

Factor Xa (FXa) has materialized as a key enzyme for the intervention of the blood coagulation cascade and for the development of new antithrombotic agents. FXa is the lone enzyme responsible for the production of thrombin and therefore is an attractive target for the control of thrombus formation. We have designed and synthesized a unique series of quinoxalinone FXa inhibitors. This series resulted in 3-[4-[5-((2S,6R)-2,6-dimethylpiperidin-1-yl)pentyl]-3-oxo-3,4-dihydroquinoxolin-2-yl]benzamidine (35) with 0.83 nM activity against FXa and excellent selectivity over similar serine proteases. An X-ray crystal structure of compound 35 bound to trypsin along with molecular modeling has led to a predicted binding conformation of compound 35 in FXa. Compound 35 has also been proven to be efficacious in vivo in both the rabbit veno-venous shunt and dog electrolytic injury models. In addition, it was shown that compound 35 did not significantly increase bleeding times in a rabbit model except at the highest doses and plasma concentrations were elevated in a dose dependent manner following a bolus dose and continuous intravenous infusion.

The discovery of fluoropyridine-based inhibitors of the factor VIIa/TF complex--Part 2.

The activated factor VII/tissue factor complex (FVIIa/TF) is known to play a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. A fluoropyridine-based series of FVIIa/TF inhibitors was discovered which utilized a diisopropylamino group for binding in the S2 and S3 binding pockets of the active site of the enzyme complex. In this series, an enhancement in binding affinity was observed by substitution at the 5-position of the hydroxybenzoic acid sidechain. An X-ray crystal structure indicates that amides at this position may increase inhibitor binding affinity through interactions with the S1'/S2' pocket.

The discovery of fluoropyridine-based inhibitors of the Factor VIIa/TF complex.

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.

In vitro and in vivo antithrombotic activity of PD-198961, a novel synthetic factor Xa inhibitor.

PD-198961, 3-(4-5-[(2R,6S)-2,6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl-3-oxo-3,4-dihydro-2-quinoxalinyl)-4-hydroxybenzenecarboximidamide, is a novel, synthetic factor Xa inhibitor with a Ki of 2.7 nM against human factor Xa. The aim of the present study was to evaluate the pharmacokinetic profile and antithrombotic efficacy of PD-198961 in rabbits. When tested in vitro, PD-198961 doubled prothrombin time (PT) and activated partial thromboplastin time (aPTT) at concentrations of 0.13 and 0.32 microM in human plasma, 0.2 and 0.09 microM in rabbit plasma, 0.3 and 0.4 microM in dog plasma, respectively. Intravenous administration of PD-198961 at 1 mg/kg over 30 minutes resulted in a maximal prolongation in PT and aPTT of 4.9 +/- 0.4 and 4.1 +/- 0.9-fold of baseline, respectively. The peak plasma concentration of PD-198961 was 977 +/- 96 ng/ml. The anticoagulant effect of PD-198961 was readily reversible; coagulation parameters and plasma concentration returned to near baseline 15 minutes after cessation of infusion. There was a good correlation between PT prolongation and plasma concentration of PD-198961 (r = 0.93). In an FeCl3-induced model of arterial thrombosis in rabbits, the antithrombotic effects of PD-198961 were compared with that of LB-30057, a direct thrombin inhibitor, and enoxaparin, a low molecular weight heparin (LMWH). PD-198961 dose dependently increased the time to occlusion (TTO), reduced thrombus weight (TW), and decreased the incidence of occlusion. When administered at 3.0 microg/kg/min IV, PD-198961 prolonged TTO from 28 +/- 5 minutes (control) to 120 +/- 0 minutes (P < 0.001) and reduced TW from 9.9 +/- 1.5 mg (control) to 2.8 +/- 0.9 mg (P < 0.01). PD-198961 also dose dependently inhibited ex vivo plasma FXa activity. At the highest dose tested, PD-198961 increased aPTT to 1.4 +/- 0.1-fold of baseline (compared with 1.5 +/- 0.1 and 2.8 +/- 0.3-fold of baseline for LB-30057 [CI-1028] and enoxaparin, respectively), and had modest effects on bleeding time (< or = 2-fold). These results indicate that PD-198961 is a potent FXa inhibitor and an effective antithrombotic agent at doses that produce only modest changes in normal hemostasis.