Fig 1. Flow diagram for in vitro and in vivo evaluation of all compounds (red and blue numbers show numbers of hits from the Pathogen and Stasis Boxes respectively, with the black numbers indicating totals).

As is seen in this figure, we started by screening 800 compounds in vitro at a concentration of 100 μM. Of those 800 compounds, 400 compounds originated from the Pathogen Box (red) and 400 compounds from the Stasis Box (blue). For the 215 compounds which were inhibited at 100 μM, IC50s were determined. IC50 was defined as the concentration of compound in μM were 50% reduction in fungal growth was obtained. In total, 13 compounds had an IC50 of 5 μM or lower, 12 originating from the Pathogen Box and 1 from the Stasis Box. Of these 13 compounds, MICs were determined against 7 clinical M. mycetomatis isolates. The MIC was defined as the first concentration at which 80% or more reduction in viable fungal mass was obtained. For the 10 most potent compounds, toxicity and efficacy in G. mellonella larvae was determined.