Novel Screen Methodologies for Identification of New Microbial Metabolites
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manner, and showed only weak activity against estrogen-receptor binding. Both
compounds were characterised as androgen-receptor antagonists by their inhibitory activities against the growth of SC-3 cells.
3.3 Cardiovascular Disease
Receptor-binding assays have also been used to screen for inhibitors of targets
associated with cardiovascular disease.
3.3.1 Inhibition of Atrial Natriuretic Factor (ANF) Binding
Atrial atriuetic factor is a cardiac hormone which is released into plasma
following atrial stretch. ANF binds with high affinity to membrane receptors
resulting in the upregulation of guanylate cyclase, and subsequent accumulation
of cyclic guanosine monophosphate (cGMP) in tissues and cells. This has many
effects, including direct potent vasodilation of the peripheral blood vessels, and
enhancement of various functions in the kidney. ANF is therefore actively
involved in blood pressure homeostasis.
A binding assay has been used to screen for microbial metabolites which
interfere with the binding of ANF to its receptors. This assay involved use of rat
[125I]-ANF and membranes from bovine adrenal cortex. The bound and free
radioligand were separated by filtration through glass fibre filters. Anantin,
a cyclic peptide produced by Streptomyces coerulescens, was discovered as
a result of this screening programme [68, 69]. Further characterisation of
anantin activity suggested it was a competitive inhibitor of ANF-binding.
Assays using bovine aorta smooth muscle showed that the ANF-induced increase in intracellular cGMP was significantly inhibited by anantin, whereas the
compound alone had no effect on intracellular cGMP levels. Anantin therefore
behaved as a functional ANF-antagonist.
3.3.2 Endothelin (ET) Binding Inhibitors
The endothelin receptor has been a productive target in terms of microbial
antagonists and binding inhibitors reported during recent years. Endothelin
(ET) is a 21 amino acid polypeptide with several isoforms (endothelin-1, -2 and
-3 : ET-1, ET-2, ET-3) which has potent, long-lasting vasoconstrictor activity in
vitro and in vivo. ET is produced from endothelial cells along with TxA2 and
angiotensin II, and is an important regulator of vascular tone. Two subtypes of
endothelin receptors, classified as the ETA and ETB receptors, have been cloned
and characterised in mammalian systems, and both have been used as the basis
of assays in screening programmes for microbial products.
91
manner, and showed only weak activity against estrogen-receptor binding. Both
compounds were characterised as androgen-receptor antagonists by their inhibitory activities against the growth of SC-3 cells.
3.3 Cardiovascular Disease
Receptor-binding assays have also been used to screen for inhibitors of targets
associated with cardiovascular disease.
3.3.1 Inhibition of Atrial Natriuretic Factor (ANF) Binding
Atrial atriuetic factor is a cardiac hormone which is released into plasma
following atrial stretch. ANF binds with high affinity to membrane receptors
resulting in the upregulation of guanylate cyclase, and subsequent accumulation
of cyclic guanosine monophosphate (cGMP) in tissues and cells. This has many
effects, including direct potent vasodilation of the peripheral blood vessels, and
enhancement of various functions in the kidney. ANF is therefore actively
involved in blood pressure homeostasis.
A binding assay has been used to screen for microbial metabolites which
interfere with the binding of ANF to its receptors. This assay involved use of rat
[125I]-ANF and membranes from bovine adrenal cortex. The bound and free
radioligand were separated by filtration through glass fibre filters. Anantin,
a cyclic peptide produced by Streptomyces coerulescens, was discovered as
a result of this screening programme [68, 69]. Further characterisation of
anantin activity suggested it was a competitive inhibitor of ANF-binding.
Assays using bovine aorta smooth muscle showed that the ANF-induced increase in intracellular cGMP was significantly inhibited by anantin, whereas the
compound alone had no effect on intracellular cGMP levels. Anantin therefore
behaved as a functional ANF-antagonist.
3.3.2 Endothelin (ET) Binding Inhibitors
The endothelin receptor has been a productive target in terms of microbial
antagonists and binding inhibitors reported during recent years. Endothelin
(ET) is a 21 amino acid polypeptide with several isoforms (endothelin-1, -2 and
-3 : ET-1, ET-2, ET-3) which has potent, long-lasting vasoconstrictor activity in
vitro and in vivo. ET is produced from endothelial cells along with TxA2 and
angiotensin II, and is an important regulator of vascular tone. Two subtypes of
endothelin receptors, classified as the ETA and ETB receptors, have been cloned
and characterised in mammalian systems, and both have been used as the basis
of assays in screening programmes for microbial products.