Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

The rate of oxygen consumption and superoxide anion formation by stimulated human neutrophils. The effect of particle concentration and size.

The respiratory burst of neutrophils was measured as a function of the ratio of the opsonised beads to neutrophils. The rate of oxygen uptake was found to be linear up to a bead:neutrophil ratio of 25. The production of the superoxide anion, as measured by the rate of reduction of cytochrome c, was negligible until a certain 'critical' value of the bead:neutrophil ratio was reached. The rate of oxygen consumption and superoxide release above the critical value varies linearly with the bead:neutrophil ratio. Both the rate of oxygen consumption and of superoxide release vary with the square of the radius of the ingested particle. It is suggested that this depends on the surface area of neutrophil membrane, activated by contact with the antagonist.

An opsonised microelectrode. Observation of the respiratory burst of a single human neutrophil.

A 10 micron diameter gold microvoltammetric electrode, opsonised with human IgG, was used to study the respiratory burst of a single human neutrophil. The electrode oxidised superoxide produced near its surface by the neutrophil back to dioxygen. It is suggested that the current so detected is proportional to the rate of superoxide production by the NADPH oxidase of a single cell. In all cases the response consisted of a relatively rapid rise in current after cell addition, followed by a 2-phase decay. It is further suggested that this complex decay results from the production of superoxide being rate-limited initially by the NADPH concentration and later by the coupled metabolism of the hexose monophosphate shunt.

An opsonised electrode. The direct electrochemical detection of superoxide generated by human neutrophils.

A pyrolytic graphite electrode was surface modified with human IgG and used as a stimulus to elicit a respiratory burst from human neutrophils. The oxidation current observed was shown to be due to re-oxidation of superoxide produced by the neutrophils. Both superoxide dismutase and N-ethylmaleimide were effective inhibitors of the oxidation current.

Napsins: new human aspartic proteinases. Distinction between two closely related genes.

cDNA sequences were elucidated for two closely related human genes which encode the precursors of two hitherto unknown aspartic proteinases. The (pro)napsin A gene is expressed predominantly in lung and kidney and its translation product is predicted to be a fully functional, glycosylated aspartic proteinase (precursor) containing an RGD motif and an additional 18 residues at its C-terminus. The (pro)napsin B gene is transcribed exclusively in cells related to the immune system but lacks an in-frame stop codon and contains a number of polymorphisms, one of which replaces a catalytically crucial Gly residue with an Arg. Consideration is given to whether (pro)napsin B may be a transcribed pseudogene or whether its putative protein product undergoes rapid intracellular degradation.

Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

Lipoprotein-associated PLA2 inhibition--a novel, non-lipid lowering strategy for atherosclerosis therapy.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a serine lipase that is associated with low density lipoprotein (LDL) in human plasma. Substrates include oxidised phosphatidylcholine (PC), which is hydrolysed by Lp-PLA2 to lyso-PC and oxidised fatty acids. Both products are bioactive and proinflammatory, and implicated in monocyte infiltration into the developing plaque, deposition of foam cells, and plaque progression and instability. Lp-PLA2 has recently been shown to be a risk factor for coronary events in previously asymptomatic, hypercholesterolaemic men. A series of azetidinones was designed as potent and selective inhibitors of this enzyme; SB-222657 inhibited release of the chemotactic cleavage products from oxidised LDL, and SB-244323 reduced atherosclerotic plaque development in a 3 month rabbit study. A series of pyrimidones has been designed from a screening hit, and nanomolar inhibitors identified. Oral efficacy in inhibiting plasma Lp-PLA2 in rabbits has been demonstrated with a variety of structural classes.

Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications.

Diphenyliodonium has been shown to be an irreversible, time-dependent inhibitor of NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4) with the Ki for diphenyliodonium chloride being 2.8 mM. Kinetic studies have indicated that diphenyliodonium interacts with the reduced enzyme and NADPH is essential for inactivation to take place. Cytochrome c acts as a competitive substrate. The use of radiolabeled diphenyliodonium has enabled two sites of covalent modification to be identified. Isolation of radiolabeled cofactor followed by mass spectrometry has shown that a phenyl group is added to FMN while the FMN is effectively trapped in the reduced state. Trypsin digestion of S-carboxymethylated P450 reductase after inhibition with radiolabeled inhibitor shows covalent modification of the protein. Purification of a single radiolabelled peptide followed by automated Edman degradation has enabled identification of the second site of covalent attachment as Trp 419.

Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains.

NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochrome P450 and catalyzes the one-electron reduction of many drugs and foreign compounds. This enzyme is a flavoprotein containing the cofactors FMN and FAD, which are essential for its function. We have expressed the putative FMN and FAD/NADPH binding domains of P450 reductase and show that these distinct peptides fold correctly to bind their respective cofactors. The FAD/NADPH domain catalyzed the one-electron reduction of a variety of substrates but did not efficiently reduce cytochrome c or cytochrome P450 (as judged by the oxidation of the CYP1A1 substrate 7-ethoxyresorufin). However, the domains could be combined to provide a functional enzyme active in the reduction of cytochrome c and in transferring electrons to cytochrome P450. Both the reconstitution of the domains and the direct binding of cytochrome c to the FMN domain were ionic-strength dependent. The FMN domain containing the hydrophobic membrane anchor sequence was a potent inhibitor of reconstituted monooxygenase activity. These data strongly support the hypothesis that FMN/FAD-containing proteins have evolved as a fusion of two ancestral genes and provide fundamental insights into how this and structurally related proteins, such as nitric oxide synthase and sulfite reductase, have evolved and function.

Identification, genomic organization, and mRNA expression of LACTB, encoding a serine beta-lactamase-like protein with an amino-terminal transmembrane domain.

Database searching with bacterial serine beta-lactamases identified mouse expressed sequence tags (ESTs) with significant similarity scores.The cloned mouse cDNA encodes a novel 551-amino-acid protein, LACTB, with a predicted amino-terminal transmembrane domain but no signal peptide. It contains an active site motif related to C-class beta-lactamases. Homologues were detected in sequence data from human, rat, cow, rabbit, pig, toad, zebrafish, and Caenorhabditis elegans, but not in Saccharomyces cerevisiae or Drosophila melanogaster. The genes were mapped to human chromosome 15q22.1 and mouse chromosome 9. Sequencing of a 14.7-kb fragment of mouse genomic DNA defined six exons. A virtual human cDNA and a 549-residue protein, predicted from unfinished genomic sequence, showed the same intron/exon structure. Northern blot analysis showed expression of the 2.3-kb mRNA predominantly in mouse liver and human skeletal muscle. This is the first reported vertebrate example of this microbial peptidase family.

Mechanism of inhibition of LDL phospholipase A2 by monocyclic-beta-lactams. Burst kinetics and the effect of stereochemistry.

Investigation of the inhibition of LDL-associated phospholipase A2 by monocyclic beta-lactams has shown that LDL phospholipase A2 is capable of hydrolyzing monocyclic-beta-lactams by a mechanism which shares many similarities to the hydrolysis of beta-lactams by beta-lactamases. We believe that this is the first demonstration of a serine-dependent lipase being able to hydrolyze an amide bond. Although 4-(phenylthio)-N-(4-phenyl-2-oxobutyl)azetidin-2-one, SB-216477, and its enantiomers are relatively modest covalent inactivators with kobs/[I] = 46 M-1 s-1 for the R enantiomer, analysis of the kinetics of inactivation and reactivation shows that these compounds act as slow-turnover substrates, presumably via an acylation-deacylation mechanism. The detection of a suprastoichiometric burst indicates that the pathway must be branched with the branching giving rise to the slow reactivation via a more stable covalent intermediate. Study of the two enantiomers of SB-216477 shows that LDL-associated phospholipase A2 is sensitive to the beta-lactam stereochemistry at C4. However, a common achiral intermediate is formed along the turnover pathway, and this must be at or immediately prior to the branch point.

Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Cloning and characterization of a novel human dual flavin reductase.

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K(m) and k(cat) values of 21 microM and 1.3 s(-1), respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.

Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins.

A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.

Structure-based design of non-peptide, carbohydrazide-based cathepsin K inhibitors.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.

Inhibition of herpes proteases and antiviral activity of 2-substituted thieno[2,3-d]oxazinones.

Cinnamyl derivatives of thieno[2,3-d]oxazinones are mechanism-based inhibitors of the HSV-2, VZV and CMV herpes proteases which demonstrate nanomolar potency. Compounds 5 and 28 inhibit protease processing in HSV-2 infected cells with a selectivity index of at least 30.

Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor.

Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.

Solid-phase synthesis of a combinatorial array of 1,3-bis(acylamino)-2-butanones, inhibitors of the cysteine proteases cathepsins K and L.

To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3-amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2-butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified.

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2).

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.