Novel Screen Methodologies for Identification of New Microbial Metabolites
97
4.1.2 Inhibition of Lipid Metabolism
Phospholipase A2 (PLA2) is a lipolytic enzyme which plays a major role in the
release of arachidonic acid from phospholipids in cell membranes. Further
metabolism of arachidonic acid leads to the formation of prostaglandins and
leukotrienes, which are potent mediators of inflammatory diseases.
Human PMNL PLA2 was assayed by incubation with [14C]-oleate labelled
Escherichia coli as substrate. After terminating the reaction, released [1-J4C]oleic acid was separated from the substrate on silicic acid columns and determined by radiometry. This assay was used in the characterisation of duramycins
B and C, complex lanthionine containing peptidic antibiotics isolated from
a Streptoverticillium strain and Streptomyces griseoluteus, respectively [94].
These compounds inhibited PLA2 from synovial fluid as well as that from
PMNLs.
Other reports of novel microbial PLA2 inhibitors include the potent inhibitor thielocin A 1 [~, a complex depside incorporating a xanthene moiety produced
by Thielavia terricola [95, 96]; the cinatrins, spiro-y-dilactones and 7-1actones
from Circinotrichum falcatisporum [97]; and folipostatin, a depsidone from
Aspergillus unguis [98]. The structure of cinatrin A is shown in Fig. 6.
The PLA2 assay used in the characterisation of the thielocins involved the
preparation of substrate by mixing 1-palmitoyl-2[1-14C] linoleoyl phosphatidylethanolamine with L-a-phosphatidylethanolamine, drying and resuspension. After the enzyme reaction, released free fatty acid was extracted and
measured radiometrically. Thielocin All3 strongly inhibited rat PLA2 II, but
weakly inhibited PLA2 I from rat pancreas. The compound was also active
against human PLA2 II from rheumatoid synovial fluid, and not human pancreas PLA2 I. Inhibition of rat PLA2 II was Ca z +-independent and independent
of substrate concentration. Thielocin Al[3 showed similar PLA2 inhibitory
activity on a variety of substrates and was shown to be a non-competitive,
reversible inhibitor.
Arachidonate 5-1ipoxygenase is another enzyme involved in arachidonic
acid metabolism. In this case the target enzyme was prepared from RBL-1 cells,
and activity measured by determining the amount of 5-hydroxyeicosatetraenoic
acid (5-HETE) produced from arachidonic acid. 5-HETE was measured by
HPLC. This assay was used in the discovery and characterisation of the
epocarbazolins, carbazole antibiotics bearing epoxide-containing side chains
from Streptomyces anulatus [99]. The structure of epocarbazolin A is shown in
Fig. 6. The activity of the epocarbazolins was stated probably to be due to their
free radical scavenging activity.
The nitrosoxacins, a family of N-nitroso-N-alkylhydroxylamines [100],
a series of 5-hydroxyanthranilic acid derivatives [101], and lagunamycin, an
unusual diazo-tetraoxoquinoline [102], were all isolated from various Streptomyces strains during the same screening programme for 5-1ipoxygenase inhibition. The activity of the nitrosoxacins was probably due to their chelating
activity.
97
4.1.2 Inhibition of Lipid Metabolism
Phospholipase A2 (PLA2) is a lipolytic enzyme which plays a major role in the
release of arachidonic acid from phospholipids in cell membranes. Further
metabolism of arachidonic acid leads to the formation of prostaglandins and
leukotrienes, which are potent mediators of inflammatory diseases.
Human PMNL PLA2 was assayed by incubation with [14C]-oleate labelled
Escherichia coli as substrate. After terminating the reaction, released [1-J4C]oleic acid was separated from the substrate on silicic acid columns and determined by radiometry. This assay was used in the characterisation of duramycins
B and C, complex lanthionine containing peptidic antibiotics isolated from
a Streptoverticillium strain and Streptomyces griseoluteus, respectively [94].
These compounds inhibited PLA2 from synovial fluid as well as that from
PMNLs.
Other reports of novel microbial PLA2 inhibitors include the potent inhibitor thielocin A 1 [~, a complex depside incorporating a xanthene moiety produced
by Thielavia terricola [95, 96]; the cinatrins, spiro-y-dilactones and 7-1actones
from Circinotrichum falcatisporum [97]; and folipostatin, a depsidone from
Aspergillus unguis [98]. The structure of cinatrin A is shown in Fig. 6.
The PLA2 assay used in the characterisation of the thielocins involved the
preparation of substrate by mixing 1-palmitoyl-2[1-14C] linoleoyl phosphatidylethanolamine with L-a-phosphatidylethanolamine, drying and resuspension. After the enzyme reaction, released free fatty acid was extracted and
measured radiometrically. Thielocin All3 strongly inhibited rat PLA2 II, but
weakly inhibited PLA2 I from rat pancreas. The compound was also active
against human PLA2 II from rheumatoid synovial fluid, and not human pancreas PLA2 I. Inhibition of rat PLA2 II was Ca z +-independent and independent
of substrate concentration. Thielocin Al[3 showed similar PLA2 inhibitory
activity on a variety of substrates and was shown to be a non-competitive,
reversible inhibitor.
Arachidonate 5-1ipoxygenase is another enzyme involved in arachidonic
acid metabolism. In this case the target enzyme was prepared from RBL-1 cells,
and activity measured by determining the amount of 5-hydroxyeicosatetraenoic
acid (5-HETE) produced from arachidonic acid. 5-HETE was measured by
HPLC. This assay was used in the discovery and characterisation of the
epocarbazolins, carbazole antibiotics bearing epoxide-containing side chains
from Streptomyces anulatus [99]. The structure of epocarbazolin A is shown in
Fig. 6. The activity of the epocarbazolins was stated probably to be due to their
free radical scavenging activity.
The nitrosoxacins, a family of N-nitroso-N-alkylhydroxylamines [100],
a series of 5-hydroxyanthranilic acid derivatives [101], and lagunamycin, an
unusual diazo-tetraoxoquinoline [102], were all isolated from various Streptomyces strains during the same screening programme for 5-1ipoxygenase inhibition. The activity of the nitrosoxacins was probably due to their chelating
activity.