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. 2013 Apr 26;76(4):630-41.
doi: 10.1021/np300834q. Epub 2013 Feb 27.

Examination of the Mode of Action of the Almiramide Family of Natural Products Against the Kinetoplastid Parasite Trypanosoma Brucei

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Free PMC article

Examination of the Mode of Action of the Almiramide Family of Natural Products Against the Kinetoplastid Parasite Trypanosoma Brucei

Laura M Sanchez et al. J Nat Prod. .
Free PMC article

Abstract

Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-molecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasite-specific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of this lead series as development candidates against T. brucei.

Figures

Figure 1
Figure 1
Current therapeutics for HAT.
Figure 2
Figure 2
Parallel target identification strategies for almiramide-based affinity purification probes. Orange triangle = biotin; Yellow star = photoaffinity benzophenone moiety; Grey sphere = Affigel resin
Figure 3
Figure 3
A) Representative SDS-page gel for protein affinity capture results. PEX11/GIM5A band enhancement outlined by dashed box. B) Venn diagram showing relative number of candidate target proteins pulled down using each approach. Relative circle areas correspond to the total number of proteins pulled down using each approach.
Figure 4
Figure 4
Synthetic route to fluorescent almiramide derivatives and control compounds for fluorescence imaging.
Figure 5
Figure 5
Fluorescence site localization results for BODIPY-labeled almiramide probe 33. A) Uptake of probe 33 at 5 µM, and distribution of staining with respect to the nucleus and kinetoplast (large and small staining regions respectively stained in DAPI channel). B) Distribution of probe 33 with respect to glycosome distributions at 50 µM. Glycosomes stained with red anti-aldolase antibody.
Figure 6
Figure 6
Fluorescence imaging for A) control compound 37 and B) almiramide probe 38 in juvenile zebrafish. Images display lateral region at the intersection of the head and the upper spine. Zebrafish eye visible as a large dark region in the center-left of each image. The fluorescent staining indicate binding of almiramide to neuromast cells. These data are also presented as a z-stack video in the supporting information (Video S1)

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