Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: A study for antiparasitic drug design

Abstract

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.

Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

“…It is lower than that for DTNB (43.4 M Ϫ1 s Ϫ1 ) and 2-carboxyethyl methanethiosulfonate (MTSCE) (7.1 M Ϫ1 s Ϫ1 ), also previously reported under neutral pH conditions (24).…”

“…We already demonstrated the role of thiol-reactive compounds on the inactivation of GlTIM (22,24). Taking into account the mechanism of action of omeprazole on proton pump enzymes, which is based on its capacity to react with Cys, we performed enzyme inactivation assays using omeprazole against recombinant GlTIM and recombinant human TIM (HsTIM).…”

“…In a previous work, we generated by sitedirected mutagenesis the single mutants for all five positions occupied by Cys in GlTIM (24). The kinetic constants were calculated and reported (24) and were in the range of those of the wild-type GlTIM (wt-GlTIM); thus, mutagenesis did not affect the activity or stability of the enzymes (24). Those mutants were used in this work.…”

“…Every 10 min (from 0 to 120 min), aliquots were removed from the samples and assayed for residual activity in the standard reaction mixture. The data were adjusted to a monoexponential decay model, A R ϭ A 0 (e Ϫkt ), where A R is the residual activity at time t, A 0 is the activity at time zero, and k is the pseudo-first-order inactivation rate constant (24).…”

“…The enzymes of the glycolytic pathway in G. lamblia have been proposed as potential targets for drug design (21,22) because the organism lacks oxidative phosphorylation (23). Our group has demonstrated the mechanism of inactivation of the giardial glycolytic enzyme triosephosphate isomerase (GlTIM) by chemical modification with thiol-reactive compounds (22,24). In line with this, omeprazole (5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole) drew our attention because it is a drug that is safely used in humans and shows a molecular mechanism of action based on the chemical modification of cysteines (Cys).…”