Examination of the Mode of Action of the Almiramide Family of Natural Products against the Kinetoplastid Parasite Trypanosoma brucei

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 disrupt 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.

Biological targets and mechanisms of action of natural products from marine cyanobacteria

“…183 The close relation of this parasite to other kinetoplastid parasites such as Trypanosoma brucei prompted its evaluation for additional biological activity. 184 Almiramides A–C and related synthetic analogs showed low micromolar IC 50 s against both Leishmania sp. and T. b. brucei , with the majority of the compounds having greater than 10-fold selectivity for the parasites than mammalian cells.…”

“…and T. b. brucei , with the majority of the compounds having greater than 10-fold selectivity for the parasites than mammalian cells. 184 The MOA of almiramides against T. brucei was interrogated using an almiramide C-biotinylated probe, photoaffinity probe and affinity-resin conjugated probe. The use of complementary techniques to search for the direct target of almiramides addressed the limitations of each technique and thereby provided greater reliability of the protein hits from affinity purification.…”

“…The use of complementary techniques to search for the direct target of almiramides addressed the limitations of each technique and thereby provided greater reliability of the protein hits from affinity purification. 184 The biotinylated probe was constructed using a commercially available biotin reporter tag with a primary amine terminus that was coupled to an almiramide C analog bearing a terminal carboxylic acid moiety. 184 Incubation of the biotinylated probe with cell lysates of T. brucei yielded 38 possible protein targets.…”

“…184 The biotinylated probe was constructed using a commercially available biotin reporter tag with a primary amine terminus that was coupled to an almiramide C analog bearing a terminal carboxylic acid moiety. 184 Incubation of the biotinylated probe with cell lysates of T. brucei yielded 38 possible protein targets. 184 The affinity resin on the other hand was prepared using a 15 Å PEG linker and a 6-aminohexanol-capped affi-gel resin and identified 12 proteins that directly bind to almiramide.…”

“…184 Incubation of the biotinylated probe with cell lysates of T. brucei yielded 38 possible protein targets. 184 The affinity resin on the other hand was prepared using a 15 Å PEG linker and a 6-aminohexanol-capped affi-gel resin and identified 12 proteins that directly bind to almiramide. 184 The photoaffinity probe was prepared like the biotinylated probe, except for the addition of a benzophenone moiety.…”