Hexokinase is the first enzyme involved in glycolysis in most organisms, including the etiological agents of Chagas disease (Trypanosoma cruzi) and African sleeping sickness (Trypanosoma brucei). The T. cruzi enzyme is unusual since, unlike the human enzyme, it is inhibited by inorganic diphosphate (PPi). Here, we show that non-hydrolyzable analogues of PPi, bisphosphonates, are potent inhibitors of T. cruzi hexokinase (TcHK). We determined the activity of 42 bisphosphonates against TcHK, and the IC(50) values were used to construct pharmacophore and comparative molecular similarity indices analysis (CoMSIA) models for enzyme inhibition. Both models revealed the importance of electrostatic, hydrophobic, and steric interactions, and the IC(50) values for 17 active compounds were predicted with an average error of 2.4x by using the CoMSIA models. The compound most active against T. cruzi hexokinase was found to have a 2.2 microM IC(50) versus the clinically relevant intracellular amastigote form of T. cruzi, but only a approximately 1-2 mM IC(50) versus Dictyostelium discoideum and a human cell line, indicating selective activity versus T. cruzi.

In this work, we report a detailed kinetic analysis of the effects of three of these bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycosomes, peroxisome-like organelles that contain most of the glycolytic pathway enzymes in this organism. We also investigated the effects of the same compounds on glucose consumption by intact and digitoninpermeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium. The bisphosphonates investigated were several orders of magnitude more active than PP i as non-competitive or mixed inhibitors of TcHK and blocked the use of glucose by the epimastigotes, inducing a metabolic shift toward the use of amino acids as carbon and energy sources. Furthermore, there was a significant correlation between the IC 50 values for TcHK inhibition and those for epimastigote growth inhibition for the 12 most potent compounds of this series. Finally, these bisphosphonates did not affect the sterol composition of the treated cells, indicating that they do not act as inhibitors of farnesyl diphosphate synthase. Taken together, our results suggest that these novel bisphosphonates act primarily as specific inhibitors of TcHK and may represent a novel class of selective anti-T. cruzi agents.Chagas disease remains the major parasitic disease burden in Latin America, despite recent advances in the control of its vectorial and transfusional transmission (1, 2). Specific chemotherapy against its etiological agent, the kinetoplastid parasite Trypanosoma cruzi, is unsatisfactory because current drugs have very limited efficacy in the prevalent, chronic phase of the disease, and there are frequent serious side effects (3). Thus, there is an urgent need for safer and more potent drugs to treat this condition, and several rational approaches are being developed, exploiting key biochemical differences between the parasite and its mammalian hosts (3, 4). In this context, it is of note that T. cruzi, and several related kinetoplastid protozoa, have several unusual characteristics in their energy metabolism, which differentiates them from other eukaryotes. 1) Most of the glycolytic pathway is compartmentalized in peroxisome-like organelles termed glycosomes (5, 6). 2) The classic, allosteric modulators of the two key regulatory enzymes of the glycolytic pathway in mammalian, fungal, and bacterial organisms, hexokinase and phosphofructokinase, do not affect the kinetoplastid enzymes (7-9). This is associated with the absence of a Pasteur effect in these parasites, in addition to their flexibility in utilizing glucose or amino acids as carbon and energy sources (10 -12). 3) Kinetoplastid parasites contain large stores of inorganic pyrophosphate (PP i ) and other short chain polyphosphates, which are by far the most abundant high energy compounds in these cells (13-15).Although earlier work had shown that T. cruzi ATP-dependent hexokinase (TcHK) 2 was not inhibited by its main regulator in vertebrates, D-glucose 6-phosphate (7, 9, 16), more rec...

The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His) was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His) did not bind either [H] cAMP or [H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA Rα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.