Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation.

Macrophages secrete matrix metalloproteinase 9 (MMP-9), an enzyme that weakens the fibrous cap of atherosclerotic plaques, predisposing them to plaque rupture and subsequent ischemic events. Recent work indicates that statins strongly reduce the possibility of heart attack. Furthermore, these compounds appear to exert beneficial effects not only by lowering plasma low-density-lipoprotein cholesterol but also by directly affecting the artery wall. To evaluate whether statins influence the proinflammatory responses of monocytic cells, we studied their effects on the chemotactic migration and MMP-9 secretion of human monocytic cell line THP-1. Simvastatin dose dependently inhibited THP-1 cell migration mediated by monocyte chemoattractant protein 1, with a 50% inhibitory concentration of about 50 nM. It also inhibited bacterial lipopolysaccharide-stimulated secretion of MMP-9. The effects of simvastatin were completely reversed by mevalonate and its derivatives, farnesylpyrophosphate and geranylgeranyl pyrophosphate, but not by ubiquinone. Additional studies revealed similar but more profound inhibitory effects with L-839,867, a specific inhibitor of geranylgeranyl transferase. However, alpha-hydroxyfarnesyl phosphonic acid, an inhibitor of farnesyl transferase, had no effect. C3 exoenzyme, a specific inhibitor of the prenylated small signaling Rho proteins, mimicked the inhibitory effects of simvastatin and L-839,867. These data supported the role of geranylgeranylation in the migration and MMP-9 secretion of monocytes.

2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase.

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure-activity studies by solid-phase synthesis.

Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.

Characterization of the two-step pathway for inhibition of thrombin by alpha-ketoamide transition state analogs.

The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Inhibition of thrombin by peptides containing Lysyl-alpha-keto carbonyl derivatives.

Several H-N-Me-D-Phe-Pro-Lysyl-alpha-keto carbonyl derivatives were shown to be potent thrombin inhibitors (Ki 0.2 to 27 nM). The inhibitory potencies of these compounds toward tissue plasminogen activator, plasmin and factor Xa were minimal; however, substantial cross-reactivity versus trypsin was observed (Ki values from 0.5 to 1500 nM). Inhibition of thrombin by alpha-keto carbonyl compounds appeared to occur via a one-step reversible reaction. The alpha-keto carbonyl inhibitors bound thrombin with a second order rate constant (k1 1-4 microM-1s-1) that was 10-100-fold slower than that expected for a diffusion-controlled reaction. Certain alpha-keto carbonyl inhibitors were as potent (on a weight basis) as hirudin when evaluated in a rat arterial thrombosis model. The modest oral bioavailability (10-19%) in rats demonstrated for three of the alpha-keto carbonyl thrombin inhibitors suggests the possibility that alpha-keto amide containing thrombin inhibitors may have utility as orally-active antithrombotic agents.

Amide and alpha-keto carbonyl inhibitors of thrombin based on arginine and lysine: synthesis, stability and biological characterization.

We report structure-activity investigations in a series of tripeptide amide inhibitors of thrombin, and the development of a series of highly potent active site directed alpha-keto carbonyl inhibitors having the side chain of lysine at P1. Compounds of this class are unstable by virtue of reactivity at the electrophilic carbonyl and racemization at the adjacent carbon (CH). Modifications of prototype alpha-keto-ester 8a have afforded analogs retaining nanomolar Ki. Optimal potency and stability have been realized in alpha-keto-amides 11b (Ki = 2.8 nM) and 11c (Ki = 0.25 nM).

Synthesis and solution conformation of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor antagonist.

The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor-selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR-derived conformational constraints were combined with computer-assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II beta-turn and an inverse gamma-turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low-temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.