Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene A4 hydrolase

The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA₄-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability.     

Discovery of novel and potent aryl diamines as leukotriene A4 hydrolase inhibitors

The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA₄-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.     

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.     

Functional expression of single-chain antibody to leukotriene C4

Leukotriene C₄ (LTC₄) is synthesized by binding of glutathione to LTA₄, an epoxide derived from arachidonic acid, and further metabolized to LTD₄ and LTE₄. We previously prepared a monoclonal antibody with a high affinity and specificity to LTC₄. To explore the structure of the antigen-binding site of a monoclonal antibody against LTC₄ (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC₄ comparable to mAbLTC. The scFvLTC also bound to LTD₄ and LTE₄ with 48% and 17% reactivities, respectively, as compared with LTC₄ binding, whereas the antibody showed almost no affinity for LTB₄.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Role of leukotriene B4 in celecoxib-mediated anticancer effect

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has anticancer effect on many cancers associated with chronic inflammation by both COX-2-dependent and COX-2-independent mechanisms. The non-COX-2 targets of celecoxib, however, are still a matter of research. Leukotriene B₄ (LTB₄) has been implicated in prostate and colon carcinogenesis, but little is known about the potential role of LTB₄ in celecoxib-mediated anticancer effect. In this study, we evaluated whether LTB₄ was involved in celecoxib-mediated inhibitory effect on human colon cancer HT-29 cells and human prostate cancer PC-3 cells. Our data showed that survival of both cell lines was obviously suppressed after celecoxib treatment for 72 h in a concentration-dependent manner. However, only in HT-29 cells, this inhibitory effect could be reversed by LTB₄, which promoted survival of HT-29 cells rather than PC-3 cells. Consistent with these results, lioxygenase (LOX) potent inhibitor nordihydroguaiaretic acid (NDGA) had a higher inhibitory effect on HT-29 cells than PC-3 cells. Additionally, ELISA results showed that celecoxib could suppress expression of LTB₄ in both cell lines, whereas, inhibition of PGE₂ was only detected in HT-29 cells. These results indicate that the anticancer effect of celecoxib is COX-2-independent in HT-29 and PC-3 cells and in HT-29 cells primarily via down-regulating LTB₄ production.     

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.     

Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema

The leukotriene A(4) hydrolase enzyme is a dual functioning enzyme with the following two catalytic activities: an epoxide hydrolase function that transforms the lipid metabolite leukotriene A(4) to leukotriene B(4) and an aminopeptidase function that hydrolyzes short peptides. To date, all drug discovery efforts have focused on the epoxide hydrolase activity of the enzyme, because of extensive biological characterization of the pro-inflammatory properties of its metabolite, leukotriene B(4). Herein, we have designed a small molecule, 4-methoxydiphenylmethane, as a pharmacological agent that is bioavailable and augments the aminopeptidase activity of the leukotriene A(4) hydrolase enzyme. Pre-clinical evaluation of our drug showed protection against intranasal elastase-induced pulmonary emphysema in murine models.     

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.     

Dual anti-inflammatory and selective inhibition mechanism of leukotriene A4 hydrolase/aminopeptidase: insights from comparative molecular dynamics and binding free energy analyses

Human leukotriene A₄ hydrolase/aminopeptidase (LTA₄H) is a zinc metalloenzyme with a dual catalytic activity; conversion of LTA₄ into LTB₄ and degradation of chemotactic tripeptide Pro-Gly-Pro (PGP). Existing inhibitors, such as SC-57461A, block both catalytic activities of the enzyme, leading to drug failures. Recently, a novel compound, ARM1, was reported to selectively inhibit the hydrolase activity of LTA₄H while sparing its aminopeptidase activity. However, the molecular understanding of such preferential inhibitory mechanism remains obscure. The discovery of ARM1 prompted us to further explore its binding theme and provide more insight into the structural and dual mechanistic features of LTA₄H protein. To accomplish this, we embarked on wide range of computational tools, including comparative molecular dynamics (MDs) simulations and postdynamic analyses for LTA₄H and in complex with ARM1, PGP, ARM1-PGP, and SC-57461A. MD analysis reveals that the binding of ARM1 exhibits a more stable active site and overall stable protein conformation when compared to the nonselective inhibitor SC-57461A. In addition, MM/GBSA-binding free energy calculation also reveals that ARM1 exhibit a lower binding affinity, when compared to the nonselective inhibitor SC-57461A – which is in a great agreement with experimental data. Per residue energy decomposition analysis showed that Phe314, Val367, Tyr378, Trp311, Pro382, and Leu369 are key residues critical for the selective inhibition of the epoxide hydrolase activity of LTA₄H by ARM1. Findings from this report will not only provide more understanding into the structural, dynamic, and mechanistic features of LTA₄H but would also assist toward the rational design of novel and selective hydrolase inhibitors of LTA₄H as anti-inflammatory drugs.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Structures and mechanisms of enzymes in the leukotriene cascade

Leukotrienes are a family of proinflammatory lipid mediators of the innate immune response and are important signaling molecules in inflammatory and allergic conditions. The leukotrienes are formed from arachidonic acid, which is released from membranes by cPLA₂, and further converted by 5-lipoxygenase to form the labile epoxide leukotriene (LT) A₄. This intermediate is converted by either of the two enzymes, LTA₄ hydrolase or LTC₄ synthase, to form LTB₄ or LTC₄, respectively. In order for 5-lipoxygenase to work efficiently in cells, five-lipoxygenase-activating protein needs to be present. LTB₄ is one of the most powerful chemotactic agents whereas LTC₄ induces smooth muscle contractions, for example in the airways causing bronchoconstriction in asthmatic patients. The leukotrienes and the five enzymes/proteins involved in their formation have been subject to intense studies including drug design programs. Compounds blocking the formation or action of leukotrienes are potentially beneficial in treatment of several acute and chronic inflammatory diseases of the cardiovascular and respiratory systems. In order to succeed with drug development studies, knowledge of the molecular characteristics of the targets is indispensable. This chapter reviews the biochemistry, catalytic, and structural properties of the enzymes in the leukotriene cascade.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D₄ (LTD₄), an active component of SRS-A would produce acute and late airway responses in allergenic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to antigen, inhalation of leukotriene D₄ aerosol (delivered dose {mean ±SE} 0.55±0.08 ug) resulted in significant early and late increases in specific lung resistance (SR_L). In three allergic sheep which only demonstrated acute responses to antigen, LTD₄ aerosol (delivered dose 0.59±0.09ug) only produced an acute increase in SR_L. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SR_L. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD₄ in the airways of sensitive animals is important for the physiologic expression of the late response.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

Role of Janus kinase-2 in IgE receptor-mediated leukotriene C4 production by mast cells

We have previously shown that Janus kinase 3, a member of the family of non-receptor protein tyrosine kinases, plays a critical role in the regulation of FcεRI-mediated mast cell responses. In the current study, we investigated the role of another JAK family member, JAK2, in these responses. Our results show that the treatment of IgE-sensitized mouse mast cells with an inhibitor of JAK2 (AG490) blocked the release of leukotriene C₄ in a dose-dependent fashion after antigen challenge. However, prostaglandin PG D₂ production and degranulation were not affected under identical experimental conditions. Transfection of RBL-2H3 mast cells with JAK-2 specific small interfering RNA resulted in a 50% reduction of LTC₄ release in response to FcεRI crosslinking, but did not inhibit mast cell degranulation or calcium ionophore-induced LTC₄ release, indicating involvement of JAK2 in IgE receptor-mediated leukotriene release. Taken together, these data suggest that JAK2 is a critical regulator of IgE/antigen-induced production of LTC₄ in mast cells.     

Processing of glutathionylcobalamin by a bovine B12 trafficking chaperone bCblC involved in intracellular B12 metabolism

Glutathionylcobalamin (GSCbl) is a biologically relevant vitamin B₁₂ derivative and contains glutathione as the upper axial ligand thought formation of a cobalt-sulfur bond. GSCbl has been shown to be an effective precursor of enzyme cofactors, however processing of the cobalamin in intracellular B₁₂ metabolism has not been fully elucidated. In this study, we discovered that bCblC, a bovine B₁₂ trafficking chaperone, catalyzes elimination of the glutathione ligand from GSCbl by using the reduced form of glutathione (GSH). Deglutathionylation products are base-off cob(II)alamin and glutathione disulfide, which are generated stoichiometrically to GSH. Although cob(I)alamin was not detected due to its instability, deglutathionylation is likely analogous to dealkylation of alkylcobalamins, which uses the thiolate of GSH for nucleophilic displacement. The catalytic turnover number for the deglutathionylation of GSCbl is ?1.62 ± 0.13 min⁻¹, which is, at least, an order of magnitude higher than that for elimination of upper axial ligands from other cobalamins. Considering the prevalence of GSH at millimolar concentrations in cells, our results explain the previous finding that GSCbl is more effective than other cobalamins for synthesis of enzyme cofactors.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Inflammatory prompts produce isoform-specific changes in the expression of leukotriene B(4) omega-hydroxylases in rat liver and kidney

Cytochrome p450 (CYP) 4Fs metabolize leukotriene B(4) and other inflammatory mediators in the arachidonic acid cascade. Here we show that lipopolysaccharide (LPS) treatment suppresses CYP4F4 and up-regulates CYP4F5 mRNA expression in rat liver whereas renal CYP4Fs are essentially unchanged. BaSO(4) treatment, in contrast, increases both hepatic and renal CYP4F expression levels. Thus, distinct regulatory mechanisms in CYP4F expression might operate under different inflammatory prompts. To examine hepatic totipotency, primary hepatocytes were treated with varying doses of LPS resulting in decrease in all the CYP4F isoforms. Treatment of hepatocytes with 5 ng/ml of interleukin-1beta mimics the in vivo effects of LPS on CYP4F expression.