JE-2147: a dipeptide protease inhibitor (PI) that potently inhibits multi-PI-resistant HIV-1.
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We designed, synthesized, and identified JE-2147, an allophenylnorstatine-containing dipeptide HIV protease inhibitor (PI), which is potent against a wide spectrum of HIV-1, HIV-2, simian immunodeficiency virus, and various clinical HIV-1 strains in vitro. Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9-11 different anti-HIV therapeutics after 32-83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all of the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains, with IC(50) values ranging from 13-41 nM (<2-fold changes in IC(50) compared with that of wild-type HIV-1). The emergence of JE-2147-resistant HIV-1 variants in vitro was substantially delayed compared with that of HIV-1 resistant to another allophenylnorstatine-containing compound, KNI-272, and other related PIs. Structural analysis revealed that the presence of a flexible P2' moiety is important for the potency of JE-2147 toward wild-type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. Further development of JE-2147 for treating patients harboring multi-PI-resistant HIV-1 is warranted. (+info)
Metalloproteinase-mediated release of the ectodomain of L1 adhesion molecule.
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The L1 adhesion molecule is an approx. 200-220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-alpha which is mediated by the membrane-bound metalloproteinase TNF-alpha converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein. (+info)
Purification, characterization, and heterologous expression in Fusarium venenatum of a novel serine carboxypeptidase from Aspergillus oryzae.
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A novel serine carboxypeptidase (EC 3.4.16.1) was found in an Aspergillus oryzae fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25 degrees C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60 degrees C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform a Fusarium venenatum host strain. The transformed strain of F. venenatum expressed an active recombinant carboxypeptidase. In F. venenatum, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from A. oryzae. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters. (+info)
Pharmacological characterisation of a new oral GH secretagogue, NN703.
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NN703 is a novel orally active GH secretagogue (GHS) derived from ipamorelin. NN703 stimulates GH release from rat pituitary cells in a dose-dependent manner with a potency and efficacy similar to that of GHRP-6. The effect is inhibited by known GHS antagonists, but not by a GH-releasing hormone antagonist. Binding of (35)S-MK677 to the human type 1A GHS receptor (GHS-R 1A) stably expressed on BHK cells was inhibited by GHRP-6 and MK677 as expected. NN703 was also able to inhibit the binding of (35)S-MK677. However, the observed K(i) value was lower than expected, as based on the observed potencies regarding GH release from rat pituitary cells. Similarly, the effect of NN703 on the GHS-R 1A-induced inositol phosphate turnover in these cells showed a lower potency, when compared with GHRP-6 and MK677, than that observed in rat pituitary cells. The effect of i.v. administration of NN703 on GH and cortisol release was studied in swine. The potency and efficacy of NN703 on GH release were determined to be 155+/-23 nmol/kg and 91+/-7 ng GH/ml plasma respectively. A 50% increase of cortisol, compared with basal levels, was observed for all the tested doses of NN703, but no dose-dependency was shown. The effect of NN703 on GH release after i. v. and oral dosing in beagle dogs was studied. NN703 dose-dependently increased the GH release after oral administration. At the highest dose (20 micromol/kg), a 35-fold increase in peak GH concentration was observed (49.5+/-17.8 ng/ml, mean+/-s.e.m.). After a single i.v. dose of 1 micromol/kg the peak GH plasma concentration was elevated to 38.5+/-19.6 ng/ml (mean+/-s.e.m.) approximately 30 min after dosing and returned to basal level after 360 min. The oral bioavailability was 30%. The plasma half-life of NN703 was 4.1+/-0.4 h. A long-term biological effect of NN703 was demonstrated in a rat study, where the body weight gain was measured during a 14-day once daily oral challenge with 100 micromol/kg. The body weight gain was significantly increased after 14 days as compared with a vehicle-treated group. In summary, we here describe an orally active and GH specific secretagogue, NN703. This compound acts through a similar mechanism as GHRP-6, but has a different receptor pharmacology. NN703 induced GH release in both swine and dogs after i.v. and/or p.o. administration, had a high degree of GH specificity in swine and significantly increased the body weight gain in rats. (+info)
Intrinsic beta-sheet propensities result from van der Waals interactions between side chains and the local backbone.
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The intrinsic secondary structure-forming propensities of the naturally occurring amino acids have been measured both experimentally in host-guest studies and statistically by examination of the protein structure databank. There has been significant progress in understanding the origins of intrinsic alpha-helical propensities, but a unifying theme for understanding intrinsic beta-sheet propensities has remained elusive. To this end, we modeled dipeptides by using a van der Waals energy function and derived Ramachandran plots for each of the amino acids. These data were used to determine the entropy and Helmholtz free energy of placing each amino acid in the beta-sheet region of phi-psi space. We quantitatively establish that the dominant cause of intrinsic beta-sheet propensity is the avoidance of steric clashes between an amino acid side chain and its local backbone. Standard implementations of coulombic and solvation effects are seen to be less important. (+info)
Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.
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Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall. (+info)
Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.
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We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin. (+info)
Liquid chromatography/mass spectrometry and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz.
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Efavirenz (Sustiva) is a potent and specific inhibitor of the HIV-1 reverse transcriptase and is approved for the treatment of HIV infection. The metabolism of efavirenz in different species has been described previously. Efavirenz is primarily metabolized in rats to the glucuronide conjugate of 8-OH efavirenz. Electrospray ionization-liquid chromatography/mass spectrometry analyses of bile samples from rats dosed with either efavirenz or with 8-OH efavirenz revealed three polar metabolites, M9, M12, and M13, with pseudomolecular ions [M-H](-) at m/z 733, 602, and 749, respectively. The characteristic mass spectral fragmentation patterns obtained for metabolites M9 and M13 suggested that these were glutathione-sulfate diconjugates, and the presence of a glutathione moiety in metabolite M9 was confirmed by liquid chromatograpy/nuclear magnetic resonance (NMR) analysis of bile extracts. Metabolite M12 was characterized by liquid chromatography/mass spectrometry as a glucuronide-sulfate diconjugate. Unambiguous structures of M9, M12, and M13 were obtained from one-dimensional proton and carbon NMR as well as proton-proton (correlated spectroscopy, two-dimensional shift correlation), proton-carbon heteronuclear multiple quantum correlation, and long-range proton-carbon (heteronuclear multiple bond correlation) correlated two-dimensional NMR analyses of metabolites isolated from rat bile. The mass spectral and NMR analyses of M10, which was isolated from rat urine, suggested a cysteinylglycine-sulfate diconjugate. The isolation of these polar metabolites for further characterization by NMR was aided by mass spectral analyses of HPLC fractions and solid phase extraction extracts during the isolation steps. The complete characterization of these novel diconjugates demonstrates that further phase II metabolism of polar conjugates such as sulfates could take place in vivo. (+info)