Indole inhibitors of human nonpancreatic secretory phospholipase A2. 2. Indole-3-acetamides with additional functionality.

As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 3. Indole-3-glyoxamides.

The preceding papers of this series detail the development of functionalized indole-3-acetamides as inhibitors of hnps-PLA2. We describe here the extension of the structure-activity relationship to include a series of indole-3-glyoxamide derivatives. Functionalized indole-3-glyoxamides with an acidic substituent appended to the 4- or 5-position of the indole ring were prepared and tested as inhibitors of hnps-PLA2. It was found that the indole-3-glyoxamides with a 4-oxyacetic acid substituent had optimal inhibitory activity. These inhibitors exhibited an improvement in potency over the best of the indole-3-acetamides, and LY315920 (6m) was selected for evaluation clinically as an hnps-PLA2 inhibitor.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 1. Indole-3-acetamides.

Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.

Porcine pancreatic phospholipase A2-induced contractions of guinea pig lung pleural strips.

The contractile nature of porcine pancreatic phospholipase A2 (PLA2) was characterized on paired pleural strips obtained from guinea pig lung. PLA2 (0.003-10 U/ml) produced concentration-related contractile responses which were sensitive to various drugs. The major component of the PLA2-induced contractions was derived from products of the cyclooxygenase pathway since a cyclooxygenase inhibitor or the combination of a thromboxane synthetase inhibitor and a thromboxane receptor antagonist produced a 54-65% reduction of the contractile responses. 5-Lipoxygenase products contributed to a smaller component of the PLA2-induced responses since 5-lipoxygenase inhibitors or the combination of a leukotriene (LT) B4 receptor antagonist and an LTD4/LTE4 receptor antagonist only suppressed the maximal responses 22-32%. PLA2-induced contractile responses were nearly abolished by altering both sides of the arachidonic acid cascade simultaneously. In contrast, a PAF receptor antagonist, a histamine (H1) receptor antagonist and an acetylcholine receptor antagonist, failed to significantly reduce PLA2-induced responses. These results demonstrate that exogenous administration of porcine pancreatic PLA2 produced concentration-dependent contractions of pleural strips mediated through the generation of eicosanoids.

Nitric oxide activates the glucose-dependent mobilization of arachidonic acid in a macrophage-like cell line (RAW 264.7) that is largely mediated by calcium-independent phospholipase A2.

Herein, we demonstrate that nitric oxide is a potent (> 20% release) and highly selective inducer of [3H]arachidonic acid mobilization in the macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one resulted in the inhibition of the large majority (86%) of nitric oxide-induced [3H]arachidonic acid release into the medium (IC50 < 0.5 microM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 exists as a catalytic complex comprised of ATP-modulated phosphofructokinase-like regulatory polypeptides and a catalytic subunit, we examined the role of glucose in facilitating nitric oxide-mediated arachidonic acid release. Nitric oxide-induced release of [3H]arachidonic acid possessed an obligatory requirement for glucose, was highly correlated with the concentration of glucose in the medium, and was dependent on the metabolism of glucose. Thus, [3H]arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. Collectively, these results identify a unifying metabolic paradigm in which the generation of lipid second messengers is coordinately linked to the signalstimulated acceleration of glycolytic flux, thereby facilitating integrated metabolic responses to cellular stimuli.

Characterization of the contractile effects of human recombinant nonpancreatic secretory phospholipase A2 (PLA2) and other PLA2s on guinea pig lung pleural strips.

Contractile activities of nPLA2, pPLA2 and hPLA2 were characterized on pleural strips of guinea pig lung. The rank order of potency for these PLA2s was nPLA2 > pPLA2 > hPLA2. The concentration-related contractions induced by nPLA2 (0.0002-0.67 micrograms/ml), pPLA2 (0.006-20 micrograms/ml) and hPLA2 (0.1-30 micrograms/ml) appear to be mediated primarily by the formation of cyclooxygenase products and to a lesser extent by 5-lipoxygenase products, as revealed by experiments using indomethacin and BW A4C. To further support a PLA2-related mechanism, the selectivity and inhibitory effects of two irreversible PLA2 inhibitors, parabromophenacyl bromide (pBPB) and manoalogue, were evaluated against the contractile responses induced by each PLA2. Various concentrations of manoalogue and pBPB were incubated with individual PLA2s for 24 hr before initiating experiments. Both agents suppressed each PLA2-induced contractile activity in a concentration-related manner. The inhibitory effects of pBPB were similar at the highest concentration, whereas manoalogue was more effective in blocking contractions induced by pPLA2 and hPLA2. Conversely, methylated manoalogue, an inactive analog, failed to reduce the PLA2-induced contractions. These results demonstrate that hPLA2 has the ability to catalytically induce the release of arachidonic acid and the formation of proinflammatory eicosanoids that contract the pleural strips. This tissue bath preparation may be a useful model for the evaluation of novel PLA2 inhibitors as potentially useful therapeutic agents.

Evidence that phospholipase A2 (PLA2) inhibitors can selectively block PLA2-mediated contractions of guinea pig lung pleural strips.

We have demonstrated previously that porcine pancreatic phospholipase A2 (PLA2)-induced contractions of guinea pig lung pleural strips can be abated by inhibitors of cyclooxygenase and 5-lipoxygenase pathways, suggesting the liberation of arachidonic acid. To validate further the involvement of a PLA2-related mechanism, the effects of three known inhibitors of PLA2 were evaluated. Manoalogue, an irreversible inhibitor of PLA2, parabromophenacyl bromide, an irreversible, active site directed inhibitor and a transition-state analog, a competitive inhibitor of PLA2 were used. Transition-state analog (3-30 microM), added to the tissues for 30 min before PLA2, shifted the PLA2 curves to the right in a concentration-related manner. In contrast, the reported inactive enantiomer of transition-state analog failed to alter the PLA2 curves. Manoalogue and para-bromophenacyl bromide, at concentrations up to 40- and 50-fold higher than the enzyme concentration, respectively, were incubated in Krebs' buffer with the enzyme for 24 hr before challenging the tissues. Under these conditions, the PLA2-induced contractions were suppressed markedly. In contrast, neither reduced nor methylated manoalogue, incubated at a 20-fold molar excess with PLA2 for 24 hr, suppressed the maximal PLA2-induced responses. These results demonstrate that inhibitors of secretory PLA2, acting by different mechanisms, can alter the contractile responses induced by PLA2 on pleural strips from guinea pig lung.