Enhanced Antigiardial Effect of Omeprazole Analog Benzimidazole Compounds.

Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 °C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14, and 17 µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M-1 s-1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Axenic cultivation and comparative phospholipase A2 activity of Giardia duodenalis in a serum-free medium.

Mammalian serum is essential for the growth of Giardia duodenalis cultivated under axenic conditions. Unfortunately, some factors present in bovine serum used as supplement in the culture medium may inhibit protozoal growth and activity. TYI-33-PACSR is a TYI medium supplemented with a serum replacement (PACSR) made up of Earle's amino acid solution, Diamond's vitamin-tween 80 mixtures and LCR (a lipid-cholesterol - rich mixture). PACSR was previously used in the culture media for axenic cultivation of Entamoeba histolytica and Trichomonas vaginalis. The main objective of this work was to demonstrate that TYI-33-PACSR is useful for axenic cultivation of G. duodenalis. Additionally, the activity of phospholipase A(2) (PLA A(2)) in the sub-cellular vesicular fraction (P30) of G. duodenalis grown in TYI-S-33 and TYI-33-PACSR was compared. All strains of Giardia grown in TYI-33-PACSR reached relative cellular densities of 91 to 95% compared to controls growing in serum-supplemented TYI-S-33 medium. Additionally, PLA A(2) activity was similar in the P30 sub-cellular fraction obtained from trophozoites growing in TYI-S-33 and TYI-33-PACSR. Thus, TYI-33-PACSR could be useful in analyzing the biological properties of G. duodenalis in the absence of serum.

Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing.

OBJECTIVES: In order to create a suitable model for high-throughput drug screening, a Giardia lamblia WB C6 strain expressing Escherichia coli glucuronidase A (GusA) was created and tested with respect to susceptibility to the anti-giardial drugs nitazoxanide and metronidazole. METHODS: GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and -- as assessed in a 96-well plate format -- to a panel of 15 other compounds to be tested for anti-giardial activity. RESULTS: GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting. CONCLUSIONS: G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia.

Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28- kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC(50) of 3 microm, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.