Influence of myeloperoxidase on colon tumor occurrence in inflamed versus non-inflamed colons of Apc(Min/+) mice.

Control of colorectal cancer needs to be tailored to its etiology. Tumor promotion mechanisms in colitis-associated colon cancer differ somewhat from the mechanisms involved in hereditary and sporadic colorectal cancer. Unlike sporadic or inherited tumors, some experimental models show that colitis-associated colon tumors do not require cyclooxygenase (COX) expression for progression, and non-steroidal anti-inflammatory drugs (NSAIDs) which prevent sporadic or inherited colon cancer do not prevent colitis-associated colon cancer. We report that myeloperoxidase (MPO), an ancestor of the COX isoenzymes, is a determinant of colitis-associated colon tumors in Apc(Min/+) mice. During experimentally induced colitis, inhibition of MPO by resorcinol dampened colon tumor development. Conversely, in the bowels of Apc(Min/+) mice without colitis, resorcinol administration or 'knockout' of MPO gene coincided with a slight, but discernible increase in colon tumor incidence. Acrolein, a by-product of MPO catalysis, formed a covalent adduct with the phosphatase tensin homolog (PTEN) tumor suppressor and enhanced the activity of the Akt kinase proto-oncogene in vitro and in vivo. Thus, MPO may be an important determinant of diet and inflammation on colon cancer risk via its effect on endogenous exposure to oxidants and acrolein. We propose a hypothetical model to explain an apparent dichotomy between colon tumor occurrence and MPO inhibition in inflamed versus non-inflamed colons.

Stream Sediment Sources in Midwest Agricultural Basins with Land Retirement along Channel.

Documenting the effects of agricultural land retirement on stream-sediment sources is critical to identifying management practices that improve water quality and aquatic habitat. Particularly difficult to quantify are the effects from conservation easements that commonly are discontinuous along channelized streams and ditches throughout the agricultural midwestern United States. Our hypotheses were that sediment from cropland, retired land, stream banks, and roads would be discernible using isotopic and elemental concentrations and that source contributions would vary with land retirement distribution along tributaries of West Fork Beaver Creek in Minnesota. Channel-bed and suspended sediment were sampled at nine locations and compared with local source samples by using linear discriminant analysis and a four-source mixing model that evaluated seven tracers: In, P, total C, Be, Tl, Th, and Ti. The proportion of sediment sources differed significantly between suspended and channel-bed sediment. Retired land contributed to channel-bed sediment but was not discernible as a source of suspended sediment, suggesting that retired-land material was not mobilized during high-flow conditions. Stream banks were a large contributor to suspended sediment; however, the percentage of stream-bank sediment in the channel bed was lower in basins with more continuous retired land along the riparian corridor. Cropland sediments had the highest P concentrations; basins with the highest cropland-sediment contributions also had the highest P concentrations. Along stream reaches with retired land, there was a lower proportion of cropland material in suspended sediment relative to sites that had almost no land retirement, indicating less movement of nutrients and sediment from cropland to the channel as a result of land retirement.

Redox signaling, alkylation (carbonylation) of conserved cysteines inactivates class I histone deacetylases 1, 2, and 3 and antagonizes their transcriptional repressor function.

Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that alpha,beta-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-PGJ(2) (15d-PGJ(2)), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys(261) and Cys(273) in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1.beta-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome.

Convergence of hormones, inflammation, and energy-related factors: a novel pathway of cancer etiology.

Colorectal cancer (CRC) is a multifactorial disease with several hypothesized etiologic factors including inflammatory processes; hormones such as estrogen, androgen, and insulin; and energy-related factors. We present evidence that integrates these elements in a pathway we call the convergence of hormones, inflammation, and energy-related factors (CHIEF). First, given the physiology of the gut, substantial epidemiologic and molecular data support the hypothesis that activation of innate immunity in the normal gut mucosa by various environmental agents (commensal bacteria, dietary antigens, mucosal irritants, pathogens) and endogenous factors such as estrogen, androgens, and insulin levels provokes basal inflammation as an underlying factor of the association of insulin, estrogen, and energy-related factors with CRC. Second, critical genes involved in this pathway, e.g., phosphatase tensin homologue on chromosome 10 (PTEN) and serine threonine kinase 11 (STK11)/LKB1, are tumor suppressor genes often mutated in intestinal cancer or CRC. Third, laboratory experiments show that cellular PTEN and STK11/LKB1 tumor suppressor enzymes are vulnerable to inactivation by redox-active species, especially chemically reactive lipid mediators of inflammation and redox stress. Epidemiologic data further support the underlying proposal that CHIEF comprises important elements of CRC risk. Although this discussion of the CHIEF pathway focuses on CRC, we believe that this pathway may play an important role in the etiology of other cancers as well.

Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor.

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes are overexpressed during inflammation and multistage tumor progression in many neoplastic disorders including lung, breast and pancreatic cancers. Here we report that the tumor suppressor phosphatase and tensin homolog (PTEN) is oxidized and inactivated during arachidonic acid (AA) metabolism in pancreatic cancer cell lines expressing COX-2 or 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased phosphatidylinositol 3,4,5-triphosphate production, activation of Akt and phosphorylation of downstream Akt targets including GSK-3beta and S6K. These effects are recapitulated with pancreatic phospholipase A(2), which hydrolyses the release of membrane-bound AA. Interference with PTEN's physiological antagonism of signals from growth factors, insulin and oncogenes may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids.

Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles.

Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF7 cells.

Reactive lipid species from cyclooxygenase-2 inactivate tumor suppressor LKB1/STK11: cyclopentenone prostaglandins and 4-hydroxy-2-nonenal covalently modify and inhibit the AMP-kinase kinase that modulates cellular energy homeostasis and protein translation.

LKB1, a unique serine/threonine kinase tumor suppressor, modulates anabolic and catabolic homeostasis, cell proliferation, and organ polarity. Chemically reactive lipids, e.g. cyclopentenone prostaglandins, formed a covalent adduct with LKB1 in MCF-7 and RKO cells. Site-directed mutagenesis implicated Cys210 in the LKB1 activation loop as the residue modified. Notably, ERK, JNK, and AKT serine/threonine kinases with leucine or methionine, instead of cysteine, in their activation loop did not form a covalent lipid adduct. 4-Hydroxy-2-nonenal, 4-oxo-2-nonenal, and cyclopentenone prostaglandin A and J, which all contain alpha,beta-unsaturated carbonyls, inhibited the AMP-kinase kinase activity of cellular LKB1. In turn, this attenuated signals throughout the LKB1 --> AMP kinase pathway and disrupted its restraint of ribosomal S6 kinases. The electrophilic beta-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2alpha, and TxB2, did not inhibit LKB1 activity (p > 0.05). Ectopic expression of cyclooxygenase-2 and endogenous biosynthesis of eicosanoids also inhibited LKB1 activity in MCF-7 cells. Our results suggested a molecular mechanism whereby chronic inflammation or oxidative stress may confer risk for hypertrophic or neoplastic diseases. Moreover, chemical inactivation of LKB1 may interfere with its physiological antagonism of signals from growth factors, insulin, and oncogenes.

Conditional expression of 15-lipoxygenase-1 inhibits the selenoenzyme thioredoxin reductase: modulation of selenoproteins by lipoxygenase enzymes.

The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFkappaB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC(50) = 13 +/- 1.5 microm and 1 +/- 0.2 microm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G(1), impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on (1)inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.

Cyclooxygenase enzymes: regulation and function.

The cyclooxygenase isoenzymes, COX-1 and COX-2, catalyze the formation of prostaglandins, thromboxane, and levuloglandins. The prostaglandins are autocoid mediators that affect virtually all known physiological and pathological processes via their reversible interaction with G-protein coupled membrane receptors. The levuloglandins are a newer class of products that appear to act via irreversible, covalent attachment to numerous proteins. COX enzymes are clinically important because they are inhibited by aspirin and numerous other non-steroidal anti-inflammatory drugs. This inhibition of COX confers relief from inflammatory, pyretic, thrombotic, neurodegenerative and oncological maladies. About one hundred years have elapsed since Hoffman designed and synthesized acetylsalicylic (aspirin) as an agent intended to lessen the gastrointestinal irritation of salicylates while maintaining their efficacy. During the past forty years systematic advances in our understanding of the structure, regulation and function of COX isoenzymes have enabled the design and synthesis of COX-2 selective inhibitors as agents intended to lessen the gastrointestinal irritation of aspirin and non-selective NSAIDs. This review discusses: 1) how two separate catalytic processes in COX - peroxidase and prostaglandin synthase - act in an integrated fashion manner to generate prostaglandins; 2) why irreversible inactivation of COX is important constitutively and pharmacologically; 3) how cells have managed to use two closely related, almost identical enzymes in ways that discriminate their physiological versus pathological roles; 4) how investigators have used these advances to formulate and test medically important uses for old drugs (i.e. aspirin) and create new ones that still seek to achieve Hoffman's original goal.

The immunopharmacology of paclitaxel (Taxol), docetaxel (Taxotere), and related agents.

Paclitaxel (Taxol) and docetaxel (Taxotere) are among the most unique, and successful, chemotherapeutic agents used for the treatment of breast and ovarian cancer. Both agents have anti-mitotic properties derived from binding to tubulin and excessive stabilization of microtubules. Their anti-neoplastic effects derive from this mechanism. Distinct from their effects on microtubule stabilization, paclitaxel, docetaxel, and related taxanes display immunopharmacological traits. In this review, we discuss their induction of pro-inflammatory genes and proteins; the current hypotheses on the molecular mechanism for this induction, especially its relationship to the lipopolysaccharide (LPS) signaling pathway. We also discuss the structure-activity relationships (SAR) that govern gene induction, especially the striking differences between the SAR for murine and human cells in vitro. Lastly, we discuss the immunopharmacological traits of paclitaxel and docetaxel in terms of their relevance to human clinical pharmacology and toxicology and their activity in animal models of autoimmune disorders.

Electrophilic prostaglandins and lipid aldehydes repress redox-sensitive transcription factors p53 and hypoxia-inducible factor by impairing the selenoprotein thioredoxin reductase.

Tumor suppressor p53 exhibits an enigmatic phenotype in cells exposed to electrophilic, cyclopentenone prostaglandins of the A and J series. Namely, cells harboring a wild-type p53 gene accumulate p53 protein that is conformationally and functionally impaired. This occurs via an unknown molecular mechanism. We report that electrophilic cyclopentenone prostaglandins covalently modify and inhibit thioredoxin reductase, a selenoprotein that governs p53 and other redox-sensitive transcription factors. This mechanism accounts fully for the unusual p53 phenotype in cells exposed to electrophilic prostaglandins. Based on this mechanism we derived, tested, and affirmed several predictions regarding the kinetics of p53 inactivation; the protective effects of selenium; the structure-activity relationships for inhibition of thioredoxin reductase and impairment of p53 by electrophilic lipids; the susceptibility of hypoxia-inducible factor to inactivation by electrophilic lipids; and the equivalence of chemical inactivation of p53 to deletion of a p53 allele. Chemical precepts dictate that other electrophilic agents should also inhibit thioredoxin reductase and impair its governance of redox-sensitive proteins. Our results provide a novel framework to understand how endogenous and exogenous electrophiles might participate in carcinogenesis; how selenoproteins and selenium might confer protection against cancer; how certain tumors might acquire their paradoxical p53 phenotype; and how chronic inflammation might heighten the risk for cancer.

Pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p53-independent cell death.

The tumor suppressor p53 is mutated in more than 50% of all cancers. Importantly, most clinically useful antineoplastic agents are less potent and efficacious in the context of mutant p53. This situation has prompted a search for agents that cause tumor cell death via molecular mechanisms independent of p53. Our recent investigations with electrophilic prostaglandins enabled us to devise a pharmacophore and mechanism of action hypothesis relevant to this problem: a cross-conjugated alpha,beta-unsaturated dienone with two sterically accessible electrophilic beta-carbons is a molecular determinant that confers activity among this class of ubiquitin isopeptidases inhibitors, and that inhibitors of ubiquitin isopeptidases cause cell death in vitro independently of p53. Here, we report the use of the National Cancer Institute's Developmental Therapeutics Database to identify compounds to test this hypothesis. Shikoccin (a diterpene), dibenzylideneacetone, and curcumin fit the pharmacophore hypothesis, inhibit cellular isopeptidases, and cause cell death independently of p53 in isogenic pairs of RKO and HCT 116 cells with differential p53 status. The sesquiterpene achillin and 2,6-diphenyl-4H-thiopyran-4-one, which have cross-conjugated dienones with sterically hindered electrophilic beta-carbons, do not inhibit isopeptidases or cause significant cell death. Furthermore, we show that a catalytic-site proteasome inhibitor causes cell death independently of p53. Combined, these data verify the p53-independence of cell death caused by inhibitors of the proteasome pathway and support the proposition that the ubiquitin-dependent proteasome pathway may contain molecular targets suitable for antineoplastic drug discovery.

Cyclooxygenase-2 induction by paclitaxel, docetaxel, and taxane analogues in human monocytes and murine macrophages: structure-activity relationships and their implications.

Paclitaxel and docetaxel can induce pro-inflammatory proteins, typified by cyclooxygenase-2 (prostaglandin H synthase). In some circumstances, this phenomenon may be relevant to the immunomodulatory actions and the adverse effects associated with paclitaxel or docetaxel. Accordingly, we compared a panel of sixteen taxanes, including paclitaxel and docetaxel, for their ability to induce cyclooxygenase-2 in a murine macrophage cell line (RAW 264.7) and in human peripheral blood monocytes. We discovered that the structure-activity relationships governing the induction of cyclooxygenase-2 protein differ markedly between the two species. Of 14 analogues evaluated, only 2 had activity comparable with paclitaxel in RAW 264.7 cells. In contrast, docetaxel and 12 of 14 analogues had activity comparable with paclitaxel in human monocytes. Our results enabled us to predict and subsequently affirm that the major human hepatic metabolite, 6alpha-hydroxypaclitaxel, would induce cyclooxygenase-2 in human cells but not in murine cells. Our structure-activity data and our experiments with combinations of taxanes suggest a provisional model for cyclooxygenase-2 induction. This model suggests that binding at a high-affinity site on tubulin and stabilization of microtubules is necessary, but not sufficient, for cyclooxygenase-2 induction. Binding at a second, lower-affinity receptor site is also necessary. However, our structure activity data are not fully compatible with two candidate proteins currently proposed as the low-affinity receptor.

Inflammation, carcinogenesis and cancer.

To fulfill their role in host-defense, granulocytes secrete chemically reactive oxidants, radicals, and electrophilic mediators. While this is an effective way to eradicate pathogenic microbes or parasites, it inevitably exposes epithelium and connective tissue to certain endogenous genotoxic agents. In ordinary circumstances, cells have adequate mechanisms to reduce the genotoxic burden imposed by these agents to a negligible level. However, inflammation persisting for a decade eventually elevates the risk of cancer sufficiently that it is discernible in case control epidemiological studies. Advances in our understanding of tumor suppressors and inflammatory mediators offer an opportunity to assess the molecular and cellular models used to guide laboratory investigations of this phenomenon. Disappointing results from recent clinical trials with anti-oxidant interventions raise questions about the risks from specific endogenous agents such as hydrogen peroxide and oxy radicals. Simultaneously, the results from the anti-oxidant trials draw attention to an alternate hypothesis, favoring epigenetic inactivation of key tumor suppressors, such as p53, and the consequent liability this places on genomic integrity.

Cyclopentenone prostaglandins of the J series inhibit the ubiquitin isopeptidase activity of the proteasome pathway.

Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated alpha,beta-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic alpha,beta-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by Delta12-prostaglandin J(2). This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents.

Inactivation of wild-type p53 tumor suppressor by electrophilic prostaglandins.

The electrophilic eicosanoids prostaglandins A(1) or A(2) impaired p53-dependent transcription of endogenous genes and exogenous p53-luciferase reporter plasmids in RKO and HCT 116 colon cancer cells. Cellular accumulation of genetically wild-type, but transcriptionally silent p53 varied as a function of exposure time and concentration of prostaglandins A(1) and A(2). Prostaglandins A(1) and A(2) induced a conformational change in wild-type p53 that corresponded with its inactivation and its aberrant redistribution from the cytosol to the nucleus. Derangement of its transcriptional activity manifested as inhibition of p53-mediated apoptosis by etoposide, a representative antineoplastic agent. We conclude that electrophilic eicosanoids impair the role of wild-type p53 as a guardian of genomic integrity by a process distinct from somatic mutation or viral oncoprotein binding. This process may pertain to malignant and premalignant conditions, such as colon carcinoma and adenoma, which often harbor a genetically wild-type, but inactive form of p53 tumor suppressor.

Cyclooxygenase-2 and carcinogenesis.

Numerous investigations have shown that COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC tumor suppressor is the initiating event. Moreover, it seems that the amount of COX-2 is important, since there is a correlation between its level of expression and the size of the tumors and their propensity to invade underlying tissue [40]. Inhibiting COX-2 at an early stage blocks the development of malignant tumors, causes pre-malignant tumors to regress and may improve the outcome once the cancer is completely established. This set of findings seems to link very strongly with the traditional observation that chronic inflammation is a precursor to a variety of types of cancer. By this formulation, inflammatory stimuli increase COX-2 and the downstream events that it induces promote tumor formation. All of these finding suggest that existing NSAIDs will be useful for the prophylaxis of colon cancer and polyps and we eagerly await clinical investigations that will generate guidelines that suggest those individuals that are the most appropriate recipients for such therapy. Although this field has progressed rapidly in the last few years, many important questions remain.

Modification of leukotriene A(4) hydrolase/aminopeptidase by sulfhydryl-blocking reagents: differential effects on dual enzyme activities by methyl-methane thiosulfonate.

The presence of a cysteine residue at or near the active site of leukotriene A(4) hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme with sulfhydryl-blocking reagents and by protection against inactivation afforded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methyl-methane thiosulfonate reacted with one thiol as judged by kinetic data and titration with 5, 5'-dithiobis-2-nitrobenzoate. Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80-85%. The inactivation rate was nonsaturable and strongly influenced by ion strength. The second-order rate constant increased from 0.9 to 4.3 M(-1) s(-1) in the presence of 0.2 M NaCl. Albumin, a stimulator of the aminopeptidase activity, increased apparent inactivation rates by shifting pK(a) for the modification from 8.2 to 7.8. The inactivated enzyme partially regained activity upon treatment with beta-mercaptoethanol. Peptide substrates and competitive inhibitors protected against inactivation. Bestatin, a competitive inhibitor, afforded complete protection with a K(D) = 0.15 microM, similar to K(i) = 0.17 microM for inhibition of peptidase activity. Treated enzyme had an unchanged K(m) but a reduced V(max). The epoxide hydrolase activity was only weakly affected by methyl-methane thiosulfonate with a maximal inactivation of 15-20% after prolonged treatment. Pretreatment of leukotriene A(4) hydrolase with the reagent did not protect against mechanism-based inactivation by its lipid substrate, leukotriene A(4). On the other hand, leukotriene B(4) was a competitive inhibitor of aminopeptidase activity and protected against modification by methyl-methane thiosulfonate. Our results suggest the presence of a cysteine at or close to subsite S'(1) of the active site of leukotriene A(4) hydrolase and that modification of this residue interferes with the function of the aminopeptidase activity, but not the epoxide hydrolase activity. This is the first report to distinguish the two catalytic activities of leukotriene A(4) hydrolase by chemical means.

Genomics and the discovery of new drug targets.

Molecular medicine and genomics technologies are inseparable for defining new molecular targets. cDNA databases and elementary informatic tools provide instantaneous glimpses of gene families or tissue-restricted expression patterns as a means of new target identification. In addition, cDNA microarrays and two-dimensional gel electrophoresis unmask the expression of genes with unassigned or unexpected functions. Depletion of mRNA with ribozymes or neutralization of proteins with intracellular antibodies enable investigators to reject or embrace new molecular hypotheses about the determinants of disease, pharmacology or toxicology.