Antiprotozoal Natural Products from Endophytic Fungi Associated with Cacao and Coffee

Abstract

Background: Collectively, leishmaniasis and Chagas disease cause approximately 8 million cases and more than 40,000 deaths annually, mostly in tropical and subtropical regions. The current drugs used to treat these diseases have limitations and many undesirable side effects; hence, new drugs with better clinical profiles are needed. Fungal endophytes associated with plants are known to produce a wide array of bioactive secondary metabolites, including antiprotozoal compounds. In this study, we analyzed endophytic fungal isolates associated with Theobroma cacao and Coffea arabica crop plants, which yielded extracts with antitrypanosomatid activity.

Methods: Crude extracts were subjected to bioassay-guided isolation by HPLC, followed by spectrometric and spectroscopic analyses via mass spectrometry (MS) and nuclear magnetic resonance (NMR), Results: Compounds 1-9 were isolated and displayed novel antitrypanosomal and antileishmanial activities ranging from 0.92 to 32 μM. Tandem liquid chromatography-mass spectrometry (LC-MS) analysis of the organic extracts from different strains via the feature-based Global Natural Products Social (GNPS) molecular networking platform allowed us to dereplicate a series of metabolites (10-23) in the extracts. Molecular docking simulations of the active compounds, using the 3-mercaptopyruvate sulfurtransferase protein from L. donovani (Ld3MST) and the cruzipain enzyme from T. cruzi as putative molecular targets, allowed us to suggest possible mechanisms for the action of these compounds.

Conclusions: The isolation of these antiprotozoal compounds confirms that crop plants like coffee and cacao harbor populations of endophytes with biomedical potential that confer added value to these crops.

Keywords: Chagas disease; Leishmania donovani; Trypanosoma cruzi; antiprotozoals; endophytes; leishmaniasis.

Figure 1

Metabolites isolated from T. cacao (1–4) and C. arabica (5–9) endophytic fungi.

Figure 2

GNPS feature-based molecular networks of T. cacao-associated endophytes. (A) C. rosea network, sorbicillinoid molecular cluster, and verticillin D. (B) W. zeylanica network, cyclodepsipeptide molecular cluster highlighting the enniatin family. The pink shadow denotes dereplicated compounds and adducts. The node color represents the m/z gradient of each feature detected.

Figure 3

GNPS feature-based molecular networks of C. arabica-associated endophytes. (A) Multiguttulispora sp. network and pyridone polyketide molecular cluster highlighting the flavipucine family, as well as 4-hydroxymellein, mellein, and fusaric acid. (B) X. grammica network, polyunsaturated fatty acid molecular cluster, and grammicin analogs. The pink shadow denotes dereplicated compounds and adducts. The node color represents the m/z gradient of each feature detected.

Figure 4

Molecular docking simulations of the interactions of isolated compounds 2–4 and 6 with cruzipain. Compounds 2 (A,E), 3 (B,F), 4 (C,G), and 6 (D,H) interact with key amino acids of cruzipain. H-bond interactions are depicted as dashed yellow lines at distances lower than 3.0 Å. Only polar hydrogens are shown for clarity. The compounds are displayed in stick representation compared with the orientation of the cocrystallized hydroxy methyl ketone noncovalent inhibitor, depicted with solid red lines.

Figure 5

Molecular docking interactions of isolated compounds 1–2, 6, and 8 with Ld3MST. Compounds 1 (A,E), 2 (B,F), 6 (C,G), and 8 (D,H) interact with critical amino acids of Ld3MST. H-bond and pi–pi stacking interactions are depicted as dashed yellow and blue lines, respectively, at distances lower than 3.0 Å. Only polar hydrogens are shown for clarity. The isolated compounds are displayed in stick representation, in contrast to the orientation of the natural substrate of Ld3MST, 3MP, depicted with red lines, and to the superposition of the sulfite ions cocrystallized in the active site of the Lm3MST template.