Synthesis and evaluation of a mechanism-based inhibitor of a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase

The first mechanism-based inhibitor of a 3-deoxy-D-arabino heptulosonate 7-phosphate (DAH7P) synthase has been synthesised in 12 steps from D-arabinose, and has been found to be a very slow binding inhibitor of Escherichia coli DAH7P synthase.     

Amelioration of mechanism-based inactivation of CYP3A4 by a H-PGDS inhibitor

This work describes the rational amelioration of mechanism-based inactivation (MBI) of Cytochrome P450 (CYP) 3A4 in a human hematopoietic prostaglandin D synthase (hH-PGDS) inhibitor (cpd 1). We utilized metabolism reports in order to check if patterns in the metabolism of 1 and similar compounds by CYP3A4 could be deciphered. Then we used structure based design, first modifying the CYP3A4 crystal structure (pdb code: 4NY4) by adding an oxyferryl moiety to the heme, followed by validating the modified structure to obtain the 1′ and 4 position oxidation products of midazolam and then recapitulating the metabolism patterns deciphered previously for 1 and analogs. We checked if the pattern deciphered could lead to a putative reactive moiety. Finally we used the docking pose of 1 into this model of the modified CYP3A4 crystal structure to guide transformation of 1 into MBI-free H-PGDS inhibitors.     

Human cystatin, a new protein inhibitor of cysteine proteinases

A new low-molecular weight protein inhibitor of cysteine proteinases, human cystatin, was isolated from sera of patients with autoimmune diseases. It inhibits papain, human cathepsin H and cathepsin B. According to its partially determined amino-acid sequence, human cystatin is highly homologous to egg white cystatin, but only distantly related to stefin, the cytosolic protein inhibitor of cysteine proteinases isolated from human polymorphonuclear granulocytes. Very probably human cystatin is identical with human gamma-trace, a microprotein of known sequence but hitherto unknown function.     

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

Reactions of glutamate 1-semialdehyde aminomutase with R- and S-enantiomers of a novel, mechanism-based inhibitor, 2,3-diaminopropyl sulfate

Glutamate semialdehyde aminomutase is a recognized target for selective herbicides and antibacterial agents because it provides the aminolevulinate from which tetrapyrroles are synthesized in plants and bacteria but not in animals. The reactions of the enzyme with R- and S-enantiomers of a novel compound, diaminopropyl sulfate, designed as a mechanism-based inhibitor of the enzyme are described. The S-enantiomer undergoes transamination without significantly inactivating the enzyme. The R-enantiomer inactivates the enzyme rapidly. Inactivation is accompanied by the formation of a 520 nm-absorbing chromophore and by the elimination of sulfate. The inactivation is attenuated by simultaneous transamination of the enzyme to its pyridoxamine phosphate form but inclusion of succinic semialdehyde to reverse the transamination leads to complete inactivation. The inactivation is attributed to further reactions arising from generation of an external aldimine between the pyridoxal phosphate cofactor and the 2,3-diaminopropene that results from enzyme-catalyzed beta-elimination of sulfate.     

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.     

Synthesis and the 5-HT6 receptor antagonistic effect of 3-arylsulfonylamino-5,6-dihydro-6-substituted pyrazolo[3,4]pyridinones for neuropathic pain treatment

A novel series of 3-arylsulfonylamino-5,6-dihydro-6-substituted-1H-pyrazolo[3,4-c]pyridine-7-ones was designed and synthesized as 5-HT₆ ligands. Among the derivatives synthesized, the lead compound, 12b, having piperidine functionality at the 6-position and (1-naphthyl)sulfonamino at the 3-position of the core structure showed the most potent 5-HT₆ inhibitory activity in vitro, good stability without CYP liability, and good neuropathic pain alleviation activity in a rat animal model.     

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.     

Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.     

On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.     

Cloning,purification and biochemical characterization of dipetarudin,a new chimeric thrombin inhibitor

The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M(r)=7560) and a thrombin inhibitory activity (K(i)=446 fM) comparable to r-hirudin.     

Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.     

SSR182289A, a selective and potent orally active thrombin inhibitor

SSR182289A 1 is the result of a rational optimisation process leading to an orally active thrombin inhibitor. The structure incorporates an original 2-(acetylamino)-[1,1'-biphenyl]-3-sulfonyl N-terminal motif, a central l-Arg surrogate carrying a weakly basic 3-amino-pyridine, and an unusual 4-difluoropiperidine at the C-terminus. Its synthesis is convergent and palladium catalysis has been employed for the construction of the key C-C bonds: Suzuki coupling for the bis-aryl fragment and Sonogashira reaction for the delta- bond of the central amino-acid chain. The compound is a potent inhibitor of thrombin's activities in vitro and demonstrates potent oral anti-thrombotic potencies in three rat models of thrombosis. The observed in vitro potency could be rationalized through the examination of the interactions within the SSR182289A 1 - thrombin crystal structure. SSR182289A 1, has been therefore selected for further development.     

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor

-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human -thrombin shows multiple surface exosites for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226 Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). _T ^–-Thrombin and thrombin Quick I (Arg 67 Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. -Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.Abbreviations used: AT, antithrombin; HC, heparin cofactor II; PCI, protein C inhibitor; serpin(s), serine proteinase inhibitor(s); FPRck, D-Phe-Pro-Arg-chloromethyl ketone; FPLck, D-Phe-Pro-Leu-chloromethyl ketone; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HNP, 20mM HEPES, 150mM NaCl, 0.1% (w/v) poly(ethyleneglycol) (M_r = 8000) buffer atpH 7.4; Unp-PLPT, unprotected pyridoxal 5phosphate modified-thrombin; HPPLPT, heparin-protected pyridoxal 5phosphate modifiedthrombin.     

Isolation and characterization of an active-site peptide from a sterol methyl transferase with a mechanism-based inhibitor.

Chemical affinity labeling of pure sterol methyl transferase (SMT) from Saccharomyces cerevisiae using the mechanism-based irreversible inhibitor, [3-3H]26,27-dehydrozymosterol, inhibited the SMT with an apparent Ki of 1.1 microM and k(inact) of 1.52 min(-1). The protein-inhibitor adduct was subjected to cleavage with trypsin and the resulting covalently modified peptide was analyzed by Edman sequencing from the N-terminus. The radiochemically labeled ca. 5.0 kDa peptide fragment of the cleavage mixture was shown to be contiguous through 17 residues to a segment that includes a highly conserved hydrophobic motif (Region I, stretching between T78 and F91) characteristic of SMT enzymes. The results confirm that Region I is the sterol binding/active site.     

Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus.

The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.     

Isolation, purification, and separation of the complex preparation of extracellular proteinases with fibrinolytic and anticoagulant properties from Aspergillus ochraceus 513]

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

Activation of the fibrinolytic system with dehydrochlormethyltestosterone

4-Chloro-1-dehydro-methyltestosterone (2 mg/kg/die) administered to rats and rabbits for three months increased the spontaneous fibrinolytic activity of blood, the release of plasminogen activator stimulated by venous stasis, and the activator activity in the kidney. Its effect in animal experiments corresponded to that of the steroid stanozolol tested for comparison.