Hemolytic and antimalarial effects of tight-binding glyoxalase 1 inhibitors on the host-parasite unit of erythrocytes infected with Plasmodium falciparum

Abstract

Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit.

The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

“…In accordance with these results, a single IC 50 experiment with 3D7Δglo1 and 3D7Δcglo2 knockout strains and the Glo1 inhibitors compound 3 and 7 of our previous study 10 showed very similar IC 50 values compared to wild-type parasites (data not shown). If human Glo1 compensated for the loss of PfGlo1 in the 3D7Δglo1 knockout strains, one would expect altered IC 50 values because the inhibitors can also slowly inactivate the human enzyme 10 . Thus, parasite killing of wild-type and knockout strains at micromolar instead of nanomolar Glo1 inhibitor concentrations can be attributed to off-target effects.…”

“…The dispensability of PFGLO1 for parasite survival also explains the high IC 50 values of tight-binding Glo1-inhibitors in previous cell culture experiments 10 in contrast to low nanomolar inhibition constants against recombinant PfGlo1 9 . In accordance with these results, a single IC 50 experiment with 3D7Δglo1 and 3D7Δcglo2 knockout strains and the Glo1 inhibitors compound 3 and 7 of our previous study 10 showed very similar IC 50 values compared to wild-type parasites (data not shown). If human Glo1 compensated for the loss of PfGlo1 in the 3D7Δglo1 knockout strains, one would expect altered IC 50 values because the inhibitors can also slowly inactivate the human enzyme 10 .…”

“…Glo1 activities form parasite extracts were determined at 25°C using a thermostatted Jasco V-550 UV-vis spectrophotometer by following the formation of S -d-lactoylglutathione and the consumption of the hemithioacetal between methylglyoxal and reduced glutathione (GSH) at 240 nm using an extinction coefficient of ε 240nm = 2.86 mM −1 cm −1 10 57 . Briefly, stock solutions of 100 mM GSH and 100 mM methylglyoxal were freshly prepared in water and stored on ice.…”

“…reported that Plasmodium falciparum -infected erythrocytes do not only consume much more glucose than uninfected erythrocytes, but also produce up to 30-times more d-lactate owing to a highly efficient glyoxalase system 1 . Since this seminal study, the P. falciparum glyoxalases have gained considerable attention as model enzymes 2 3 4 5 and as potential targets for rational drug development 6 7 8 9 10 . The glyoxalase system, which usually consists of glutathione (or trypanothione in kinetoplastida), the isomerase glyoxalase 1 (Glo1) and the thioesterase glyoxalase 2 (Glo2), is found in most eukaryotes and several prokaryotes 5 11 12 13 .…”

“…We previously characterized the effects of non-glutathione as well as glutathione-derived inhibitors on the two different active sites of recombinant PfGlo1 4 9 . Two tight-binding Glo1 inhibitors and a variety of ester derivates were also characterized in cell culture experiments revealing micromolar IC 50 values that were three to four orders of magnitude higher than the IC 50 values with recombinant PfGlo1 9 10 . Whether the glyoxalase system of the erythrocyte- P. falciparum host-parasite unit is indeed a suitable drug target remained to be shown.…”