Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

“…This result is different with that previously reported for the fused G6PD::6PGL protein from G. lamblia, where the enzyme reached a maximum value at pH 8.75. In this way, the optimal pH determined for TvG6PD::6PGL agrees with that of the other previously purified G6PDs, where pH values of 8.0 were found in G6PDs (in Homo sapiens, B. malayi, buffalo liver, camel liver, dog liver, Taenia crassiceps, Trypanosoma cruzy, Aspergillus Oryzae, Thermotoga maritima, Pseudomonas aeruginosa, and E. coli DH5α [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

“…With respect to the K m value for G6P determined in TvG6PD::6PGL protein from T. vaginalis, it is similar with the K m value reported for G6PD from T. brucei [43], and higher than the previously reported values in G6PDs from G. lamblia [23], P. falciparum [22], P. vivax [41], T. cruzi [40], and Homo sapiens [42], respectively. Moreover, as shown in Table 1, the fused TvG6PD::6PGL protein presented a high catalytic constant (k cat ) value (147 s −1 ) with respect to the fused G6PD::6PGL proteins from parasites such as G. lamblia (31 s −1 ) [23], P. falciparum (8 s −1 ) [22], and G6PD from T. cruzi (53 s −1 ) [40]. However, the catalytic constant (k cat ) value determined for the TvG6PD::6PGL protein was lower compared to human G6PD (233 s −1 ) (non-fused) [34] and non-fused G6PD from G. diazotrophicus (293,181 s −1 ) [44].…”

“…We observed that the Km value for NADP + obtained in TvG6PD::6PGL protein is similar with the Km value of NADP + on the G6PD from T. cruzi [40] and is higher than with the previously reported in G6PDs from G. lamblia [23], P. falciparum [22], P. vivax [41], and Homo sapiens [42]. With respect to the K m value for G6P determined in TvG6PD::6PGL protein from T. vaginalis, it is similar with the K m value reported for G6PD from T. brucei [43], and higher than the previously reported values in G6PDs from G. lamblia [23], P. falciparum [22], P. vivax [41], T. cruzi [40], and Homo sapiens [42], respectively. Moreover, as shown in Table 1, the fused TvG6PD::6PGL protein presented a high catalytic constant (kcat) value (147 s −1 ) with respect to the fused G6PD::6PGL proteins from parasites such as G. lamblia (31 s −1 ) [23], P. falciparum (8 s −1 ) [22], and G6PD from T. cruzi (53 s −1 ) [40].…”

“…Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0. [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

Potential Drug Targets in the Pentose Phosphate Pathway of Trypanosomatids

“…2A) were shown to inhibit the TbG6PDH in an uncompetitive fashion with Ki values about 6-fold lower than those reported for the human enzyme and exhibit in vitro anti-parasitic activity against bloodstream forms T. brucei [251]. However, DHEA failed to decrease T. cruzi in vitro growth, but its brominated derivatives, which are more potent inhibitors, displayed EC50 values in the low-mid micromolar range, similarly to benznidazole [289][290][291]. Interestingly, DHEA and EA did not inhibit L.…”