Sonic hedgehog (Shh) signaling has been implicated in the pathogenesis of pancreatic cancer. Our laboratory is developing drugs that block modification of Shh proteins with palmitate and thereby prevent Shh mediated growth of human pancreatic cancer cells. In order to signal correctly, Shh must be modified by attachment of the 16-carbon fatty acid palmitate to its N-terminus. We aim to exploit Shh palmitoylation as a potential Achilles heel by targeting Hhat (Hedgehog acyltransferase), the enzyme that catalyzes attachment of palmitate to Shh. Since palmitoylation is required for Shh function, Hhat inhibitors that block Shh palmitoylation could be developed into novel chemotherapeutics that will be efficacious in the treatment of pancreatic cancer. My laboratory developed an in vitro Shh palmitoylation assay that is dependent on Hhat and used this assay for High Throughput Screening (HTS) to identify small molecule inhibitors of Hhat. We recently completed the HTS, identified and validated 99 “hits” and picked 4 lead compounds that inhibit Hhat and Shh-mediated cell growth. Our current focus is to determine the effect of Hhat inhibitors on the growth of human pancreatic cancer cells. We are treating pancreatic cancer cell lines with each of the Hhat inhibitors and analyzing the effects on Shh palmitoylation and signaling, cell growth, migration and invasion, and paracrine signaling. In addition, we are actively engaged in preclinical testing of the Hhat inhibitors. We collaborate with several Memorial Sloan-Kettering Cores to optimize “druggability” of the lead compounds. The ability of the lead compounds to block tumorigenesis in mouse xenograft models and genetically engineered mouse models of pancreatic cancer is being assessed. The most effective inhibitors will be optimized for further therapeutic development.