Novel Screen Methodologies for Identification of New Microbial Metabolites
103
benzo[alnaphthacene skeleton and are produced by the same Streptomyces sp.
This same Streptomyces sp. also produced an unrelated fluorene hydroquinone
derivative, cysfluoretin, which also inhibits GST [132].
The complex hydroxamic acid-containing matlystatins from Actinomadura
atramentaria were discoverd as type IV collagenase inhibitors [133, 134]. Type
IV collagenase can break down collagen in basement membranes leading to
tumour invasion.
Levels of N-acetyl-13-D-glucosaminidase (NAGase), an enzyme responsible
for release of N-acetylglucosamine from glycoproteins and glycolipids, are
known to increase in conditions such as leukaemia and cancer. Nagstatin
(Fig. 7), a substrate-competitive inhibitor of NAGase which potentiates cellular
immune response in normal mice and reactivates the depressed immune response in tumour bearing mice, was discovered as a metabolite of Streptomyces
amakusaensis in a NAGase screening programme [135, 1361.
4.3 Cardiovascular Disease
4.3.1 Inhibition of Cholesterol Metabolism
Acyl CoA: cholesterol acyltransferase (ACAT) plays an important role in
atherogenesis and cholesterol adsorption from the intestines. ACAT inhibitors
may therefore be useful in the treatment of atherosclerosis and hypercholesterolemia. ACAT has been a fruitful enzyme target in terms of novel microbial
metabolites detected and identified during recent years.
ACAT activity can be assayed using cholesterol, rat liver microsomal protein
and [14C]-oleolyl-CoA. After incubation and termination of the reaction, total
lipids are separated using TLC, and activity of the cholesteryl oleate region of
the chromatogram determined radiometrically. Assays of this type have been
used in the discovery of the purpactins, glisoprenins, pyripyropenes, terpendoles,
AS-183, lateritin and gypsetin.
The purpactins are depsidones produced by Penicillum purpurogenum
[137, 138]. Purpactin A was demonstrated to inhibit cholesterol formation in
J774 macrophages. In this assay, the cells were dispensed in 24 well microtitre
plates and incubated with cholesterol rich liposomes and [3H]-oleate. After
incubation, cholesteryl-1-aH]-oleate formation was measured using TLC. The
compound was one hundred times more potent in this assay than in the in vitro
enzyme assay. The purpactins had no antimicrobial activity and purpactin
A was not toxic in vivo.
The glisoprenins, hydroxylated nonaprenol derivatives from a Gilocladium
sp., inhibited ACAT activity in the microsomal enzyme assay and in J774
macrophages, and were not toxic in vivo [139, 1401.
The pyripyropenes are produced by Aspergillusfumigatus and are the most
potent ACAT inhibitors of microbial origin [141-143]. They possess steroidlike skeletons consisting of pyridine, ~-pyrone and sesquiterpene moieties.
103
benzo[alnaphthacene skeleton and are produced by the same Streptomyces sp.
This same Streptomyces sp. also produced an unrelated fluorene hydroquinone
derivative, cysfluoretin, which also inhibits GST [132].
The complex hydroxamic acid-containing matlystatins from Actinomadura
atramentaria were discoverd as type IV collagenase inhibitors [133, 134]. Type
IV collagenase can break down collagen in basement membranes leading to
tumour invasion.
Levels of N-acetyl-13-D-glucosaminidase (NAGase), an enzyme responsible
for release of N-acetylglucosamine from glycoproteins and glycolipids, are
known to increase in conditions such as leukaemia and cancer. Nagstatin
(Fig. 7), a substrate-competitive inhibitor of NAGase which potentiates cellular
immune response in normal mice and reactivates the depressed immune response in tumour bearing mice, was discovered as a metabolite of Streptomyces
amakusaensis in a NAGase screening programme [135, 1361.
4.3 Cardiovascular Disease
4.3.1 Inhibition of Cholesterol Metabolism
Acyl CoA: cholesterol acyltransferase (ACAT) plays an important role in
atherogenesis and cholesterol adsorption from the intestines. ACAT inhibitors
may therefore be useful in the treatment of atherosclerosis and hypercholesterolemia. ACAT has been a fruitful enzyme target in terms of novel microbial
metabolites detected and identified during recent years.
ACAT activity can be assayed using cholesterol, rat liver microsomal protein
and [14C]-oleolyl-CoA. After incubation and termination of the reaction, total
lipids are separated using TLC, and activity of the cholesteryl oleate region of
the chromatogram determined radiometrically. Assays of this type have been
used in the discovery of the purpactins, glisoprenins, pyripyropenes, terpendoles,
AS-183, lateritin and gypsetin.
The purpactins are depsidones produced by Penicillum purpurogenum
[137, 138]. Purpactin A was demonstrated to inhibit cholesterol formation in
J774 macrophages. In this assay, the cells were dispensed in 24 well microtitre
plates and incubated with cholesterol rich liposomes and [3H]-oleate. After
incubation, cholesteryl-1-aH]-oleate formation was measured using TLC. The
compound was one hundred times more potent in this assay than in the in vitro
enzyme assay. The purpactins had no antimicrobial activity and purpactin
A was not toxic in vivo.
The glisoprenins, hydroxylated nonaprenol derivatives from a Gilocladium
sp., inhibited ACAT activity in the microsomal enzyme assay and in J774
macrophages, and were not toxic in vivo [139, 1401.
The pyripyropenes are produced by Aspergillusfumigatus and are the most
potent ACAT inhibitors of microbial origin [141-143]. They possess steroidlike skeletons consisting of pyridine, ~-pyrone and sesquiterpene moieties.