Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012:6:93-102.
doi: 10.2174/1875397301206010093. Epub 2012 Dec 31.

A homogenous luminescence assay reveals novel inhibitors for giardia lamblia carbamate kinase

Catherine Z Chen  1 , Noel Southall, Andrey Galkin, Kap Lim, Juan J Marugan, Liudmila Kulakova, Paul Shinn, Danielle van Leer, Wei Zheng, Osnat Herzberg
Affiliations

A homogenous luminescence assay reveals novel inhibitors for giardia lamblia carbamate kinase

Catherine Z Chen et al. Curr Chem Genomics. 2012.

Abstract

The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z' factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.

Keywords: Carbamate kinase; Giardia; assay development.; high throughput screening.

PubMed Disclaimer

Figures

Fig. (1)
Fig. (1)
Assay Principles.
Fig. (2)
Fig. (2)
Assay optimization. (a) Titration of ADP. The Km was determined with 2 mM CP and 2.5 nM glCK as 58.3 mM. (b) Titration of CP. The Km was determined with 333 mM ADP and 1.7 nM glCK as 52.5 mM. (c) The time course of the reaction was determined with 150 mM ADP, 150 mM CP and 0.5 nM glCK. The signal is linear up to 30 min incubation at room temperature.
Fig. (3)
Fig. (3)
Assay miniaturization. (a) Plate map for 1536-well format. Columns 1-2: no enzyme DMSO control, columns 3-4: 0.5 nM glCK DMSO control and columns 5-48: 0.5 nM glCK compound area. (b) Scatter plot of DMSO plate test. DMSO was pin-transferred as 23 nl/well as a solvent control.
Fig. (4)
Fig. (4)
Concentration response curves for (a) Disulfiram, with IC50 = 0.58 mM, (b) Gallic acid, with IC50 = 0.65 mM and (c) Succimer, with IC50 = 0.65 mM in the glCK assay.
See this image and copyright information in PMC

References

    1. Upcroft JA, Upcroft P. Drug susceptibility testing of anaerobic protozoa. Antimicrob Agents Chemother. 2001;45(6 ):1810–4. - PMC - PubMed
    1. Wright JM, Dunn LA, Upcroft P, et al. Efficacy of antigiardial drugs. Expert Opin Drug Saf. 2003;2(6 ):529–41. - PubMed
    1. McPhee SJ, Papadakis MAE. Current medical diagnosis & treatment 2010. The McGraw-Hill Companies, Inc; 2010.
    1. Farbey MD, Reynoldson JA, Thompson RC. In vitro drug susceptibility of 29 isolates of Giardia duodenalis from humans as assessed by an adhesion assay. Int J Parasitol. 1995;25(5 ):593–9. - PubMed
    1. Upcroft JA, Upcroft P, Boreham PF. Drug resistance in Giardia intestinalis. Int J Parasitol. 1990;20(4 ):489–96. - PubMed

LinkOut - more resources