The long-term goal of this proposal is to identify small molecule inhibitors of a novel drug target, Mcm2-7, and develop these inhibitors into both experimental probes and human therapeutic agents. DNA replication utilizes many proteins of similar enzymology; however, few experimental inhibitors are available to help distinguish their function. In addition, although inhibitors of this process have traditionally been used against cancer, most such chemotherapeutics act non-specifically, and often cause significant DNA damage to both normal and cancerous cells. Relevant to both issues, our laboratory studies an essential replication protein, the Mcm2-7 replicative helicase, for which no useful inhibitors are currently available. Our recent studies identify various novel genetic interactions between Mcm2-7 and other replication factors, providing a means to identify informative inhibitors as well as specifically target abnormal DNA replication. As we use budding yeast, we propose to first develop screening approaches in this organism while working to transition to a human cell system. The following aims will be pursued in conjunction with the Pittsburgh Drug Discovery Institute, which has well-developed resources for assay design and library screening: 1) We propose to develop high-throughput cell-based assays to identify test compounds that inhibit either Mcm2-7 or proteins that interact with it. Two basic budding yeast screens are proposed: one based upon synthetic lethality against a specific Mcm mutation in the query strain, and a second that identifies molecules that suppress the lethality of a strain that over-expresses Mcm2-7. Successful development of these two assays will culminate in pilot screening of a small chemical library, which, with additional validation, will generate a small number of candidate compounds for additional screening. 2) We propose secondary screens to identify inhibitor targets and their mechanism of inhibition. Given the ease of use of budding yeast, several genetic screens will be developed to define the molecular target of the inhibitors. In addition, we plan to subject a number of these hits to additional biochemical and genetic assays previously developed in our lab to determine their mechanism of inhibition. 3) The screening approaches proposed in Aim 1 depend upon very specific budding yeast mutations. As DNA replication is much less well studied in human cells, we propose to test if our above approaches will work and it so to build human cell query strains for future screening purposes.
/Public Health Relevance Uncontrolled cell division, resulting in tumor formation, is a major feature of cancer that depends upon mis-regulated DNA replication. Although regulated replication is essential in normal tissues, most current chemotherapeutic agents are relatively non-specific and block both normal and cancer- related DNA replication. We propose here to develop chemical inhibitors to a previously neglected DNA replication factor ? Mcm2-7 ? that will specifically block cancerous but not normal DNA replication.
