Application of proteomics to the study of cardiovascular biology.

Proteomics involves the integration of a number of technologies with the aim of analyzing the complete complement of proteins expressed by a biological system in response to various stimuli and/or under different physiological or pathophysiological conditions. Recent technical improvements to the methods employed for protein separation and protein identification have resulted in a dramatic increase in the number of proteomics-based research projects. More importantly, it has become readily apparent that examining changes in the proteome offers insight into understanding cellular and molecular mechanisms that cannot be obtained through genomic analysis. There are numerous examples of cardiovascular functions whose molecular pathways are mediated through post-translational processes such as phosphorylation. The use of proteomics offers the ability to simultaneously monitor the changes in protein expression and/or cell signaling pathways in response to such conditions as cardiac hypertrophy and heart failure. Together with complementary genomic data, proteomics-based research can greatly increase our understanding of cardiovascular biology.

Rational design, synthesis, and biological activity of benzoxazinones as novel factor Xa inhibitors.

Inappropriate thrombus formation within blood vessels is the leading cause of mortality in the industrialized world. Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade and represents an attractive target for anticoagulant drug development. From a high-throughput in vitro mass screen of our chemical library, we identified 4-[5-[(2R,6S)-2, 6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-2-phenyl-2H-1, 4-benzoxazin-3(4H)-one (1a) as an inhibitor of FXa with an IC(50) of 27 microM. Through a combination of SAR studies and molecular modeling, we synthesized 3-(4-[5-[(2R,6S)-2, 6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-3-oxo-3,4-dihydro-2H- 1,4-benzoxazin-2-yl)-1-benzenecarboximidamide (1n) which was a potent FXa inhibitor with an IC(50) of 3 nM. This compound exhibited high selectivity for FXa over other related serine proteases and was efficacious when dosed intravenously in rabbit and dog antithrombotic models.

Cardiac sarcoplasmic reticulum and sarcolemmal proteins separated by two-dimensional electrophoresis: surfactant effects on membrane solubilization.

We evaluated the use of the alkyaryl amidosulfobetaine zwitterionic detergent, designated as C8psi, to facilitate the solubilization of cardiac subcellular, membrane-associated proteins. Hearts from 7-week-old male Sprague-Dawley rats were isolated, and the left ventricles dissected and subsequently homogenized. The sarcolemma (SL) and the sarcoplasmic reticulum (SR) were isolated from different homogenate preparations originating from rat hearts by ultracentrifugation methods. The isolated membrane preparations were solubilized and the proteins precipitated. After resuspension, protein separation was achieved in first-dimensional IEF using an immobilized (pH 4-7) gradient and in the second dimension using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then stained, images analyzed, and protein spots excised for subsequent identification. Protein identification from both SR and SL samples did not identify any of the known major membrane-associated proteins. Solubilization of whole tissue lysates with C8psi resulted in no increase in the total number of proteins detected relative to samples solubilized in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulforate (CHAPS). The data suggest the utility of newer surfactants such as C8psi to improve both the resolution of (2-D) protein profiles and increase the number of proteins extracted from subcellular organelle fractions.

Structural basis of the thrombin selectivity of a ligand that contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl- piperidin-3-yl)-propanoic acid at P1.

We have studied the thrombin and trypsin complexed structures of a pair of peptidomimetic thrombin inhibitors, containing different P1 fragments. The first has arginine as its P1 fragment, and the second contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl-piperidin-3-yl)-propano ic acid (SAPA), a fragment known to enhance thrombin/trypsin selectivity of inhibitors. On the basis of an analysis of the nonbonded interactions present in the structures of the trypsin and thrombin complexes of the two inhibitors, the calculated accessible surfaces of the enzymes and inhibitors in the four complexes, data on known structures of trypsin complexes of inhibitors, and factor Xa inhibitory potency of these compounds, we conclude that the ability of this arginine mimic to increase thrombin selectivity of an inhibitor is mediated by its differential interaction with the residue at position 192 (chymotrypsinogen numbering). Thrombin has a glutamic acid at residue 192, and trypsin has a glutamine. The analysis also suggests that this constrained arginine mimic, when present in an inhibitor, might enhance selectivity against other trypsin-like enzymes that have a glutamine at residue position 192.

The design of potent and selective inhibitors of thrombin utilizing a piperazinedione template: part 1.

Utilizing X-ray crystallography and molecular modeling, highly potent and selective peptidomimetic thrombin inhibitors have been designed containing a rigid piperazinedione template. The synthesis and biological activity of these compounds will be described.

The design of potent and selective inhibitors of thrombin utilizing a piperazinedione template: part 2.

Potent and selective thrombin inhibitors have been prepared with a piperazinedione template and L-amino acids. Likewise, incorporation of D-amino acids led to potent inhibitors with a novel mode of binding. Herein, the structure activity relationships and structural aspects of these compounds will be described.

Potent and selective bicyclic lactam inhibitors of thrombin: Part 3: P1' modifications.

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors are described. We have explored the SAR around the P1' site. Modification of the P1' site has been found to affect potency and selectivity.

Effects of a factor Xa inhibitor, DX-9065a, in a novel rabbit model of venous thrombosis.

The objective of this study was to evaluate the effects of DX-9065a, a nonpeptide, direct inhibitor of factor Xa (FXa), in a novel experimental model of venous thrombosis. The experiments were conducted on anesthetized rabbits in which a veno-venous shunt with cotton threads was inserted into the vena cava. DX-9065a was administered intravenously to the rabbits as an initial bolus followed by a maintenance infusion using the following dosing schedules: DX-I: 0.25 mg/kg + 3 micrograms/kg/min.; DX-II: 0.75 mg/kg + 9 micrograms/kg/min.; DX-III: 1.5 mg/kg + 18 micrograms/kg/min.; DX-IV: 3.0 mg/kg + 36 micrograms/kg/min.; DX-V: 6.0 mg/kg + 72 micrograms/kg/min. DX-9065a induced a dose-dependent increase in the time to occlusion and a dose-dependent decrease in thrombus weight. Because of the unique character of the model, we were also able to show a dose-dependent increase in blood flow through the shunt. In addition, there were dose-dependent increases in prothrombin time (PT) and activated coagulation time (ACT) with more variable responses in the activated partial thromboplastin time (APTT). DX-9065a had little effect on thrombin time (TT) or bleeding time at all doses tested. In conclusion, dose-dependent antithrombotic efficacy was documented with DX-9065a in this new model of venous thrombosis. Although the in vivo potency of the compound was not striking, the results support the utility of FXa inhibition in venous thrombosis and demonstrate the utility of this experimental model for evaluating the efficacy of novel anticoagulants.

Cardiac protein phosphorylation: functional and pathophysiological correlates.

Protein phosphorylation acts a pivotal mechanism in regulating the contractile state of the heart by modulating particular levels of autonomic control on cardiac force/length relationships. Early studies of changes in cardiac protein phosphorylation focused on key components of the excitation-coupling process, namely phospholamban of the sarcoplasmic reticulum and myofibrillar troponin I. In more recent years the emphasis has shifted towards the identification of other phosphoproteins, and more importantly, the delineation of the mechanistic and signaling pathways regulating the various known phosphoproteins. In addition to cAMP- and Ca(2+)-calmodulin-dependent kinase processes, these have included regulation by protein kinase C and the ever-emerging family of growth factor-related kinases such as the tyrosine-, mitogen- and stress-activated protein kinases. Similarly, the role of protein dephosphorylation by protein phosphatases has been recognized as integral in modulating normal cardiac cellular function. Recent studies involving a variety of cardiovascular pathologies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins may underlie cardiac dysfunction in disease states. The emphasis of this comprehensive review will be on discussing the role of cardiac phosphoproteins in regulating myocardial function and pathophysiology based not only on in vitro data, but more importantly, from ex vivo experiments with corroborative physiological and biochemical evidence.

Potent and selective bicyclic lactam inhibitors of thrombin: Part 2: P1 modifications.

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors is described. We have explored the SAR with modifications to the P1 site. The introduction of arginine mimetics at the P1 site led to potent and selective thrombin inhibitors.

Exploring the binding preferences/specificity in the active site of human cathepsin E.

Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to other mechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generated to elucidate the specificity in the individual binding pockets with systematic substitutions in the P5-P2, and P2'-P3' based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, a second series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined for both sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, and mouse and human renin. Important specificity-determining interactions were found in the S3 (Glu-13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites.

Nonpeptidic potent HIV-1 protease inhibitors: (4-hydroxy-6-phenyl-2-oxo-2H- pyran-3-yl)thiomethanes that span P1-P2' subsites in a unique mode of active site binding.

Using molecular modeling and the information derived from the X-ray crystal structure of HIV-1 protease (HIV PR) complexed with the pyran-2-one 1, a series of (4-hydroxy-6-phenyl-2-oxo-2H-pyran-3-yl)thiomethanes was designed and analyzed as novel, nonpeptidic inhibitors of HIV PR. Structure-activity studies led to the discovery of inhibitor 19 having (RS)-1-(cyclopentylthio)-3-methylbutyl functionalization at the C-3 position, which exhibited a Kc of 33 nM. A X-ray crystallographic structure of 19 bound to HIV PR showed that structural water-301 (inhibitor-flap-bridging water) was displaced by the inhibitor. Interestingly, the enol moiety of the pyran-2-one formed a hydrogen bond directly with Asp125 and with Asp25 via a bridging water molecule, thus illustrating a unique mode of active site binding by an HIV PR inhibitor. The pendant cyclopentyl and isobutyl groups of 19 occupied the S1' and S2' binding sites, respectively, whereas the 6-phenyl group occupied a region in between the S1 and S3 pockets of HIV PR. Selected compounds were tested for antiviral activity on H9 cells infected with HIV-1IIIb. A correlation between enzymatic activity and antiviral activity was not found in this series. The best antiviral compound in this series, 18, contained (RS)-3-[cyclopentyl(cyclopentylthio)methyl] functionalization at the C-3 position of the pyran-2-one ring and exhibited a CIC50 of 14 microM and TC50 of 70 microM. These studies demonstrate that potent enzyme inhibition can be achieved by inhibitors that span only three subsites.

Effect of an orally active renin inhibitor Cl-992 on blood pressure in normotensive and hypertensive monkeys.

Cl-992, a novel potent inhibitor of primate renin, was tested for blood pressure-lowering efficacy in sodium-restricted, furosemide-treated conscious normotensive cynomolgus monkeys and conscious renal hypertensive monkeys. The hypertensive monkey model provided an opportunity to determine the response to a renin inhibitor in a pathological nonhuman primate model of hypertension without concurrent diuretic treatment or dietary sodium restriction and on repeated oral administration. Cl-992 has IC50 values of 0.58 +/- 0.06 (n = 4) and 0.36 +/- 0.03 nM (n = 8) against human and monkey renin, respectively. In normotensive monkeys, oral Cl-992 at doses of 3, 10 and 30 mg/kg reduced mean arterial blood pressure (MABP) by 8 +/- 2, 15 +/- 7 and 29 +/- 7 mm Hg (n = 5 animals per dose level, P < .05), respectively (base line, 103 +/- 3 mm Hg). Intravenous Cl-992 (0.0001 to 0.1 mg/kg) also caused dose-dependent decreases in MABP and a maximum reduction of 23 +/- 4 mm Hg. The decrease in MABP after Cl-992 was paralleled by an inhibition of plasma renin activity (PRA) and a reduction in immunoreactive angiotensin II. In renal hypertensive monkeys, oral Cl-992 at doses of 3, 10 and 30 mg/kg reduced MABP by 6 +/- 2, 18 +/- 6 and 37 +/- 8 mm Hg (n = 3 or 4, P < .05), respectively (base line, 134 +/- 4 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of endothelins as chemoattractants for human neutrophils.

The chemotactic response of human neutrophils to endothelins (ET) and ET-derived peptides was examined. ET-1, ET-2, and ET-3 elicited maximum responses at 10(-7), 3.3 x 10(-8) and 10(-7) M, respectively. Relative activities of the peptides at their optimal concentrations were: ET-1, ET-2 > ET-3. The chemotactic activity of ET-1 was localized to its Leu6-Met7-Asp8 segment. Conformation of the disulfide-linked Cys3-Cys11 loop appears to be critical for proper orientation of the chemotactic epitope. In comparison, ET-1 failed to stimulate the neutrophil respiratory burst, degranulation or arachidonic acid metabolism. These results demonstrate the selective chemoattractant activity of endothelins for human neutrophils.

Structure-activity relationships of C-terminal endothelin hexapeptide antagonists.

The discovery of selective endothelin (ET) receptor antagonists will facilitate identification of the physiological and pathological roles for ET and its isopeptides. Structure-activity studies of the C-terminal hexapeptide of ET have been carried out to elucidate those amino acids important for receptor binding and agonist or antagonist activity. Binding studies were performed in rat heart ventricle, rabbit renal artery vascular smooth muscle cells, and rat cerebellum. In addition, many of the compounds have been evaluated functionally for their effects on endothelin-1-induced arachidonic acid release and inositol phosphate accumulation in specific cell lines. Selected compounds have been evaluated in a functional bioassay in tissue preparations specifically expressing either ETA or ETB receptors. We have previously described the structure-activity relationships in the hydrophobic C-terminal hexapeptide of ET, a region known to be highly important for receptor recognition. A mono-D-amino acid scan of the ET[16-21] revealed that substitution at His gave rise to analogs with significantly enhanced binding affinity. We have further evaluated the C-terminal region and will describe the design, synthesis, and pharmacological evaluation of several novel and potent ET peptide receptor antagonists.

Specificity in the binding of inhibitors to the active site of human/primate aspartic proteinases: analysis of P2-P1-P1'-P2' variation.

To understand the differences in the binding specificities within the aspartic proteinase family of enzymes, we have carried out studies to determine the inhibition constants of a set of related compounds with various members of the human enzyme family. The inhibition constants (Ki values) were determined by competitive inhibition of the hydrolysis of chromogenic octapeptide substrates in the pH range of 3-5. For comparison, inhibition of monkey renin was studied by RIA at pH 6.0. All inhibitors were based on the general structure 4-(morpholinylsulfonyl)-L-Phe-P2-(cyclohexyl)Ala psi[isostere]-P1'-P2'. The isosteric replacements of the scissile peptide bond included difluorohydroxyethylene, 1,2-diols, 1,3-diols, and difluoroketones. Side chain substituents in P2 include hydrogen, allyl, ethylthio, (methoxycarbonyl)methyl, N-methylthiouridobutyl, imidazolylmethyl, and 4-amino-2-thiazolylmethyl. Our measurements have identified potent and selective inhibitors which are useful in evaluating the differences in the specificities among selected enzymes of this family.

Disparity between blood pressure and PRA inhibition after administration of a renin inhibitor to anesthetized dogs: methodological considerations.

A dissociation between changes in blood pressure (BP) and plasma renin activity (PRA) has been noted after administration of renin inhibitors. In the present study, the renin inhibitor PD 132002 was given to salt-deplete, anesthetized dogs. PRA was measured at pH 6.0 by a conventional angiotensin I (ANG I) RIA method (PRA-C) and by an ANG I antibody-trapping RIA method (PRA-AT) performed at pH 7.4. PD 132002 at 0.01, 0.1, 1, and 10 mg/kg IV, reduced BP by 3 +/- 2, 9 +/- 2, 24 +/- 4, and 39 +/- 4 mm Hg, respectively, (baseline of 136 +/- 8 mm Hg, N = 5), when infused IV over 30 minutes with a 30 minute recovery between doses. The BP response at 10 mg/kg equaled that of saralasin (20 micrograms/kg/min IV). PRA-AT (baseline of 20 +/- 6 ng ANG l/ml/hr, N = 4) was inhibited by 0%, 28% +/- 12%, 75% +/- 10%, and 97% +/- 1% at 0.01, 0.1, 1, and 10 mg/kg, respectively. Plasma concentrations of immunoreactive ANG II were also reduced dose-dependently and paralleled changes in BP. In contrast, PRA-C (baseline of 13 +/- 4 ng ANG l/ml/hr, N = 4) was inhibited by 82% +/- 8% at 0.01 mg/kg and by > 98% at higher doses. After a single dose of PD 132002 at 10 mg/kg infused over 30 minutes, BP recovery paralleled changes in immunoreactive ANG II and PRA-AT, yet PRA-C inhibition showed no recovery over the same time course. Our data support the conclusion that BP relates better to PRA-AT than PRA-C. Thus the dissociation sometimes observed in studies with renin inhibitors between changes in BP and PRA may be attributed to the assay used to determine PRA.

Renin inhibitors containing alpha-heteroatom amino acids as P2 residues.

A series of renin inhibitors having alpha-heteroatom amino acids as P2 substitutions has been prepared. Examples where the heteroatom is oxygen, sulfur, or nitrogen are described. Many of the compounds exhibit subnanomolar potency when tested in vitro against monkey renin. When selected compounds were tested orally in conscious, salt-depleted, normotensive, Cynomolgus monkeys, low to moderate blood pressure lowering was observed. At an oral dose of 30 mg/kg, compound 53a lowered blood pressure by a maximum of 18 mmHg at 2.5 h post-dose.