DiscussionGarcinol is a polyisoprenylated benzophenone natural product with reported activity against multiple histone acetyltransferases (HATs), including human p300 and PCAF. In the past decade, nearly 100 published papers have utilized garcinol for epigenetic-based experiments as well potential therapeutic applications, including cancers. Often cell-based and in vivo phenomena in these experiments are attributed to specific compound-target interactions. During a drug discovery project targeting Rtt109 − a HAT critical for fungal replication-coupled nucleosome assembly − we observed that garcinol inhibited multiple HATs at low micromolar compound concentrations in vitro, including yeast Rtt109 and Pneumocystis Rtt109. Herein, we present data showing garcinol also inhibits Aspergillus fumigatus Rtt109 (AfRtt109) and is cytotoxic versus A. fumigatus in vivo. As garcinol contains chemical moieties associated with assay interference, we sought to further characterize its mechanism(s) of inhibition in vitro. Our studies show it is unlikely that garcinol exerts its anti-HAT effects by thiol-reactive or redox-active mechanisms. However, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) suggests garcinol inhibits AfRtt109 activity and other targets in vitro by non-specific compound-protein interactions, and may therefore exert its promiscuous bioactivity in vitro by a therapeutically uninteresting mechanism. Our results suggest caution in attributing target-specific effects in cell-based and in vivo assays to garcinol, especially when tested at relatively high compound concentrations.