A Homogenous Luminescence Assay Reveals Novel Inhibitors for Giardia Lamblia Carbamate Kinase

Send Orders of Reprints at [email protected] Current Chemical Genomics, 2012, 6, 93-102 93 Open Access A Homogenous Luminescence Assay Reveals Novel Inhibitors for Giardia Lamblia Carbamate Kinase Catherine Z. Chen1, Noel Southall1, Andrey Galkin2, Kap Lim2, Juan J. Marugan1, Liudmila Kulakova2, Paul Shinn1, Danielle van Leer1, Wei Zheng1,# and Osnat Herzberg2,3,*,# 1National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA 2Institute for Bioscience and Biotechnology Research, University of Maryland 9600, Rockville MD 20850, USA 3Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA 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 use- ful for high throughput screening of large compound collection for identification of the inhibitors for drug development. Keywords: Carbamate kinase, Giardia, high throughput screening, assay development. INTRODUCTION evasion [11, 12]. Of the three enzymes involved in this pathway, the G. lamblia CK (glCK) was found to be the The protozoan parasite Giardia lamblia is a human pathogen with worldwide health impact of 280 million an- most active, with a maximal activity in cell free extracts that nual cases and 10000 deaths [1, 2]. The parasite causes the was 5- to 10-fold greater than those of arginine deiminase diarrheal disease giardiasis, which can lead to acute and and ornithine transcarbamoylase [10]. In addition, glCK has chronic diarrhea, malnutrition and failure to thrive. The stan- been shown to be essential for the viability of G. lamblia dard of care for giardiasis is either metronidazole or tinida- WB trophozoites [13]. The essentiality of glCK and the lack zole, with single course cure rates of 80–95%, while other of the arginine dihydrolase pathway in higher eukaryotes drugs such as paromomycin, albendazole and nitazoxanide place glCK as a potential anti-giadial target. are used to a lesser extent with similar and/or lower success CK catalyzes the reversible conversion of carbamoyl rates [3]. Current drugs are predominantly of the nitroimida- phosphate and ADP to ATP, ammonia and CO (Fig. 1). zole or benzimidazole classes and are susceptible to mecha- 2 Several assays are available for both the forward and reverse nisms such as down-regulation of pyruvate:ferredoxin oxi- CK reaction, but are limited by throughput. For the forward doreductase (PFOR) and mutations of !-tubulin [1]. Al- reaction, CO , one of three products, can be measured di- though these drugs are generally effective, treatment failures 2 and drug resistant strains have been increasingly reported [2, rectly as a gas released with limited assay throughput as a 4, 5], highlighting the need for novel drug targets. specialized apparatus is needed for the measurement [14]. Measurement of another product ATP has been achieved by The three-step arginine dihydrolase pathway, involving coupling the CK reaction to hexokinase and glucose-6- arginine deiminase, ornithine transcarbamoylase and carba- phosphate dehydrogenase to produce NADPH, which is then mate kinase (CK, EC 2.7.2.2), is used as a major source of detected by 340 nm absorbance [13, 15]. For the reverse CK energy generation by a number of anaerobic prokaryotes [6, reaction, two enzyme coupled assays are available: (1) the 7] and some parasitic protozoa such as trichomonads [8] and CP product can be assayed by coupling to ornithine transcar- G. lamblia [9, 10]. In G. lamblia, the arginine dihydrolase bamylase to produce citrulline, which is monitored col- pathway is not only a major source for energy production, orimetrically [15], and (2) the ADP product can be assayed but also plays a role in host colonization and immune system by coupling to pyruvate kinase and lactate dehydrogenase to oxidize NADH, which is monitored by 340 nm absorbance *Address correspondence to this author at the Institute for Bioscience and [13]. However, absorbance and colorimetric assays typically Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, MD 20850, USA; Tel: (240) 314-6245; E-mail: [email protected] have limited sensitivity and robustness and are not ideal for #Both authors contribute equally HTS in miniaturized plate formats. We have developed a 1875-3973/12 2012 Bentham Open 94 Current Chemical Genomics, 2012, Volume 6 Chen et al. O Carbamate kinase ( glCK) H2N O HO P O + ADP NH3 + CO 2 + ATP OH Carbamoyl Phosphate (CP) COOH O N Luciferase N + PPi + AMP +CO + Light ATP + S + O 2 S 2 S Mg 2+ S H O N H O N D-Luciferin Oxyluciferin Fig. (1). Assay Principles. new luminescence-based assay for the forward CK reaction were prepared as 4.47 mM stock solutions due to solubility that detects the production of ATP. This assay has been suc- limitations. All library compounds were serially diluted in cessfully miniaturized to 1536-well format and validated in a DMSO via 1:5 inter-plate titrations for primary screens, then pilot screen of 4096 small molecule compounds. formatted to 1536-well compound plates using an Evolution P3 system (PerkinElmer, Waltham, MA) [19]. After format- MATERIALS AND METHODS ting, compound plates were stored desiccated at room temperature for as long as 6 months when in use, or heat sealed Reagents and stored at -80 oC for long-term storage. Compound Adenosine 5’-diphosphate potassium salt (ADP, EMD cherry-picking for hit confirmation was done as follows: 10 Millipore, Darmstadt, Germany), Adenosine 5’-triphosphate mM compound solutions were thawed from storage, diluted disodium salt (ATP) and carbamoyl phosphate dilithium salt in DMSO as 12-point 1:3 intra-plate titrations, and then for- (CP, Sigma-Aldrich, St. Louis, MO) were purchased as matted to 1536-well compound plates. powder samples and dissolved in deionized water. AT- TM Luminescence-Based Carbamate Kinase Assay in 384- PLite assay kit (PerkinElmer, Waltham, MA) for quanti- Well Format tating amount of ATP was reconstituted according to vendor directions. Recombinantly expressed and purified glCK was All assay optimizations were carried out in white, 384- prepared as previously described [13]. All micro-titer plates well, low volume, white, solid bottom, medium binding used in this study were purchased from Greiner Bio One plates (Greiner Bio-one, Monroe, NC). Unless otherwise (Monroe, NC). noted, 4 µl/well of 1 nM glCK enzyme in assay buffer (20 Small Molecule Libraries and Compound Management mM Tris pH 8, 30 mM MgCl2, 0.1% BSA, 0.05% Tween 20) was dispensed with a BioRAPTR FRD Microfluidic Work- The library of pharmacologically active compounds station (Beckman Coulter, Brea, CA). Subsequently, the re- 1280 (LOPAC ) consists of a collection of 1,280 small mole- action was started by simultaneously dispensing 2 l/well of 1280 µ cules with known biological activities. The LOPAC li- substrate mix (600 µM ADP and 600 µM carbamate phos- brary has been extensively used for HTS assay validations phate in assay buffer) and 2 µl/well of the detection reagent [16, 17] and was purchased from Sigma-Aldrich. The NIH (ATPLiteTM) with the FRD Workstation. The reaction was Chemical Genomics Center (NCGC) Pharmaceutical collec- allowed to proceed at room temperature for 20 min, and then tion (NPC) was constructed internally through a combined the signal was read on a ViewLux CCD-based imaging plate effort of compound purchasing and custom synthesis [18]. reader (PerkinElmer, Waltham, MA) using a clear filter. At the time of screening, the NPC library consisted of 2816 small molecule compounds, 52% of which were drugs ap- The steady-state kinetics were determined with 2.5 nM proved for human or veterinary use by the United States glCK, 2 mM of the saturating substrate and the variable sub- Food and Drug Administration (FDA), 22% were drugs ap- strate at concentrations of 0.076, 0.23, 0.69, 2.1, 6.2, 18.5, proved in Europe, Canada or Japan, and the remaining 25% 55.6, 167 µM. The reaction signals were read at every min- were compounds that entered clinical trials or were research ute for 5 min to calculate the initial velocities (V0). The compounds commonly used in biomedical research. Com- Michaelis constant (Km) and Kcat values were calculated pounds from both libraries were obtained as powder samples from the V0 values as a function of the concentration of the and dissolved in DMSO as 10 mM stock solutions, except varied substrate, [S] by fitting with GraphPad Prism to the for several hundred compounds from the NPC library that equation V0 = Vmax [S] / (Km + [S]) to obtain the maximum A Homogenous Luminescence Assay Reveals Novel Inhibitors Current Chemical Genomics, 2012, Volume 6 95 Table 1. Assay Protocol Step Parameter Value Description 1 Reagent 2 ml 1 nM glCK in assay buffer 2 Reagent 23 nl Pin transfer compounds in DMSO 3 Time 15 min Room temperature incubation 4a Reagent 1 ml 600 mM CP, 600 mM ADP in assay buffer 4b Reagent 1 ml ATPLiteTM detection reagent 5 Time 20 min Room temperature incubation 6 Detection ViewLux plate reader luminescence mode velocity (Vmax) and the Km.

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