[Show abstract][Hide abstract] ABSTRACT: Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).
No preview · Article · Apr 1997 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Further development of N-carboxyalkyl dipeptide inhibitors of stromelysin-1 (MMP-3) led to the discovery of C-carboxyalkyl dipeptide analogs with improved oral bioavailability. An in vivo assay of human MMP-3 mediated degradation of a macromolecular substrate in an extravascular space is described and inhibition studies are reported.
[Show abstract][Hide abstract] ABSTRACT: The matrix metalloproteinase stromelysin-1 (MMP-3) is inhibited more strongly by peptidyl phosphinic acid 7 than by its corresponding phosphonamidate and phosphonate analogs. Extending a benzyl group at P′1 to a phenylethyl group in 8 further increases the potency (Ki = 1.4 nM). Enhanced potency with an extended substituent into the P3 region was observed.
No preview · Article · Feb 1996 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Aroyl and arylacyl aminoalkyl substitutents at the P1 position of N-carboxyalkyl dipeptides were found to enhance potency and selectivity for stromelysin-1 (MMP-3). In particular, the phthalimidobutyl and phenylpropanoylaminopropyl groups offered inhibitors of MMP-3 with Ki's of ∼ 10nM.
No preview · Article · Feb 1996 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: A series of N-carboxyalkyl dipeptides were synthesized to evaluate their inhibitory activities against human stromelysin-1(MMP-3), collagenase(MMP-1), and gelatinase-A(MMP-2). Structures with a homophenylalanine residue at P1′ substituted at the para position with small alkyl groups are potent inhibitors of (MMP-3) and (MMP-2) (Ki′ s 2–40 nM), but weak inhibitors of (MMP-1).
No preview · Article · Oct 1995 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: A series of N-carboxyalkyl peptides were prepared to test their inhibitory activity against human stromelysin (MMP-3), collagenase (MMP-1), and gelatinase A (MMP-2). Linear alkyl and ω-aminoalkyl residues were employed as replacements for a phenethyl group yielding inhibitors with in vitro activities comparable to their corresponding aromatic analogs.
No preview · Article · Mar 1995 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: A series of phosphinic acid-containing peptide inhibitors of human stromelysin-1 (MMP-3) were prepared. The P1 through P3 subsites were varied in a systematic manner on analogs possessing an invariant P1′-P3′ segment. The in vitro activity of these compounds as inhibitors of stromelysin and collagenase is discussed.
No preview · Article · May 1994 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.
No preview · Article · Jan 1994 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: To probe the specificity of the metalloendoproteinase stromelysin toward peptide substrates, we determined kc/Km values for the stromelysin-catalyzed hydrolyses of peptides whose design was based loosely on the structure of a known SLN substrate, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, hydrolysis at Gln-Phe, kc/Km = 1700 M-1 s-1). Several noteworthy points emerge from this study: (i) Catalytic efficiency is dependent on peptide chain length with N-terminal truncation of substance P resulting in more pronounced rate-constant reductions than C-terminal truncation. These results suggest the existence of an extended active site for stromelysin. (ii) Preferences at positions P3, P2, P1, P1', and P2' are for the hydrophobic amino acids Pro, Leu, Ala, Nva, and Trp, respectively. (iii) Investigation of specificity at P3' supports our earlier hypothesis that SLN has a requirement for a hydrogen-bond donor at this position in its substrates. Based on these observations, we designed and had synthesized the fluorogenic substrate N-(2,4-dinitrophenyl)Arg-Pro-Lys-Pro-Leu-Ala-Nva-TrpNH2, whose stromelysin-catalyzed hydrolysis can be monitored continuously (kc/Km = 45,000 M-1 s-1).
[Show abstract][Hide abstract] ABSTRACT: To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2,Arg-Pro-Ala-Pro-Gln-Gln- Phe-Phe - Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.
[Show abstract][Hide abstract] ABSTRACT: The cyclosporin A binding protein, cyclophilin (CyP), and the FK-506 binding protein, FKBP, catalyze the cis-to-trans isomerization of Xaa-Pro bonds in peptides. To probe the mechanism of these reactions and their nonenzymatic counterparts, we determined the following: (1) substrate specificities of CyP and FKBP; (2) dependencies of k(c)/K(m) on pH and solvent deuterium; (3) secondary deuterium isotope effects; and (4) temperature-dependencies. The results indicate that (1) for cis-to-trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA, values of k(c)/K(m) for the CyP-catalyzed reactions show little dependence on Xaa. In contrast, for FKBP, k(c)/K(m) displays a marked dependence on Xaa with a preference for hydrophobic residues. (2) For both enzymes, k(c)/K(m) is independent of both pH and isotopic composition of the solvent. (3) For the CyP-catalyzed cis-to-trans isomerization of Suc-Ala-Gly(L,L)-cis-Pro-Phe-pNA (L = H, D), H(k(c)/K(m))/D(k(c)/K(m)) = 1.13 +/- 0.01 and is independent of temperature between 2 and 30-degrees-C (2-degrees-C, (H)k/(D)k = 1.14 +/- 0.02; 10-degrees-C, (H)k/(D)k = 1.13 +/- 0.01; 30-degrees-C, (H)k/(D)k = 1.14 +/- 0.03). A secondary deuterium isotope effect of 1.13 suggests that, in the transition state of this reaction, the Xaa-Pro bond is twisted out of planarity. This value is inconsistent with mechanisms involving nucleophilic catalysis. (4) Eyring plots of In [(k(c)/K(m))/T] vs 1/T for reactions of FKBP are linear, while Eyring plots for CyP catalysis are curved and display a maximum around 30-degrees-C. The data for CyP were fit to a model involving a temperature-dependent, reversible isomerization of active to an inactive enzyme. This model is supported by the temperature-independence of the secondary deuterium isotope effect (see 3 above) and the independence of the equilibrium of the two enzyme forms on substrate structure. The activation parameters that were calculated from the Eyring plots indicated enthalpy-entropy compensation for both CyP and FKBP with the critical temperature, T(c), equal to 287 and 260 K, respectively. We interpret the compensation in terms of a simple mechanism in which stronger transition-state interactions between enzyme and substrate (more positive DELTA-H double dagger) are accompanied by greater restrictions of translational and rotational freedom (more negative DELTA-S double dagger).
No preview · Article · Apr 1992 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.
[Show abstract][Hide abstract] ABSTRACT: Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond.