Structure-based approach to the identification of a novel group of selective glucosamine analogue inhibitors of Trypanosoma cruzi glucokinase

Abstract

Glucokinase and hexokinase from pathogenic protozoa Trypanosoma cruzi are potential drug targets for antiparasitic chemotherapy of Chagas’ disease. These glucose kinases phosphorylate D-glucose with co-substrate ATP and yields glucose 6-phosphate and are involved in essential metabolic pathways, such as glycolysis and the pentose phosphate pathway. An inhibitor class was conceived that is selective for T. cruzi glucokinase (TcGlcK) using structure-based drug design involving glucosamine having a linker from the C2 amino that terminates with a hydrophobic group either being phenyl, p-hydroxyphenyl, or dioxobenzo[b]thiophenyl groups. The synthesis and characterization for two of the four compounds are presented while the other two compounds were commercially available. Four high-resolution X-ray crystal structures of TcGlcK inhibitor complexes are reported along with enzyme inhibition constants (Ki) for TcGlcK and Homo sapiens hexokinase IV (HsHxKIV). These glucosamine analogue inhibitors include three strongly selective TcGlcK inhibitors and a fourth inhibitor, benzoyl glucosamine (BENZ-GlcN), which is a similar variant exhibiting a shorter linker. Carboxybenzyl glucosamine (CBZ-GlcN) was found to be the strongest glucokinase inhibitor known to date, having a Ki of 0.71 ± 0.05 μM. Also reported are two biologically active inhibitors against in vitro Trypanosoma cruzi culture that were BENZ-GlcN and CBZ-GlcN, with intracellular amastigote growth inhibition IC50 values of 16.08 ± 0.16 μM and 48.73 ± 0.69 μM, respectively. These compounds revealed little to no toxicity against mammalian NIH-3T3 fibroblasts and provides a key starting point for further drug development with this class of compound.

Diglycosyl diselenides alter redox homeostasis and glucose consumption of infective African trypanosomes

“…Although carbohydrates, natural and synthetic derivatives alike, constitute one of the largest and most variegated group of biologically active compounds, these moieties occur only in some nucleoside derivatives with trypanocidal activity, such as Genzyme 644131/MDL-738 ( Bacchi et al., 2009b ) or cordycepin and analogs ( Vodnala et al., 2013 ). Antitrypanosomal activities were recently recorded for auranofin (see below), a gold-containing thiosugar ( Ilari et al., 2012 ) and for some N -acyl-glucosamine derivatives ( D'Antonio et al., 2015 ). Particularly, carbohydrate metabolism deserves attention as a potential target for the inhibition of trypanosomes because these pathogens depend on glucose as a preferred source of energy ( Yorke et al., 1929 , Bringaud et al., 2006 ) and asbuilding block of several metabolic and structural macromolecules ( Creek et al., 2015 ).…”

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

“…The NfGlck structure is highly similar to that of TcGlck. The T. cruzi enzyme was noted for having a structure consistent with that described for E. coli glucokinase (EcGlck), a group A Glck, which is a member of the group A ribokinase enzymes (19). Like other group A enzymes, NfGlck was limited to using ATP as a phosphoryl donor.…”

“…The closest homolog by sequence in the PDB is the T. cruzi Glck (TcGlck) with 27% sequence identity. This protein is also the source of the closest structural homologs in the PDB, which are structures of the TcGlck in complex with β- D -glucose and ADP (PDB code 2Q2R, 1.7Å RMSD), in complex with CBZ-GLCN (5BRE, 1.8Å RMSD), in complex with BENZ-GLCN (5BRD, 2.0Å RMSD), HPOP-GLCN (5BRF, 2.0Å RMSD), and DBT-GLCN (5BRH, 2.0Å RMSD) (19). The structure of HsGlck (3FGU) superimposes with an RMSD of 3.0Å.…”

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

“…The NfGlck structure is highly similar to that of TcGlck. The T. cruzi enzyme was noted for having a structure consistent with that described for E. coli glucokinase (EcGlck), a group A Glck, which is a member of the group A ribokinase enzymes (10). Like other group A enzymes, NfGlck was limited to using ATP as a phosphoryl donor.…”

“…The closest homolog by sequence in the PDB is the T. cruzi Glck (TcGlck), with 27% sequence identity (Data File S1). This protein is also the source of the closest structural homologs in the PDB, which are structures of the TcGlck in complex with ␤-D-glucose and ADP (PDB entry 2Q2R, 1.7-Å root mean square deviations [RMSD]), in complex with carboxybenzyl glucosamine (CBZ-GLCN, 5BRE, 1.8-Å RMSD), and in complex with benzoyl glucosamine (BENZ-GLCN, 5BRD, 2.0-Å RMSD), hydroxyphenyloxopropyl glucosamine (HPOP-GLCN, 5BRF, 2.0-Å RMSD), and dioxobenzylthiophenyl glucosamine (DBT-GLCN, 5BRH, 2.0-Å RMSD) (10). The structure of HsGlck (3FGU) superimposes with an RMSD of 3.0 Å.…”