This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Plasminogen activators (PAs) are proteases of immense clinical importance as they convert plasminogen into plasmin which dissolves fibrin clots (thrombus). PAs are thus used for the treatment of thromboembolic diseases, myocardial infarction, and cerebral apoplexy. The major PAs in use include streptokinase, urinary type plasminogen activator (uPA) and tissue type plasminogen activator (tPA). Each of these is however limited in its application due to one or more of the following factors: low potency, side effects (generalized bleeding due to low fibrin specificity), low bioavailability, immunogenicity and high cost due to inadequate source materials as well as inefficient upstream and downstream processes. In the proposed research, different mammalian cell lines and micro organisms will be screened for secretion of novel potent and fibrin-specific PAs. The PAs so detected will be purified and characterized. Fibrin specificity of these enzymes will be determined by performing a bioassay in the presence of different quantities of fibrin. The broth will also be screened for the presence of any fibrinolytic agents without PA activity. New affinity filtration membranes and other affinity matrices will be prepared for the separation of PA from broth. Affinity ligands will be synthesized on the basis of latest reports available in literature on inhibitors of PAs. uPA will be used as the model PA for this purpose. uPA has also been implicated as a key mediator of cellular invasion and metastasis of tumor cells, angiogenesis and chronic wounds. Inhibitors of uPA are thus good candidates for use as drugs in treatment of cancer. Plants and plant products will be selected on the basis of information available in literature and traditionally known therapeutic effects and screened for presence of inhibitors of uPA in their extracts. Inhibitors so detected will be isolated, purified and characterized. One of the long term goals is to use these inhibitors as lead molecules for synthesis of drugs for cancer and also use these inhibitors as affinity ligands for separation of uPA. The project will be executed through a close collaboration between a protein biochemist (the Principal investigator), an organic chemist (for synthesis of ligands and chemical modification of membranes, characterization of bioactive compounds), and plant pathologist (whose experience with microbial work will be applied). a.
Specific Aims Plasminogen Activators (PAs) activate plasminogen by cleaving a specific Arg-Val peptide bond located within the protease domain. The resulting plasmin dissolves clots (thrombus)1. These activators are majorly derived from mammals, the two major PAs being urinary-type and tissue-type plasminogen activators (uPA and tPA). Several bacterial species also secrete PAs', such as streptokinase and staphylokinase. Owing to the thrombolytic ability of the PAs, these are used for the treatment of thromboembolic disease, a common disorder that tends to increase in severe conditions such as myocardial infarction, and cerebral apoplexy. PAs are also used for cleaning blocked catheters in hospitals. The major PAs in use include streptokinase, uPA and tPA. Each of these is however limited in its application due to one or more of the following factors: low potency, side effects (generalized bleeding), low bioavailability, immunogenicity and high cost due to inadequate source materials as well as inefficient upstream and downstream processes2""""""""5. There is also an emerging interest in the physiological role of uPA as it has been implicated as a key mediator of cellular invasion and metastasis of tumor cells, angiogenesis and chronic wounds6'7. Inhibitors of uPA are thus good candidates for use as drugs in treatment of cancer and other disease situations where uPA-driven degradation of extra cellular matrix or uPA-dependent cell migration is thought to be important8. The Principal investigator's doctoral studies were focused on PAs, especially uPA, and our research led to the isolation and characterization of uPA secreted by human kidney cell line HT-1080 and development of: (a) a high efficiency production process for uPA in hollow fiber reactor9, (b) new bioreactors for uPA production using human cell lines4'10p 11, and (c) novel strategy for uPA separation from bioreactor in continuous recycle mode11""""""""13. However, there is still a lot of scope for the discovery of more potent PAs as well as their inhibitors, each being a vital clinical drug. Pathogenic microbes secrete PAs to aid the invasion of host tissues while many human tissues secrete PAs inherently. And the PAs isolated and characterized from different sources so far are different with respect to potency and fibrin affinity14, but still restricted by undesirable side-effects. The working hypotheses for this proposal are: (a) we believe that there is scope for discovery of new PAs which will provide better alternatives to the ones currently in use;(b) the current high costs of the thrombolytic drugs can be brought down if downstream processes are improved, and finally;(c) the versatility of PA system can be turned to an advantage by isolating new inhibitors of PAs which could become lead molecules for drugs against cancer. Hence this research is being proposed with the following goals in mind: (a) exploring other sources for PAs with improved potency and selectivity;(b) providing a rapid, simple, and commercially viable method of isolating a PA compound from a PA compound-containing solution;(c) screening different plant sources for selectively potent inhibitors of PAs. To achieve these goals, the following specific aims will be focused upon: 1. To screen selected mammalian cell lines and microbial cells for new plasminogen activators. Cell lines derived from human tissues known to secrete PAs and microorganisms will be screened for secretion of PAs using a bioassay and a colorimetric assay. 2. To characterize the plasminogen activators so identified. The PAs so detected in mammalian/microbial broths will be characterized with respect to activity, fibrin specificity, and molecular size. 3. To purify thus identified plasminogen activators. If the PAs appear promising after initial characterization in the crude solutions, they will be processed to obtain pure preparations through size exclusion and affinity chromatography. The kinetics of the pure preparation for human plasminogen substrate will be studied. 4. To develop new techniques based on affinity interactions for the purification of plasminogen activators. Highly selective ligands of uPA will be selected from reports available in literature. These inhibitors will be synthesized (if not available commercially) and attached to filtration membranes. The affinity membranes so developed will be used to check the efficiency of purification of uPA (our model PA) from the mammalian cell culture broth. 5. To screen different plant sources for inhibitors of plasminogen activators. The extracts from different plants and plant products (selected on the basis of reports available in literature and traditional knowledge) will be screened for presence of inhibitors of uPA. The first test for detection of PAs will be done in crude extracts using bioassay. 6. To isolate the inhibitors so identified, determine their structure. In case of detection of uPAinhibitor activity in crude extracts, the extracts will be processed for isolation of the pure inhibitors. The structure of pure inhibitors will be determined using Mass spectrometry/ NMR spectroscopy.
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