Novel Screen Methodologies for Identification of New Microbial Metabolites
107
inhibitor (serpin) superfamily of protease inhibitors. Elevated plasma concentrations of PAI-1 are thought to be important risk factors in thrombotic disease,
and modulators of PAI-1 inhibition of tPA have therefore been sought as
potential antithrombotic agents. An amidolytic assay of tissue plasminogen
activator mediated plasmin generation has been used to screen micorbial samples at Xenova. The assay mixture contained a concentration of PAI-1 which
was just sufficient to inhibit tPA activity. Inhibition of PAI-1 activity resulted in
the tPA mediated clearance of plasminogen. The action of the released plasmin
on a chromogenic substrate ws then measured spectrophotometrically.
Two diketopiperazines from an unindentified Strepotomyces sp. were characterised by their ability to inhibit PAI-1 activity in this assay [-156]. The structure
of one of these is shown in Fig. 8. Their structures were confirmed by chemical
synthesis, and further synthetic studies generated analogues with superior activities in vitro and in vivo. Preclinical studies have confirmed that this series of
compounds is effective in preventing clot formation in laboratory models of
thrombosis. These compounds do not inhibit normal blood coagulation, suggesting that they will not cause excessive bleeding. They are the first compounds of
a novel therapeutic class which, although discovered in a enzyme assay, inhibit
an interaction between two large proteins.
4.4 CNS Disease
4.4.1 General Screen Methodologies and Targets
Screening programmes reported in the literature for enzyme inhibitors targeted
at CNS disease have included: aminopeptidase-M (AP-M), an enkephalin inactivating enzyme key to opioid peptide metabolism, and prolylendopeptidase
(PEP), which degrades a range of biologically active peptides and is found at
elevated levels in the occipital lobe of Alzheimer patients. Probestin, and tetrapeptide from Streptomyces azureus [157] and leuhistin, an imidazole derivative
produced by Bacillus laterosporus [158] were discovered in an AP-M screening
programme. Poststatin, a pentapeptide metabolite of Streptomyces viridochromogenes [159, 160] and eurystatins A and B, cyclic tripeptides from Streptomyces eurythermus [161, 162] were discovered during a PEP screening programme.
5 Discussion and Conclusions
5.1 Review of Compounds Identified
The microbial metabolites described in the previous sections illustrate the
continuing importance of micro-organisms as a source of chemical diversity for
107
inhibitor (serpin) superfamily of protease inhibitors. Elevated plasma concentrations of PAI-1 are thought to be important risk factors in thrombotic disease,
and modulators of PAI-1 inhibition of tPA have therefore been sought as
potential antithrombotic agents. An amidolytic assay of tissue plasminogen
activator mediated plasmin generation has been used to screen micorbial samples at Xenova. The assay mixture contained a concentration of PAI-1 which
was just sufficient to inhibit tPA activity. Inhibition of PAI-1 activity resulted in
the tPA mediated clearance of plasminogen. The action of the released plasmin
on a chromogenic substrate ws then measured spectrophotometrically.
Two diketopiperazines from an unindentified Strepotomyces sp. were characterised by their ability to inhibit PAI-1 activity in this assay [-156]. The structure
of one of these is shown in Fig. 8. Their structures were confirmed by chemical
synthesis, and further synthetic studies generated analogues with superior activities in vitro and in vivo. Preclinical studies have confirmed that this series of
compounds is effective in preventing clot formation in laboratory models of
thrombosis. These compounds do not inhibit normal blood coagulation, suggesting that they will not cause excessive bleeding. They are the first compounds of
a novel therapeutic class which, although discovered in a enzyme assay, inhibit
an interaction between two large proteins.
4.4 CNS Disease
4.4.1 General Screen Methodologies and Targets
Screening programmes reported in the literature for enzyme inhibitors targeted
at CNS disease have included: aminopeptidase-M (AP-M), an enkephalin inactivating enzyme key to opioid peptide metabolism, and prolylendopeptidase
(PEP), which degrades a range of biologically active peptides and is found at
elevated levels in the occipital lobe of Alzheimer patients. Probestin, and tetrapeptide from Streptomyces azureus [157] and leuhistin, an imidazole derivative
produced by Bacillus laterosporus [158] were discovered in an AP-M screening
programme. Poststatin, a pentapeptide metabolite of Streptomyces viridochromogenes [159, 160] and eurystatins A and B, cyclic tripeptides from Streptomyces eurythermus [161, 162] were discovered during a PEP screening programme.
5 Discussion and Conclusions
5.1 Review of Compounds Identified
The microbial metabolites described in the previous sections illustrate the
continuing importance of micro-organisms as a source of chemical diversity for