Emerging evidence implies that protein acetyltransferases play a crucial role in diverse biological processes and various human diseases including cancer. Protein N-terminal acetyltransferase D (NatD), also known as Naa40, Nat4, or Patt1, is a unique member of protein N-terminal acetyltransferases because it only acetylates histones H2A and H4 that share the identical N-terminal sequence of SGRGK. NatD has been reported to play an important role in a variety of processes including remodeling of chromatin structure, cell migration and invasion, and apoptosis. The elevated level of NatD in human lung and colorectal cancer tissues correlates with poor clinical outcomes. Moreover, loss of NatD suppresses human lung cancer cell invasion and decreases the tumor growth in colorectal cancer xenograft mice models. Hence, we hypothesize that NatD is a compelling target for the development of novel cancer therapeutics for lung and colorectal cancers. However, there are no specific small molecule probes available for NatD to decipher the functions of NatD acetyltransferase activity in cancer. To fill this gap, our long-term goal is to discover novel, potent, and selective small molecule NatD inhibitors. For this application, we will employ a series of facile and reproducible high-throughput screening (HTS) assays with orthogonal readouts to screen 400,000 diverse compounds from selected chemical libraries at the Chemical Genomics Facility at Purdue Institute for Drug Discovery. Then we will characterize active compounds in structural, mechanistic, selectivity, and cell-based studies. Upon completion of this project, we expect to identify potent and selective first-in-class NatD small molecule inhibitors as chemical probes for NatD function in cells. The knowledge gained from this project would expedite the development of NatD modulators and our understanding of NatD-regulated pathways in cancer patients.