Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

Insights into the ubiquinol/dioxygen binding and proton relay pathways of the alternative oxidase.

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7 Å, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase.

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).

Structure of the trypanosome cyanide-insensitive alternative oxidase.

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.