Chemistry and structure--activity relationships of leukotriene receptor antagonists.

Several strategies have been employed by medicinal chemists in the design of potent and selective leukotriene receptor antagonists-leukotriene structural analogs, FPL 55712 analogs, and random screening of corporate compound banks. Lead compounds were optimized, often through the exchange of ideas with groups working on other chemical series of leukotriene antagonists. Pranlukast can likely be traced to a lead compound identified by random screening that was initially modified by incorporating structural components present in FPL 55712. Montelukast originated from an early quinoline lead, which was modified with leukotriene structural elements. Zafirlukast is based on a lead compound that incorporated structural components from both FPL 55712 and the leukotrienes. Therefore, each medicinal chemistry strategy that was originally employed has successfully identified clinically effective leukotriene receptor antagonists.

Characterization of macrophage proteins bearing the functional leukotriene D4-binding site of an anti-cysteinylleukotriene monoclonal antibody.

Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti-idiotypic IgG (AIAb) against an anti-LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1:1000 against F(ab')2 from the LTmAb (in an enzyme-linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non-immunized rabbit serum was not reactive. The specific reactivity of Fc-purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT-recognizing domain of the LTmAb (in enzyme-linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT-binding site of LTmAb (in radioimmunoassay). Using the antisera, Western-blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18 kDa. Dithiothreitol-reduced proteins were detected at 25 kDa and 18 kDa. In general, this suggested recognition of a 236-kDa oligomeric protein composed of subunits with molecular masses of 25 kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75-kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western-blot analysis, the membrane-enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane-associated proteins bearing the LTD4-binding site of LTmAb, which may include identification of the putative LT receptor.

Strategies in the design of peptidoleukotriene antagonists.

The research of the last two decades in the field of SRS-A and peptidoleukotriene (pLT) antagonists has provided information for the design of potent pLT antagonists, which share some or all of the following structural elements: (1) a lipophilic anchor, which fits into the lipophilic pocket of the LTD4 receptor; (2) a central lipophilic unit mimicking the tetraene system of LTD4; (3) one or two acidic groups, as mimics of the cysteinyl-glycine unit and/or the carboxylic group in the eicosanoid backbone of LTD4; (4) spacers connecting these elements. Potent pLT antagonists lacking a second polar binding group compensate by stronger interaction in other regions of the receptor. Identification of pLT antagonists is based on lead optimisation, preparation of pLT analogs and on the knowledge of the pLT receptor.

Glutathione S-transferase-dependent conjugation of leukotriene A4-methyl ester to leukotriene C4-methyl ester in mammalian skin.

The glutathione S-transferase (GST)-dependent conjugation of reduced glutathione (GSH) with leukotriene A4 (LTA4)-methyl ester in rodent and human skin was investigated. Incubation of [3H]LTA4-methyl ester (1 nmole, approximately 200,000 dpm) with cytosol prepared from rat, mouse and human skin or with affinity purified GST from rat skin cytosol in the presence of GSH resulted in the formation of LTC4-methyl ester. Maximum enzyme activity was observed in rat skin followed by mouse and human skin. With heat-denatured cytosol or in the absence of GSH, the product formation was negligible. GST purified from rat skin cytosol by GSH-agarose affinity chromatography exhibited a several-fold increase in the specific activity of enzyme with 1-chloro-2,4-dinitrobenzene (55-fold), ethacrynic acid (67-fold) and LTA4-methyl ester (12-fold) as substrates. Western blot analysis of the affinity purified GST indicated a predominant expression of the Pi class of GST isozyme followed by Mu and Alpha classes of isozymes. The formation of LTC4-methyl ester was established by its radioactivity profile on high pressure liquid chromatography and absorption spectroscopy. These results suggest that, in addition to xenobiotic metabolism, cutaneous GSTs may also be capable of metabolizing physiological substrates such as LTA4.

Eicosanoids in hypoxic insult to neonatal rabbit bowel.

Eicosanoids, derivatives of arachidonic acid, play a role in several inflammatory diseases of the bowel. To determine whether prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and leukotriene C4D4E4 (LTC4D4E4), have a role in hypoxic insult to the intestine, we examined the levels of these mediators in a hypoxic neonatal rabbit model. One group of animals underwent hypoxic insult postnatally, the second group did not undergo hypoxia and served as a control. The levels of PGE2, LTB4, and LTC4D4E4 were determined by radioimmunoassay. PGE2 in the hypoxic group was 1,779 +/- 142 pg/mg protein (mean +/- SD) as opposed to 2,380 +/- 197 pg/mg protein in the control group (p less than 0.02). LTB4 level was 5,446 +/- 3,492 pg/mg protein in the hypoxic rabbits and 3,362 +/- 2,570 pg/mg protein in the control group (p less than 0.03). There was no statistically significant difference in the level of LTC4D4E4 between the two groups. Our study shows that hypoxia shifts the arachidonic acid metabolism toward enhanced lipoxygenase activity with a resultant increase in LTB4 levels and a concomitant decrease in cyclooxygenase activity with reduced PGE2 levels in the bowel. The shift in the balance between these eicosanoids may play a role in the pathogenesis of ischemic-hypoxic bowel diseases by enhancing the inflammatory response in the intestine, and simultaneously, diminishing cytoprotection.

Syntheses of leukotriene-derivatives.

Some general synthetic routes for the synthesis of cysteinyl-leukotriene derivatives derived from stable building blocks are described. D6-LTE4, a metabolically stable isotopically labelled mass spectrometric internal standard, 20-hydroxy-LTE4, the unnatural 6-epi-LTE4; LTE3, a LT-derivative with 2-amino-thiophenol as a modified "amino-acid" and 14,15-dehydro-LTA4 were prepared. The compounds were tested in a LT-inhibition assay using a monoclonal antibody.

OVID and SUPER: two overlap programs for drug design.

Two programs, OVID and SUPER, for exploring the similarity of molecules with respect to their action at a receptor are described. OVID accepts two molecules as input and optimizes the three-dimensional overlap of specified atoms in one molecule with specified atoms in the second molecule. The result is expressed as a percent of the theoretical maximum. OVID gives a quantitative measure of the extent of a guessed correspondence between two molecules based on volume overlap of selected atoms. The Achilles' heel of OVID is that the correspondence between the two molecules has to be guessed. We realized that it would be better to systematically examine all possible correspondences of two structures to minimize the chance of overlooking a superior correspondence. We created SUPER to satisfy this need. SUPER accepts two molecules as input and finds the top twenty correspondences of their surfaces and charge distributions, giving a quantitative measure of the extent of each correspondence. An instructive example of the application of OVID and SUPER to the design of leukotriene D4 receptor antagonists is described. SUPER appears to be a practical brain-storming tool for the medicinal chemist trying to understand how molecules whose structures may not resemble one another in an obvious way can bind to the same site.

Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver.

Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation.

Direct airway injury results in elevated levels of sulfidopeptide leukotrienes, detectable in airway secretions.

Sulfidopeptide leukotrienes (LTC4/D4/E4) are suspected to be important lipid mediators in inflammatory responses in the lung. Previous investigations have provided evidence to support enhanced synthesis and secretion of these eicosanoids into bronchoalveolar lavage fluid in patients with Adult Respiratory Distress Syndrome (ARDS). We have prospectively examined the relationship between sulfidopeptide leukotriene levels in tracheal aspirates of 14 intubated and mechanically ventilated patients. When compared with the aspirate from one patient who required ventilation because of respiratory muscle weakness, the tracheal aspirates from eight ARDS patients had elevated leukotriene levels (range 2020-2052 pg/aspirate). However, the aspirates from four of the five patients with direct airway injury [inhalational burn (n = 3) and massive aspiration of gastric contents (n = 2)] contained significantly higher amounts of sulfidopeptide leukotrienes (range 10309-52244 pg/aspirate). Three of the five patients with direct airway injury did not develop ARDS. We conclude that simple aspiration of tracheal secretions can be used to monitor airway leukotriene biosynthesis in patients with lung injury and that elevated airway leukotriene levels may reflect airway epithelial damage, but may not predict the development of ARDS.

Capillary isotachophoretic determination of cysteinyl leukotrienes.

The cysteinyl leukotrienes (LTs) C4, D4 and E4 are among the most potent lipid mediators of anaphylaxis and inflammation. A capillary isotachophoretic method is described for the determination of these cysteinyl LTs. The method is based on anionic separation and detection using UV (254 nm) and conductivity detectors. The total analysis time is of the order of 30 min. The limit of detection of the method was determined to be 0.5 nmol of LTE4. Despite of similar chemical structures, all three cysteinyl LTs can be determined simultaneously.

Inhibition of leukotriene omega-oxidation by omega-trifluoro analogs of leukotrienes.

omega-Oxidation with subsequent beta-oxidation from the omega-end is the major pathway for inactivation and degradation of leukotrienes. Oxidative degradation of leukotriene E4 (LTE4), N-acetyl-LTE4, and LTB4 was inhibited by the omega-trifluoro analogs of LTE4, omega-trifluoro-LTE4 (omega-F3-LTE4), and (1S,2R)-5-(3-[1-hydroxy-15,15,15-trifluoro-2-(2-1H- tetrazol-5-ylethyl-thio)pentadeca-3(E),5(Z)-dienyl+ ++]phenyl)-1H-tetrazole (LY 245769). The latter substance inhibited the oxidative degradation of LTE4 and N-acetyl-LTE4 in the rat in vivo by 50% at a dose of 7 mumol/kg body weight. In rat hepatocyte cultures both omega-trifluoro analogs interfered with the omega-oxidation of N-acetyl-LTE4 and LTB4 with IC50 values of about 4 microM. Both analogs inhibited the omega-hydroxylation in isolated rat liver microsomes with IC50 values between 16 and 37 microM. This inhibition is apparently competitive. In addition, in liver cytosol, the conversion of the omega-hydroxylated leukotrienes to omega-carboxy-LTE4 and omega-carboxy-LTB4 was inhibited by both compounds. omega-Trifluoro analogs of leukotrienes provide a new tool for interfering with the inactivation of leukotrienes in the omega-oxidation pathway.

Preparation and tandem mass spectrometric analyses of deuterium-labeled cysteine-containing leukotrienes.

Leukotrienes (LT) C4, E4 and N-acetyl-E4, their respective monomethyl esters and 14,15-2H2 analogs have been synthesized. The collisionally activated decompositions of the [M + H]+ and [M - H]- ions formed by fast atom bombardment (FAB) have been studied by tandem mass spectrometry using a hybrid sector/quadrupole instrument. Structurally informative product ion spectra were obtained for each analyte; the fragmentation pathways proposed are consistent with the parallel data obtained for labeled and derivatized species. Fragmentation of [M + H]+ ions occurs prominently via cleavage of the thioether linkage with charge retention on the cysteine-containing (predominant for LTC4) or lipid-derived (predominant for LTE4) moieties. More pronounced differences were observed between the fragmentations of [M - H]- ions derived from LTC4 and LTE4; the preference for charge retention, however, parallels that observed for the fragmentation of [M + H]+ ions. Selected ion monitoring during continuous-flow FAB mass spectrometric analysis of authentic LTC4 indicated a low-picogram detection limit.