Development of micellar approach for inhibition analysis of galactonolactone oxidase from Trypanosoma cruzi
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Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow, Russia
Publication date: 2022-05-27
Public Health Toxicol 2022;2(Supplement 2):A1
Trypanosoma cruzi is a parasite causing Chagas disease, which is nowadays treated with only two drugs: benznidazole and nifurtimox. However, these drugs are effective only in the early or acute infection phases and have side effects. It should be added that about 7 million people all over the world suffer from the disease and thus the development of new drugs against Chagas disease is a significant task. The action of new drugs may be based on their inhibitory effect on the biosynthesis of compounds vital for the parasite, for example vitamin C (antioxidant). Galactonolactone oxidase from Trypanosoma cruzi (TcGAL) is an enzyme which catalyzes the final stage of the biosynthesis of vitamin C, and so the inhibition of TcGAL may be a suitable way to suppress the growth of the parasite. However, the development of a technique for the analysis of activity and, hence, the search for an effective inhibitor of the enzyme has been impossible until now due to its membranotropic nature and, accordingly, the tendency to aggregate in aqueous solutions. Thus, the expression of the recombinant enzyme TcGAL from Trypanosoma cruzi in E. coli cells leads to the formation of an aggregated and inactive protein, the so-called inclusion bodies. Attempts to restore the catalytic function of TcGAL using various methods traditionally used for enzyme refolding have not been successful.
In this project, an innovative technology has been developed for obtaining genetically engineered TcGAL in a soluble and catalytically active form using a system of reverse micelles of surfactants.
The developed approach makes it possible to create a high-throughput screening system for the search for a selective TcGAL inhibitor, on the basis of which it is possible to conduct a targeted search for new drugs against diseases caused by trypanosomal infection.
We have developed a micellar approach for measuring TcGAL activity and successfully applied it for inhibition analysis of TcGAL. The main point of the approach is the use of a reverse micellar system as a membrane model. Membrane has many important functions (transition of ions, proteins, fusion and compartmentalization in membranes and so on) and micellar non-bilayer structures were found in mitochondria so the used model is reliable. The use of bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles system allowed us to learn the influence of a number of compounds on TcGAL activity including lycorine (which is known to suppress the growth of T. cruzi), chalcones (they have lycorine-like structure) and derivates of allylbenzene and apiole (some of them also suppresses T. cruzi growth).
Inhibition analysis of TcGAL showed that the inhibitory effect of apiole, dillapiole, lycorine and chalcones is probably associated with a common structural fragment, benzodioxol. The effect of eugenol, estragole and tetramethoxyallylbenzene may be explained by a common structural fragment namely allylbenzene which is an inhibitor itself. In order to enhance the inhibitory effect, two inhibitors (tetramethoxyallylbenzene and dillapiole) were conjugated with triphenylphosphonium (TPP) cations. TPP provides the selective delivery of the desired compounds into the mitochondria driven by the inner negative transmembrane potential according to literature data, and so we observed a similar effect in the case of micellar reverse system too. The conjugates of tetramethoxyallylbenzene and dillapiole with TPP showed 100% inhibition at 100–200 µM, which was not obtained using the compounds without TTP fragment.
Thus, the approach to inhibition analysis of TcGAL has been developed using AOT reverse micelles system as a mitochondrial membrane model and a number of inhibitors of TcGAL were characterized. The dependence of the inhibition effect on the structure of the inhibitor was investigated and allylbenzene and benzodioxol were found to be necessary structural fragments for TcGAL inhibition.
The work was performed using equipment (FTIR spectrometer Bruker Tensor 27 and Jasco J-815 CD Spectrometer) of the program for the development of Moscow State University.