2011 Annual Report 1a.Objectives (from AD-416) Discover and develop potent, safe and soluble inhibitors of botulinum neurotoxins (BoNTs). 1b.Approach (from AD-416) Microfungal colonists (fungicolous fungi) of long-lived fungal sporocarps collected in forested habitats within the continental United States and Hawaii were isolated into pure culture. Individual fungi are grown in both liquid and solid culture fermentation and extracted with organic solvents. Extracts containing fungal produced compounds that neutralize botulism, in assays performed by the United States Army Medical Research Institute of Infectious Diseases, will be delivered to a cooperating chemist at the University of Iowa for isolation and characterization of the bioactive compound(s). Cultures producing compounds that neutralize botulism neurotoxins will be identified based on morphological characteristics and DNA sequencing of the internal transcribed spacer (ITS) region and domains D1 and D2 of the nuclear large subumit (28S) rDNA geme and comparison with sequences deposited in GenBank (National Center for Biotechnology Information). Viable cultures of the small molecule producing fungal strains of interest will be maintained. 3.Progress Report The Authorized Departmental Officer’s Designated Representative (ADODR) monitors this agreement by e-mails, telephone calls, providing samples for testing, and reviewing text for poster presentation. Four structurally related compounds, each produced by unrelated fungicolous fungi, were shown to inhibit botulism neurotoxin serotype A based on in vitro tests. This class of fungal metabolites is one of a very small number of natural products from fungi reported to inhibit neurotoxin serotype A.