The bloodstream stage of Trypanosoma brucei and probably the intracellular (amastigote) stage of Trypanosoma cruzi derive all of their energy from glycolysis. Inhibiting glycolytic enzymes may be a novel approach for the development of antitrypanosomatid drugs provided that sufficient parasite versus host selectivity can be obtained. Guided by the crystal structures of human, T. brucei, and Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase, 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, IC 50 Ϸ 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 compounds do not inhibit the human glycolytic enzyme when tested up to their solubility limit (Ϸ40 M). When tested against cultured bloodstream T. brucei and intracellular T. cruzi, N 6 -(1-naphthalenemethyl)-2-(3-chlorobenzamido)adenosine inhibited growth in the low micromolar range. Within minutes after adding this compound to bloodstream T. brucei, production of glucose-derived pyruvate ceased, parasite motility was lost, and a mixture of grossly deformed and lysed parasites was observed. These studies underscore 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 binding competitive inhibitor of an enzyme in the glycolytic pathway.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD + interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N 6 -(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (6e), N 6 -(substituted-naphthalenemethyl)-2′-deoxy-2′-(substituted-benzamido)adenosine analogues were investigated. N 6 -(1-Naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (6m), N 6 -[1-(3-hydroxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (7m), N 6 -[1-(3-methoxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (9m), N 6 -(2-naphthalene-methyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (11e), and N 6 -(2-naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2-to 3-fold improvement over 6e and a 7100-to 25000-fold improvement over the adenosine template. IC 50 's of these compounds were in the range 2-12 μM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N 6 -(substituted)-2′-deoxy-2′-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N 6 and C2′ substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5′-amido substituents and found that 2′,5′-