Fungus-elicited metabolites from plants as an enriched source for new leishmanicidal agents: antifungal phenyl-phenalenone phytoalexins from the banana plant (Musa acuminata) target mitochondria of Leishmania donovani promastigotes

Abstract

Two antifungal phenyl-phenalenone phytoalexins isolated from the banana plant (Musa acuminata) elicited with the fungus Fusarium oxysporum, together with a methoxy derivative of one of them and two epoxide precursors of their chemical synthesis, were tested for leishmanicidal activity on Leishmania donovani promastigotes and L. infantum amastigotes. Drugs inhibited proliferation of both forms of the parasite with a 50% lethal concentration range between 10.3 and 68.7 micro g/ml. Their lethal mechanism was found linked to the respiratory chain by a systematic approach, including electron microscopy, measurement of the oxygen consumption rate on digitonin-permeabilized promastigotes, and enzymatic assays on a mitochondrial enriched fraction. Whereas the whole set of compounds inhibited the activity of fumarate reductase in the mitochondrial fraction (50% effective concentration [EC(50)] between 33.3 and 78.8 micro g/ml) and on purified enzyme (EC(50) = 53.3 to 115 micro g/ml), inhibition for succinate dehydrogenase was only observed for the two phytoalexins with the highest leishmanicidal activity: anigorufone and its natural analogue 2-methoxy-9-phenyl-phenalen-1-one (EC(50) = 33.5 and 59.6 micro g/ml, respectively). These results provided a new structural motif, phenyl-phenalenone, as a new lead for leishmanicidal activity, and support the use of plant extracts enriched in antifungal phytoalexins, synthesized under fungal challenge, as a more rational and effective strategy to screen for new plant leishmanicidal drugs.

FIG. 1.

Chemical structures of the compounds assayed for leishmanicidal activity: anigorufone, 2-hydroxy-9-phenyl-phenalen-1-one (molecular weight = 272.31); REF5, 2-hydroxy-9-(p-methoxyphenyl)- phenalen-1-one (molecular weight = 302.33); REF20, 2-methoxy-9-phenyl-phenalen-1-one (molecular weight = 286.33); EP5, 2,3-epoxy-9-(p-methoxyphenyl)phenalen-1-one (molecular weight = 302.33); and EP6, 2,3-epoxy-9-phenyl-phenalen-1-one (molecular weight = 272.31).

FIG. 2.

In vivo variation of the luminescence of 3-Luc L. donovani parasites after drug addition at their respective LC₅₀. Parasites (2 × 10⁷ cells/ml) were incubated with 25 μM DMNPE-luciferin. When luminescence reached a plateau, drug was added (t = 0) and luminescence was monitored as described in Materials and Methods. Drugs were administered as follows: 12 μg of anigorufone/ml (•), 24 μg of REF5/ml (▾), 10 μg of REF20/ml (○), 38 μg of EP5/ml (▿), and 69 μg of EP6/ml (▪). The inset shows the dose-dependent variation of luminescence with anigorufone at 2.5 μg/ml (▴), 5 μg/ml (⋄), 10 μg/ml (▪), 15 μg/ml (▿), and 20 μg/ml (•). The results were normalized relative to the luminescence variation in control parasites.

FIG. 3.

Electron microscopy of L. donovani promastigotes treated with phenyl-phenalenones at their respective LC₅₀s. (A) Control parasites; (B) anigorufone; (C) REF5; (D) REF20; (E) EP5. (F) EP6. k, kinetoplast. Bar, 0.5 μm.

FIG. 4.

Inhibition of FRD activity in the mitochondrial fraction of L. donovani promastigotes, with different phenyl-phenalenones. The results for anigorufone (•), REF5 (▾), REF20 (○), EP5 (▿), and EP6 (▪) are given. The data represent the average of at least two different experiments.

FIG. 5.

Inhibition of SDH activity in the mitochondrial fraction of L. donovani promastigotes. SDH was assayed by determining the reduction of DCPIP as described in Materials and Methods. The results for anigorufone (•), REF5 (▾), REF20 (○), EP5 (▿), and EP6 (▪) are shown. Maximal inhibition (100%) was considered to be that caused by 12 mM malonate.