Project Summary/Abstract Chemical modification of RNAs opens another avenue to regulate gene expression at the post-transcriptional level. Many mRNAs are modified with N6-methyladenosine (m6A), and controlled modification is important to maintain proper RNA function throughout its life cycle, including processing, translation, splicing, and degradation. METTL3/METTL14 RNA methyltransferase complex is responsible for creating m6A marks on many mRNAs. The catalytic activity of the METTL3/METTL14 complex is essential to most of its known functions. Dysregulation of METTL3/METTL14 activity has been linked to many types of cancer. In numerous types of malignancies, hyperactivity of the METTL3/METTL14 complex promotes disease. Therefore, potent and specific small molecule inhibitors of METTL3/METTL14 are likely to have therapeutic benefit. Moreover, the mechanisms through which a change in METTL3/METTL14 activity influences various biological processes including oncogenesis need to be investigated with more rigor. Chemicals that can specifically switch off METTL3/METTL14 on demand will be valuable probes to dissect the diverse pathways that involve m6A, including carcinogenesis. Here we propose to identify small drug-like molecules that block the methyltransferase activity of METTL3/METTL14, prioritizing the inhibitory effect on the gain-of-function mutant. We will use an unbiased screen of a large chemical library to find compounds that can effectively inhibit METTL3/METTL14 activity. In Aim 1, we will establish the primary high-throughput assay and implement it to test all the compounds in our chemical library. In Aim 2, we will efficiently and effectively prioritize the cherry-picked compounds to identify the top hits through a streamlined approach that uses multiple high-throughput secondary and counter screens. In Aim 3, we will use multiple orthogonal assays and mechanism of action studies to prioritize the top hits further to arrive at potential lead compounds that are specific for METTL3/METTL14, and not for other enzymes including other m6A methyltransferases. In addition we will initiate lead optimization by using our structural expertise to analyze the structure-activity relationship. The proposed study will yield small molecules inhibitors of METTL3/METTL14 that have been rigorously characterized by using biochemical, biophysical, and cell-based methods and will establish the groundwork for future development through the NCI Experimental Therapeutics (NExT) program.