The rational design of therapeutic agents for cancer requires an understanding of the biochemical basis by which cancer cells arise and progress. While these mechanisms are many and diverse, recent studies have suggested that small classes of genes may be involved in the process of tumorigenesis. Our first purchase on these genes arose from the study of oncogenic retroviruses, and the discoveries that many of the transforming genes of these viruses were tyrosine kinases, and that transformed cells had increased content of proteins containing phosphotyrosine. If specific inhibitors for the viral protein tyrosine kinases (PTK) could be prepared, we would be able to test the role of protein phosphorylation in neoplastic transformation and determine general principles for the future design of chemotherapeutic agents-no matter what their biologic targets are. Our premise is that drugs can be developed that will selectively block individual PTKs without affecting Ser/Thr kinases or other tyrosine kinases within the host. This is likely since while PTKs thus far discovered share common features (homology of the catalytic region), they differ in amino acid sequences that will confer unique structural features that, in turn, should limit access to the caralytic site unless inhibitor drugs are conformationally specific to that PTK. We believe that knowledge of the 1) structure of unique chemicals that inhibit PTK activity, 2) canons for chemical interactions with the active site of PTKs, 3) tertiary structure of PTKs, and 4) computer-aided drug modeling will lead to the development of drugs to selectively block specific PTKs. Our approach to finding PTK inhibitors is two-pronged. First, we will attempt to determine specific features of individual kinases which are likely targets for enzyme inhibition and, therefore, for therapeutic drugs. Central to this aspect of the proposal is that we will obtain large amounts of 4-5 purified PTK (i.e., pp60(v- src), pp60(c-src), p210(bcr-abl), two polypeptides from the c-src catalytic region) for study using Spodoptera frugiperda calls infected with an appropriate baculovirus vector. This material will be used to do quantitative enzyme kinetics, obtain crystal structure, conduct solution phase NMR analysis, and evaluate phosphoryl transfer using stereochemical techniques. Our second approach will be to find PTK inhibitors whose mechanism of action cannot be predicted a priori. Our efforts in this approach will be in high through put (Briston Myers) screening using sensitive cell-based and cell-free ELISA screening methods developed in our laboratories. The assays will detect inhibitors which both directly and indirectly affect PTK activity. An additional emphasis of this proposal will be on screening techniques of putative PTK inhibitors. Inhibitors of potential interest will be examined by standard enzymology. Additionally, they will be evaluated for their ability to inhibit c-src, v-src and bcr-abl kinase activity in neoplastic cell lines that have abnormally high kinase activity. In total, our proposal should provide a rationale framework to discover and develop specific substrates and inhibitors of different PTK associated with the maintenance of the malignant phenotype.
