Eumycetoma is a chronic infectious disease characterized by a large subcutaneous mass, often caused by the fungus Madurella mycetomatis. A combination of surgery and prolonged medication is needed to treat this infection with a success rate of only 30%. There is, therefore, an urgent need to find more effective drugs for the treatment of this disease. In this study, we screened 800 diverse drug-like molecules and identified 215 molecules that were active in vitro. Minimal inhibitory concentrations were determined for the 13 most active compounds. One of the most potent compounds, a fenarimol analogue for which a large analogue library is available, led to the screening of an additional 35 compounds for their in vitro activity against M. mycetomatis hyphae, rendering four further hit compounds. To assess the in vivo potency of these hit compounds, a Galleria mellonella larvae model infected with M. mycetomatis was used. Several of the compounds identified in vitro demonstrated promising efficacy in vivo in terms of prolonged larval survival and/or reduced fungal burden. The results presented in this paper are the starting point of an Open Source Mycetoma (MycetOS) approach in which members of the global scientific community are invited to participate and contribute as equal partners. We hope that this initiative, coupled with the promising new hits we have reported, will lead to progress in drug discovery for this most neglected of neglected tropical diseases.

Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.

Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the formation of grains. However, the processes leading to grain formation are not known. In this study, we employed a proteomic approach to characterise M. mycetomatis grain formation in Galleria mellonella larvae and map the processes leading to grain formation over time. For this, at 1 day, 3 days and 7 days post-inoculation, proteins from grains and hemolymph were extracted and analysed by label-free mass spectrometry. A total of 87, 51 and 48 M. mycetomatis proteins and 713, 997, 18 G. mellonella proteins were found in grains on day 1, 3 and 7 post-inoculation respectively. M. mycetomatis proteins were mainly involved in cellular metabolic processes and numerous enzymes were encountered. G. mellonella proteins were primarily involved in the nodulation process. The proteins identified were linked to nodulation and grain formation and four steps of grain formation were identified. The results of this proteomic approach could in the future be used to design novel strategies to interfere with mycetoma grain formation and to combat this difficult to treat infection.

Objectives Eumycetoma is currently treated with a combination of itraconazole therapy and surgery, with limited success. Recently, olorofim, the lead candidate of the orotomides, a novel class of antifungal agents, entered a Phase II trial for the treatment of invasive fungal infections. Here we determined the activity of olorofim against Madurella mycetomatis, the main causative agent of eumycetoma. Methods Activity of olorofim against M. mycetomatis was determined by in silico comparison of the target gene, dihydroorotate dehydrogenase (DHODH), and in vitro susceptibility testing. We also investigated the in vitro interaction between olorofim and itraconazole against M. mycetomatis. Results M. mycetomatis and Aspergillus fumigatus share six out of seven predicted binding residues in their DHODH DNA sequence, predicting susceptibility to olorofim. Olorofim demonstrated excellent potency against M. mycetomatis in vivo with MICs ranging from 0.004 to 0.125 mg/L and an MIC90 of 0.063 mg/L. Olorofim MICs were mostly one dilution step lower than the itraconazole MICs. In vitro interaction studies demonstrated that olorofim and itraconazole work indifferently when combined. Conclusions We demonstrated olorofim has potent in vitro activity against M. mycetomatis and should be further evaluated in vivo as a treatment option for this disease.

Background Eumycetoma is a neglected tropical disease most commonly caused by the fungus Madurella mycetomatis. Identification of eumycetoma causative agents can only be reliably performed by molecular identification, most commonly by species-specific PCR. The current M. mycetomatis specific PCR primers were recently discovered to cross-react with Madurella pseudomycetomatis. Here, we used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification. Methodology Predicted-protein coding sequences unique to M. mycetomatis were first identified in BLASTCLUST based on E-value, size and presence of orthologues. Primers were then developed for 16 unique sequences and evaluated against 60 M. mycetomatis isolates and other eumycetoma causing agents including the Madurella sibling species. Out of the 16, only one was found to be specific to M. mycetomatis. Conclusion We have discovered a predicted-protein coding sequence unique to M. mycetomatis and have developed a new species-specific PCR to be used as a novel diagnostic marker for M. mycetomatis.