106
D.C. Hill et al.
A showed potent activity (50% inhibitory dose of 200 gg kg- 1 body weight) in
an acute in vivo model of hepatic cholesterol synthesis in mice [152].
4.3.2 Smooth Muscle Contraction Inhibitors
Myosin light chain kinase (MLCK) is a regulatory enzyme involved in smooth
muscle contraction. MLCK catalyses transfer of the y-phosphate of ATP to
ser-9 of the 20 kDa myosin light chain. Phosphorylated myosin can then
interact with myosin and generate contractile force. Since smooth muscle cells
are distributed in arteries and bronchi, it is postulated that MLCK inhibitors
would be potential vasodilators and bronchodilators. MLCK has been the
target of a recent microbial screening programme which resulted in the identification of MS-347a, MS-282a and MS-282b. Chicken gizzard smooth muscle
MLCK activity was monitored by assaying phosphorylation of a peptide
substrate in the presence of calmodulin. After incubation, the reaction was
terminated by addition of acid and product measured by HPLC analysis.
MS-374a, a novel xanthene epoxide, was isolated from an Aspergillius sp.
together with MS-347b, the known fungal metabolite syndowinin B [153].
MS-347a inhibited Ca 2 + and calmodulin-dependent and independent activity of
chicken gizzard MLCK in a dose-dependent manner. MS-347b did not inhibit
the enzyme. MS-347a also inhibited PKC, but did not inhibit cAMP-dependent
protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent
cyclic nucleotidase phosphodiesterase (PDE). MLCK inhibition was dependent
on the length of a pre-incubation period, suggesting irreversible inhibition.
MS-282a and MS-282b, macrocyclic dilactones from Streptomyces tauricus,
inhibited the calmodulin-dependent, but not the calmodulin-independant, activity
of MLCK, suggesting that these compounds block MLCK activity by antagonising calmodulin [ 154]. MS-282a also inhibited calmodulin-dependent PDE.
A screening programme targeted at calmodulin-dependent PDE inhibitors
led to the discovery of SCH 45752 [155]. Calmodulin-dependent bovine heart
PDE was assayed by hydrolysis of cyclic AMP, or cyclic GMP. SCH 4572 is
a symmetrical bis-naphthopyrone from a culture of an unidentified fungus. It is
identical to the known Verticillium sp. metabolite cephalochromin.
SCH 4572 was a potent inhibitor of bovine heart and bovine aorta PDE. The
compound was much less active against calmodulin independent PDE, MLCK,
cAMP-dependent and cGMP-dependent protein kinase, and protein kinase C.
SCH 4572 stimulated carotid artery smooth muscle contraction
4.3.3 Inhibition of Plasminogen Activator Inhibitor-I
A key event in the regulation of thrombus formation and clearance is the
generation of plasmin by tissue plasminogen activator (tPA). tPA activity is
controlled by PAI-1, a fast acting inhibitor belonging to the serine protease
D.C. Hill et al.
A showed potent activity (50% inhibitory dose of 200 gg kg- 1 body weight) in
an acute in vivo model of hepatic cholesterol synthesis in mice [152].
4.3.2 Smooth Muscle Contraction Inhibitors
Myosin light chain kinase (MLCK) is a regulatory enzyme involved in smooth
muscle contraction. MLCK catalyses transfer of the y-phosphate of ATP to
ser-9 of the 20 kDa myosin light chain. Phosphorylated myosin can then
interact with myosin and generate contractile force. Since smooth muscle cells
are distributed in arteries and bronchi, it is postulated that MLCK inhibitors
would be potential vasodilators and bronchodilators. MLCK has been the
target of a recent microbial screening programme which resulted in the identification of MS-347a, MS-282a and MS-282b. Chicken gizzard smooth muscle
MLCK activity was monitored by assaying phosphorylation of a peptide
substrate in the presence of calmodulin. After incubation, the reaction was
terminated by addition of acid and product measured by HPLC analysis.
MS-374a, a novel xanthene epoxide, was isolated from an Aspergillius sp.
together with MS-347b, the known fungal metabolite syndowinin B [153].
MS-347a inhibited Ca 2 + and calmodulin-dependent and independent activity of
chicken gizzard MLCK in a dose-dependent manner. MS-347b did not inhibit
the enzyme. MS-347a also inhibited PKC, but did not inhibit cAMP-dependent
protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent
cyclic nucleotidase phosphodiesterase (PDE). MLCK inhibition was dependent
on the length of a pre-incubation period, suggesting irreversible inhibition.
MS-282a and MS-282b, macrocyclic dilactones from Streptomyces tauricus,
inhibited the calmodulin-dependent, but not the calmodulin-independant, activity
of MLCK, suggesting that these compounds block MLCK activity by antagonising calmodulin [ 154]. MS-282a also inhibited calmodulin-dependent PDE.
A screening programme targeted at calmodulin-dependent PDE inhibitors
led to the discovery of SCH 45752 [155]. Calmodulin-dependent bovine heart
PDE was assayed by hydrolysis of cyclic AMP, or cyclic GMP. SCH 4572 is
a symmetrical bis-naphthopyrone from a culture of an unidentified fungus. It is
identical to the known Verticillium sp. metabolite cephalochromin.
SCH 4572 was a potent inhibitor of bovine heart and bovine aorta PDE. The
compound was much less active against calmodulin independent PDE, MLCK,
cAMP-dependent and cGMP-dependent protein kinase, and protein kinase C.
SCH 4572 stimulated carotid artery smooth muscle contraction
4.3.3 Inhibition of Plasminogen Activator Inhibitor-I
A key event in the regulation of thrombus formation and clearance is the
generation of plasmin by tissue plasminogen activator (tPA). tPA activity is
controlled by PAI-1, a fast acting inhibitor belonging to the serine protease