Structure-based design of submicromolar, biologically active inhibitors oftrypanosomatid glyceraldehyde-3-phosphate dehydrogenase

The bloodstream stage of Trypanosoma brucei and probably the intracellular (amastigote) stage of Trypanosoma cruzi derive all of their energy from gly colysis, Inhibiting glycolytic enzymes may be a novel approach for the deve lopment of antitrypanosomatid drugs provided that sufficient parasite versu s host selectivity can be obtained. Guided by the crystal structures of hum an, T. brucei, and Leishmania mexicana glyceraldehyde-3-phosphate dehydroge nase, we designed adenosine analogs as tight binding inhibitors that occupy the pocket on the enzyme that accommodates the adenosyl moiety of the NAD( +) cosubstrate. Although adenosine is a very poor inhibitor, IC50 approxima te to 50 mM, addition of substituents to the 2' position of ribose and the N-6-position of adenosine led to disubstituted nucleosides with micromolar to submicromolar potency in glyceraldehyde-3-phosphate dehydrogenase assays , an improvement of 5 orders of magnitude over the lead. The designed compo unds do not inhibit the human glycolytic enzyme when tested up to their sol ubility limit (approximate to 40 mu M). When tested against cultured bloods tream T. brucei and intracellular T, cruzi, N-6-(1-naphthalenemethyl)-2'-(3 -chlorobenzamido)adenosine inhibited growth in the low micromolar range. Wi thin minutes after adding this compound to bloodstream T, brucei, productio n of glucose-derived pyruvate ceased, parasite motility was lost, and a mix ture of grossly deformed and lysed parasites was observed. These studies un derscore the feasibility of using structure-based drug design to transform a mediocre lead compound into a potent enzyme inhibitor. They also suggest that energy production can be blocked in trypanosomatids with a tight bindi ng competitive inhibitor of an enzyme in the glycolytic pathway.