Novel Compound MMV1804559 from the Global Health Priority Box Exhibits In Vitro and In Vivo Activity against Madurella mycetomatis

Abstract

Objectives: Eumycetoma is a neglected tropical disease (NTD) characterized by subcutaneous lesions and the formation of grains. Attempts to treat eumycetoma involve a combination of antifungal treatment and surgery, although the outcome is frequently disappointing. Therefore, there is a need to identify novel antifungal drugs to treat eumycetoma. In this respect, Medicines for Malaria Venture (MMV) has assembled libraries of compounds for researchers to use in drug discovery research against NTD. Therefore, we screened two MMVOpen compound libraries to identify novel leads for eumycetoma.

Methods: A total of 400 compounds from the COVID Box and the Global Health Priority Box were screened in vitro at 100 µM and 25 µM against the most common causative agents of eumycetoma, namely Madurella mycetomatis and Falciformispora senegalensis, and the resulting IC₅₀ and MIC₅₀ values were obtained. Compounds with an IC₅₀ < 8 µM were identified for possible in vivo efficacy studies using an M. mycetomatis grain model in Galleria mellonella larvae.

Results: Out of the 400 compounds, 22 were able to inhibit both M. mycetomatis and F. senegalensis growth at 100 µM and 25 µM, with compounds MMV1593278, MMV020335, and MMV1804559 being selected for in vivo testing. Of these three, only the pyrazolopyrimidine derivative MMV1804559 was able to prolong the survival of M. mycetomatis-infected G. mellonella larvae. Furthermore, the grains in MMV1804559-treated larvae were significantly smaller compared to the PBS-treated group.

Conclusion: MMV1804559 shows promising in vitro and in vivo activity against M. mycetomatis.

Keywords: COVID box; Global Health Priority Box; MMV open access boxes; Madurella mycetomatis; eumycetoma; mycetoma; treatment.

Figure 1

Flow diagram for in vitro and in vivo evaluation of the MMV compounds tested. In total, 400 compounds were tested for growth inhibition of both M. mycetomatis and F. senegalensis at 100 µM and 25 µM. Fifteen of the 160 compounds present in the MMV COVID Box (green) inhibited the growth of both M. mycetomatis and F. senegalensis. A total of 7 of the 240 compounds from the MMV Global Health box (blue) were able to inhibit the growth of both fungi. The concentrations at which these 22 compounds were able to achieve a 50% (IC₅₀) reduction in fungal growth for M. mycetomatis and F. senegalensis were determined, with three compounds from the MMV Global Health box achieving IC₅₀ at a concentration below 8 µM for M. mycetomatis. Additionally, the MIC of these three compounds was determined against eight other M. mycetomatis isolates, and all three compounds were able to inhibit the growth of eight additional isolates, namely CBS132419, CBS132588, CBS132589, p1, PARIJS 15580 AL1, SO1, Peru72012, and CBS247.48. After subsequent toxicity and efficacy testing in G. mellonella larvae, it was found that one compound, compound MMV1804559, was able to prolong larval survival and, therefore, was considered a new lead compound.

Figure 2

The activities of compounds from the MMV COVID Box and MMV Global Health Priority Box against the two main causative agents of eumycetoma, i.e., M. mycetomatis MM55 and F. senegalensis CBS197.79. In panel (A), the percentage growth of M. mycetomatis exposed to 100 µM of each compound is shown. In panel (B), the percentage growth of F. senegalensis exposed to 100 µM of each compound is shown. Each dot represents a single compound. A green dot indicates a compound that originated from the MMV COVID Box, and a blue dot indicates a compound that originated from the MMV Global Health Priority Box. Growth inhibition was indicated when the percentage growth of fungi was inhibited by ≥80%. (indicated by a dashed line). In panel (C), the percentage growth of M. mycetomatis (x-axis) and F. senegalensis (y-axis) exposed to a compound concentration of 25 µM is plotted. In total, 22 compounds had a percentage growth inhibition of ≥80% for both M. mycetomatis and F. senegalensis. In panel (D), the IC₅₀ values in µM of these 22 selected compounds against M. mycetomatis (x-axis) and F. senegalensis (y-axis) are shown. The dashed line shows the 8 µM boundary, i.e., the IC₅₀ concentration at which a compound would be selected for in vivo testing.

Figure 3

Chemical structures of compounds MMV020335, MMV1804559, and MMV1593278 from the MMV Global Health box with an IC₅₀ < 20% for M. mycetomatis.

Figure 4

Survival curve of treatment therapy against M. mycetomatis-infected larvae using three selected compounds. In panel (A), the toxicity (dashed lines) and efficacy (solid lines) of MMV1593278 (red), MMV020335 (green), and MMV1804559 (blue) in G. mellonella larvae are shown. PBS was used as a growth control (black). Toxicity testing was performed in uninfected larvae at 20 µM/larva for all three compounds and 10 µM/larva for MMV1804559 (dark blue). Efficacy testing was performed using 20 µM/larva for MMV1593278 and MMV020335 and at 10 µM/larva for MMV1804559 in M. mycetomatis-infected larvae. All treated groups were compared with the PBS group via the Log-Rank test. Only MMV1804559 prolonged the survival of M. mycetomatis-infected larvae after 4 days (Log-Rank, p = 0.0223) and after 10 days (Log-Rank, p = 0.0313). Prolonged survival is indicated with an * at day 4 and day 10. No enhanced survival was noted for MMV1593278 and MMV020335. In panel (B), the efficacy of the combination of MMV1804559 with itraconazole (ITZ) is shown. The survival curves of larvae treated with PBS (black), MMV1804559 at 10 µM/larva (blue), 5.7 mg/kg ITZ (red), and the combination of MMV1804559 at 10 µM/larvae with 5.7 mg/kg ITZ (green) are shown. Significantly enhanced survival is indicated with an * and observed for larvae treated with MMV1804559 only (Log-Rank, p = 0.0313), the combination of MMV1804559 and ITZ after 4 days (Log-Rank, p = 0.0227), and the combination of MMV1804559 and ITZ after 10 days (Log-Rank, p = 0.0465). * indicates a p-value between 0.01 and 0.05.

Figure 5

Grains in histology slides of four treatment groups in M. mycetomatis-infected larvae. In this figure, H&E-stained (panels (A–D)) and Grocott-stained (panels (E–H)) M. mycetomatis grains formed in G. mellonella larvae are shown. Slides were prepared after 72 h of infection and treatment with PBS (panels (A) and (E)), 5.7 mg/kg ITZ treatment (panels (B) and (F)), 10 µM/larva MMV1804559 treatment (panels (C) and (G)), or the combination of both (panels (D) and (H)). The scale bars stand for 250 µm in each image. The grains were grouped into large (black arrow), medium (white arrow), and small (dashed arrow) grains. The number of grains that were classified as large (orange), medium (purple), and small (blue) in each slide is depicted in panel (I). The Mann–Whitney U-test was used to compare the difference in the total number of grains/larva (panel (J)), the total number of large grains (panel (K)), and the total size of the grains (panel (L)). No significant difference in total grain number (panel (J)) was noted. However, a significantly lower number of large grains was noted in larvae treated with MMV1804559 (Mann–Whitney, p = 0.0159) (Panel (K)). This also resulted in a significantly lower total grain size for larvae treated with MMV1804559 (red) (Mann–Whitney, p = 0.0079) or the combination MMV1804559 with ITZ (blue) (Mann–Whitney, p = 0.0317) (Panel (L)). Statistical significance is displayed as * (0.01 < p < 0.05) and ** (0.0001 < p < 0.01).