Small, free-living amoebae (FLA) are ubiquitous in nature and are found in soil, fresh water, and marine environments. Most FLA feed on bacteria and are of no medical importance, yet several FLA are known to cause serious, usually fatal disease in humans and animals. Naegleria fowleri and Acanthamoeba spp. are the best known examples of pathogenic FLA. N. fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a disease characterized by a fulminant, rapidly fatal encephalitic disease that most often afflicts healthy young humans. Acanthamoeba spp. cause granulomatous amoebic encephalitis (GAE), a chronic disease seen most often in immune-compromised hosts and those at risk of opportunistic infections. Acanthamoeba spp. also cause amoebic keratitis, skin, nasopharyngeal, and disseminated infections. The major problem for infections with pathogenic FLA is the lack of effective therapeutics and the absence of drug discovery research for this emerging infectious disease. Treatment of PAM is empirical and based upon the first few cases of successful treatment with amphotericin B combination therapy. Importantly, most victims with neurological disease die from PAM or GAE despite treatment with amphotericin B combined with other antimicrobial drugs. Despite the severity of infections caused by FLA, there are few data available on new drugs and very few modern drug discovery or development efforts. The majority of the research literature on drug discovery for FLA consists of limited in vitro or in vivo studies with only a few drugs already approved for other uses. Most of the new chemical entities (NCE's) approved by the Food and Drug Administration in the past 25 years are from natural products research. Among treatments for parasites, only 4 of the 13 small molecule drugs approved for treatment of parasites in the last 25 years are totally synthetic; all others are either natural products or their derivatives, or are based on a natural product template. Whether as native natural products or as the basis for synthetic analogues, it is clear that drug discovery continues to be dominated by small molecules found in nature. Despite the promise of natural products research, there have been no large- scale screens of natural products for discovery of new drugs to treat pathogenic FLA infections. We hypothesize that natural products are a rich source of chemical diversity that can be used to identify new chemical scaffolds with potential for development as new drugs for FLA. Herein we propose to discover new drugs and drug leads that can be used in combination with amphotericin B for the treatment of pathogenic FLA. We will apply modern tenets of drug discovery and natural products research for the first time with pathogenic FLA to discover novel hits that can serve as the basis for lead optimization and development of new drugs to treat pathogenic FLA infections. Many new chemotypes have emerged from natural products research, yet most efforts have focused on the same terrestrial regions of high biodiversity. Conversely, many other regions of the world and other sources of remarkable biodiversity have been understudied. We will investigate several niche sources of microbial biodiversity in the search for new chemotypes to treat FLA. These include the world's largest collection of filamentous fungi (Mycosynthetix), endophytic fungi from mangrove plants, and marine invertebrate-associated eubacteria from Antarctica (Baker Lab at the University of South Florida). In Phase I we will screen 30,000 extracts for activity against N. fowleri and de-replicat the active hits (>50% inhibition at 5 g/ml and cytotoxicity index > 10). In Phase II we will conduct bioassay guided fractionation of the hits to identify novel, natural product chemical scaffolds for lead optimization as new therapies for pathogenic FLA. In addition we will expand the screening campaign to increase the biodiversity being sampled to identify new drugs for these deadly diseases.