Identification of natural compounds inhibiting Trypanosoma brucei Glyceraldehyde-3-phosphate dehydrogenase by in-silico screening and a spectrophotometric enzyme inhibition assay

Continuing our in-silico study [1] focusing on the identification of promising natural compounds acting against neglected protozoan infections by means of molecular docking, the present communication reports on a first experimental confirmation of the hits returned by the mentioned approach. Validation of the in-silico hits obtained by docking various natural product databases into the active site of Trypanosoma brucei brucei Glyceraldehyde-3-dehydrogenase (TbbGAPDH) was achieved by a photometric inhibition assay as described in [2]. The enzyme was produced recombinantly in Escherichia coli as described in [3] and the assay conducted as previously reported [2]. The enzyme's activity in the absence of inhibitors was considered to mark 100% while the percent inhibition of the tested natural compounds was calculated accordingly. Virtual screening of a commercially available natural product database (AnalytiCon Discovery GmbH, Potsdam, Germany; database subset used for virtual screening consisted of 700 compounds) resulted in 17 hits which were chosen for experimental testing. Eight of these hits showed significant inhibitory activity (> 10% inhibition at 10µM), out of which three compounds inhibited TbbGAPDH by more than 50% at 10µM. These natural products are reported to inhibit TbbGAPDH for the first time, potentially revealing promising new lead structures for further inhibitor design. Determination of their IC₅₀-values and evaluation of the antitrypanosomal activity in cellular tests is in progress.

Fig. 1: Most active identified natural compounds; Values in brackets represent the percent inhibition of TbbGAPDH at 10µM concentration.

Acknowledgements: Bacteria containing the expression plasmid for TbbGAPDH were kindly provided by M.L. Bolognesi and S. Piretti (Bologna, Italy) and P. Michels (Edinburgh, U.K.). Support of CCG, Montreal, is gratefully acknowledged. This work is part of the activities of ResNetNPND: http://www.uni-muenster.de/ResNetNPND/

References:

[1] Herrmann F, Schmidt T J. Planta Med. 2013; 79 1132

[2] Lambeir A-M, et al. Eur. J. Biochem. 1991; 198 429 – 435

[3] Piretti S, et al. PLoS Negl. Trop. Dis. 2013; 7(1) e2012