Biochemistry & Molecular Biology
- Gene Therapy
- Protein Chemistry/Enzymology
- Cancer Treatment
- Signal Transduction
- Cardiovascular & Skeletal Muscle Diseases
- Anti-cancer and Anti-angiogenic Therapy
Research OverviewDIRECT ACTING BLOOD CLOT DISSOLVING ENZYME:
Pharmacologic dissolution of an established thrombus has become an accepted therapeutic approach for many patients who develop arterial or venous thrombotic occlusive disease. The major hypothesis of our work is that direct-acting fibrinolytic enzymes offer a unique alternative approach for thrombolytic therapy with theoretical, as well as practical, advantages over presently available plasminogen activator (PA)-based thrombolytic agents, such as tissue plasminogen activator. Fibrolase is a 23 kDa, direct-acting, fibrinolytic metalloproteinase, found in southern copperhead snake venom, that cleaves fibrin independently of plasmin(ogen). It is not inactivated by serine proteinase inhibitors in the blood, but is inhibited by α2-macroglobulin. Fibrolase lyses fibrin by selective cleavage of peptide bonds in the alpha and beta chains. The thrombolytic actions of fibrolase have been demonstrated in animal models of arterial and venous thrombosis (in the canine and the rabbit). Fibrolase does not activate platelets or coagulation factors, nor does it lyse red blood cells. The recombinant version of fibrolase, named alfimeprase, has been produced by a large Biotech Company using a yeast expression system and the recombinant version which is identical to fibrolase except for the truncation of two amino-terminal amino acids and an aditional change in the new amino-terminus, is being tested in patients for peripheral arterial occlusive disease (PAO) and stroke in two Phase III trials. The clinical trials were recently abondened due to failure to reach established endpoints. However, several colleagues and I are trying to provide experimental evidence to support the reestablishment of the trials.
We have grown crystals of fibrolase that diffract to 1.7Ao and recently published the three-dimensional structure of fibrolase in collaboration with a colleague on the University Park Campus. We are also synthesizing a chimera of fibrolase, which possess both clot dissolving and platelet aggregation inhibitory activities. We should be able to deliver the modified form of the enzyme and specifically localize it at the site of a coronary thrombus (or heart attack). Our hope is that this would lead to opening of the obstructed coronary artery and prevent platelet-mediated reocclusion, thereby serving as an effective form of therapy for myocardial infarction.
DISINTEGRINS - EFFECTIVE INHIBITORS OF CANCER GROWTH AND ANGIOGENESIS:
Spread of cancer to remote sites, e.g. bone, lungs, liver, brain, is a characteristic of malignancy and often leads to inoperable disease. Control of metastasis offers an important avenue for cancer treatment. The first step in metastasis involves adhesion of the cancer cells to tissue around the primary tumor site. The cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade into these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. Agents that block any of the above steps should act to inhibit metastasis. In order for a tumor mass to grow beyond a size of 1-2 mm3 the development of a vascular network is required. This process is called angiogenesis or neovascularization. Tumor cells at either a primary site or at metastatic sites induce the formation of new blood vessels, and these vessels supply nutrients and growth factors to the tumor and, importantly, serve as a route for tumor dissemination (metastasis). Since cancer-induced angiogenesis is essential for progressive growth of cancer, therapies that block new blood vessel growth into the tumor will also inhibit tumor growth and are of considerable interest to the clinical community since they may provide a unique and practical way for long term control of cancer. Anti-angiogenic therapy promotes long-term dormancy of the tumor and is non-cytotoxic thereby avoiding side effects, such as gastrointestinal problems, loss of hair and bone marrow suppression, that accompany chemotherapy. Further, successfully blocking development of the vascular network may provide a useful alternative to chemotherapy that acts directly on tumor cells; attacking vascular cells would avoid the problem of acquired resistance to chemotherapy that results from genetic instability of tumor cells. We have been studying a protein from southern copperhead snake venom possessing potent anti-tumor activity that we call contortrostatin. Contortrostatin (CN) is a member of a family of peptides called disintegrins that are found in snake venoms. Many members of this family are distinguished by the presence of an amino acid sequence, arginine-glycine-aspartic acid (RGD), that enables them to bind to cell surface receptors called integrins found on cancer cells and newly growing (angiogenic) blood vessels in the tumor. Integrins mediate interactions between cells and their surroundings, and on cancer cells they play important roles in tumor invasion and dissemination. We postulated that since contortrostatin disrupts integrin interactions, it should block both cancer cell and newly growing blood vessel cell integrins and may, therefore, have significant anti-angiogenic and anti-metastatic activity. We have shown that CN acts as an effective inhibitor of breast cancer progression. Importantly, CN displays impressive inhibitory activity on the growth of new blood vessels into the breast cancer. The RGD sequence in contortrostatin plays a critical role in binding to and inhibiting growth and spread of breast cancer cells. CN is a homodimer with two identical chains held together by two inter-chain disulfide bonds. We recently succeeded in producing a recombinant version of CN using an E. coli expression system. The recombinant protein is a monomer, called vicrostatin (VCN), designed with a slightly different amino acid sequence. Using a delivery system in which VCN is encapsulated in unilamellar lipid particles (liposomes), we have shown (see figure below) that intravenous delivery of the liposomal formulation (LVCN) twice weekly in an animal model of human, metastatic breast cancer leads to close to 80% inhibition of tumor growth and over 90% inhibition of angiogenesis. Recently we have been examining the effect of VCN on the growth of human ovarian cancer cells introduced intraperitoneally in mice and have observed significant inhibition of tumor growth and dissemination. Presently we are studying human breast, prostate and ovarian cancer and glioma (a devastating brain tumor) animal models to demonstrate the anti-tumor and anti-angiogenic activities of VCN. We are also examining the mechanism by which VCN dramatically disrupts the actin cytoskeleton of cancer and angiogenic blood vessel cells leading to an abrupt halt to the locomotor apparatus of these cells.
In separate studies we are examining another class of related proteins derived from members of the ADAM (A Disintegrin and Metalloproteinase domain) family. We are able to recombinantly produce large quantities of the disintegrin domain (DD) of the ADAM proteins using a highly engineered bacterial expression system. One of these ADAM DDs has interesting antitumor activity based on its interaction with integrins on the tumor cell surface. Since members of the ADAMs have quite different structures of their DDs, we are interested in characterizing binding specificity and affinities of these ADAM DDs and characterizing their biological activities with potenital application to human disease.