Development of High-Throughput screening assays for identification of small molecule inhibitors of the Mcm2-7 replicative helicase

开发用于鉴定 Mcm2-7 复制解旋酶小分子抑制剂的高通量筛选测定法

• 批准号: 9238087
• 负责人: ANTHONY SCHWACHA
• 金额: $ 33.58万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238087
• 关键词: Alleles; Biochemical; Biochemical Genetics; Biological; Biological Assay; Cancerous; Cell Line; Cell division; Cells; Chemicals; Collaborations; Complex; DNA Damage; DNA Replication Factor; DNA biosynthesis; Development; Disease; Drug Targeting; Engineering; Enzymatic Biochemistry; Eukaryota; Future; Genetic; Genetic Screening; Genomic Instability; Goals; Growth; Human; Human Cell Line; Institutes; Laboratory Study; Libraries; Malignant Neoplasms; Molecular Target; Mutation; Normal tissue morphology; Organism; Pathologic; Pathway interactions; Peptide Initiation Factors; Phenotype; Process; Protein Overexpression; Proteins; Publishing; Regulation; Replication Initiation; Resistance; Resources; Saccharomycetales; Small Interfering RNA; Stream; System; Testing; Therapeutic Agents; Universities; Validation; Work; Yeasts; assay development; base; chemotherapeutic agent; cytotoxic; design; drug discovery; experience; genetic approach; helicase; high throughput screening; human disease; inhibitor/antagonist; killings; mutant; neglect; new therapeutic target; novel; overexpression; public health relevance; screening; small molecule inhibitor; small molecule libraries; therapeutic target; tumor

Project Summary 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.

项目总结