Adenosine 5’-diphosphate potassium salt (ADP, EMD Millipore, Darmstadt, Germany), Adenosine 5’-triphosphate disodium salt (ATP) and carbamoyl phosphate dilithium salt (CP, Sigma-Aldrich, St. Louis, MO) were purchased as powder samples and dissolved in deionized water. ATPLite^TM assay kit (PerkinElmer, Waltham, MA) for quantitating amount of ATP was reconstituted according to vendor directions. Recombinantly expressed and purified glCK was prepared as previously described [13Galkin A, Kulakova L, Wu R, Nash TE, Dunaway-Mariano D, Herzberg O. X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia Acta Crystallogr Sect F Struct Biol Cryst Commun 2010; 66(4 ): 386-90.]. All micro-titer plates used in this study were purchased from Greiner Bio One (Monroe, NC).

The library of pharmacologically active compounds (LOPAC¹²⁸⁰) consists of a collection of 1,280 small molecules with known biological activities. The LOPAC¹²⁸⁰ library has been extensively used for HTS assay validations [16Lea WA, Xi J, Jadhav A, et al. A high-throughput approach for identification of novel general anesthetics PLoS One 2009; 4(9 ): e7150., 17Titus SA, Li X, Southall N, et al. A cell-based PDE4 assay in 1536-well plate format for high-throughput screening J Biomol Screen 2008; 13(7 ): 609-18.] and was purchased from Sigma-Aldrich. The NIH Chemical Genomics Center (NCGC) Pharmaceutical collection (NPC) was constructed internally through a combined effort of compound purchasing and custom synthesis [18Huang R, Southall N, Wanget Y, et al. The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics Sci Transl Med 2011; 3(80 ): 80ps16.]. At the time of screening, the NPC library consisted of 2816 small molecule compounds, 52% of which were drugs approved for human or veterinary use by the United States Food and Drug Administration (FDA), 22% were drugs approved in Europe, Canada or Japan, and the remaining 25% were compounds that entered clinical trials or were research compounds commonly used in biomedical research. Compounds from both libraries were obtained as powder samples and dissolved in DMSO as 10 mM stock solutions, except for several hundred compounds from the NPC library that were prepared as 4.47 mM stock solutions due to solubility limitations. All library compounds were serially diluted in DMSO via 1:5 inter-plate titrations for primary screens, then formatted to 1536-well compound plates using an Evolution P3 system (PerkinElmer, Waltham, MA) [19Yasgar A, Shinn P, Jadhav A, et al. Compound Management for Quantitative High-Throughput Screening JALA Charlottesv Va 2008; 13(2 ): 79-89.]. After formatting, compound plates were stored desiccated at room temperature for as long as 6 months when in use, or heat sealed and stored at -80 ^oC for long-term storage. Compound cherry-picking for hit confirmation was done as follows: 10 mM compound solutions were thawed from storage, diluted in DMSO as 12-point 1:3 intra-plate titrations, and then formatted to 1536-well compound plates.

All assay optimizations were carried out in white, 384-well, low volume, white, solid bottom, medium binding plates (Greiner Bio-one, Monroe, NC). Unless otherwise noted, 4 µl/well of 1 nM glCK enzyme in assay buffer (20 mM Tris pH 8, 30 mM MgCl₂, 0.1% BSA, 0.05% Tween 20) was dispensed with a BioRAPTR FRD Microfluidic Workstation (Beckman Coulter, Brea, CA). Subsequently, the reaction was started by simultaneously dispensing 2 µl/well of substrate mix (600 µM ADP and 600 µM carbamate phosphate in assay buffer) and 2 µl/well of the detection reagent (ATPLite^TM) with the FRD Workstation. The reaction was allowed to proceed at room temperature for 20 min, and then the signal was read on a ViewLux CCD-based imaging plate reader (PerkinElmer, Waltham, MA) using a clear filter.

The steady-state kinetics were determined with 2.5 nM glCK, 2 mM of the saturating substrate and the variable substrate at concentrations of 0.076, 0.23, 0.69, 2.1, 6.2, 18.5, 55.6, 167 µM. The reaction signals were read at every minute for 5 min to calculate the initial velocities (V₀). The Michaelis constant (Km) and K_cat values were calculated from the V₀ values as a function of the concentration of the varied substrate, [S] by fitting with GraphPad Prism to the equation V₀ = V_max [S] / (Km + [S]) to obtain the maximum velocity (V_max) and the Km. Kcat was calculated from the ratio of V_max and the pMol enzyme used in each reaction.

The glCK assay was carried out in 1536-well, medium-bind, white, solid bottom plates (Greiner Bio-one, Monroe, NC) as listed in Table 1. Briefly, 2 µl/well of 1 nM glCK enzyme in assay buffer was dispensed with the FRD Workstation. Compounds dissolved in DMSO were added from a source plate to an assay plate at a volume of 23 nl/well by an automated pin-tool station (Wako-Kalypsys, San Diego, CA). The enzyme was allowed to equilibrate with compounds for 15 min at room temperature. Subsequently, the reaction was started with the simultaneous dispensing of 1 µl/well of substrate mix (600 µM ADP and 600 µM carbamate phosphate in assay buffer) and 1 µl/well of the detection reagent (ATPLite^TM) with the FRD Workstation. The reaction was allowed to proceed at room temperature for 20 min and the signal was read in the luminescence mode on a ViewLux plate reader.

Table 1

Assay Protocol

The counter-screen assay was carried out in 1536-well format by replacing the glCK enzyme with ATP product at a concentration that was experimentally determined to be generated by the glCK assay conditions used. Briefly, 2 µl/well of 20 µM ATP in assay buffer was dispensed using the FRD Workstation. Compounds dissolved in DMSO were added from a source plate to the assay plate at a volume of 23 nl/well using an automated pin-tool station, and was allowed to equilibrate with the solution for 15 min at room temperature. Then, the detection reagent ATPLite^TM diluted 1:2 with assay buffer was dispensed as 2 µl/well with the FRD Workstation. The plates were incubated at room temperature for 5 min before the signal was read on a ViewLux plate reader.

Z’ factor was calculated with the equation of Z’ = 1 – 3 (SD_Total + SD_Basal) / (Mean_Total – Mean_Basal), where SD_Total is the standard deviation of DMSO treated wells with 0.5 nM glCK, SD_Basal is the standard deviation of no-enzyme control wells treated with DMSO, Mean_Total is the mean signal of DMSO treated wells with 0.1 nM glCK and Mean_Basal is the mean signal of no-enzyme control wells treated with DMSO [20Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays J Biomol Screen 1999; 4(2 ): 67-73.]. Coefficient of variation (CV) was calculated with the equation of CV = SD_Total / Mean_Basal, expressed as a percentage. Data normalization and curve fitting was performed as previously described [21Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries Proc Natl Acad Sci USA 2006; 103(31 ): 11473-8.]. Briefly, raw plate reads for each titration point were first normalized relative to DMSO only wells (0% activity) and no enzyme control wells (100% activity), and then corrected by applying a pattern correction algorithm using compound-free control plates (DMSO plates). Concentration–response titration points for each compound were fitted to the Hill equation, yielding concentrations of half-maximal inhibition (IC₅₀) and maximal response (efficacy) values. Data from the counter-screen underwent the same initial analysis, with the exception that raw luminescence counts were normalized relative to DMSO only wells (0% activity) and wells lacking ATP (100% activity).

The coupled assay described previously [13Galkin A, Kulakova L, Wu R, Nash TE, Dunaway-Mariano D, Herzberg O. X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia Acta Crystallogr Sect F Struct Biol Cryst Commun 2010; 66(4 ): 386-90.] served as a confirmatory assay. The conversion of ADP + carbamoyl phosphate to ATP + ammonium carbamate was coupled to the conversion of NADP⁺ to NADPH using hexokinase, glucose-6-phosphate (G6P) dehydrogenase, and glucose. All chemical and biochemical components (except glCK) were purchased from Sigma-Aldrich. Briefly, the assay was performed in 96-well plates in 200 μL reaction solutions. 20 μL solutions containing 400 µM ADP, 400 µM carbamoyl phosphate, 30 mM MgCl₂, 10 mM D-glucose, 1 mM NADP, 10 units hexokinase, 10 units G6P dehydrogenase, and 20 mM Tris-HCl (pH 7.5) were dispensed into the wells. Solutions containing glCK and 20 mM Tris-HCl (pH 7.5) were incubated for 15 minutes with different concentration of inhibitors, such that the final enzyme and inhibitor concentrations after adding 180µL aliquots to the wells were 5 nM glCK and 0.002, 0.02, 0.2, 1, 2, 20, and 200 μM inhibitor. The reactions were performed at room temperature and the reaction progress was monitored at 340 nm (Δε = 6.2 mM^-1 cm^-1) using a SPECTRAmax spectrophotometer (Molecular Devices Corp, Sunnyvale, California). IC₅₀ values were calculated from the initial velocity data. Counter assays, omitting glCK and supplying ATP, were performed to confirm that the hexokinase and G6P dehydrogenase were not inhibited by the compounds.

In order to increase the assay sensitivity, a luminescence-based ATP content assay was applied to the forward CK reaction. The assay principles are illustrated in Fig. (1). The glCK enzyme converts the carbamoyl phosphate (CP) and ADP into ammonia, CO₂ and ATP. The amount of ATP produced is then monitored via coupling to a firefly luciferase enzyme in the ATPLite^TM assay kit. Using this assay format, the kinetics of glCK were experimentally determined at room temperature for each of the substrates (Fig. 2a, b). The Km values of ADP and CP were 58.3 ± 2.4 µM and 52.5 ± 4.3 µM, respectively. These values are slightly lower than previously reported by an absorbance-based enzyme-coupled assay [13Galkin A, Kulakova L, Wu R, Nash TE, Dunaway-Mariano D, Herzberg O. X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia Acta Crystallogr Sect F Struct Biol Cryst Commun 2010; 66(4 ): 386-90.]. However, the previously reported values were determined at 37^oC, while the current assay conditions room temperature. For the purpose of HTS, room temperature is preferred to prevent temperature gradients in assay plates associated with incubation at elevated temperatures. Therefore, to optimize the glCK assay for HTS, the steady-state kinetics was determined at room temperature.

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.

In order for the assay to remain sensitive to competitive inhibitors while still providing wide signal range, the concentrations of substrates were kept at 150 µM each. A time course study of enzyme activity showed a linear increase in enzyme activity (0.5 nM glCK) up to 30 min at room temperature (Fig. 2c). An incubation time of 20 min was then selected as optimal condition that falls within the linear region of the time course curve and yields sufficient signal.

The glCK assay was miniaturized to 1536-well plate format from the initial 384-well low volume plate by a 2-fold reduction in assay volume. Because DMSO is the solvent of all compounds screened, a DMSO plate was used initially to validate the 1536-well assay. The signal-to-basal ratio (S/B), Z’ factor and correlation of variation (CV) obtained from this DMSO plate test were 11.5-fold, 0.8 and 5.7%, respectively (Fig. 3). These values indicate that the assay in 1536-well plate format is robust for HTS.

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.

Using these optimized conditions, the glCK assay was used to screen both the LOPAC¹²⁸⁰ and NPC libraries in quantitative HTS (qHTS) format [21Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries Proc Natl Acad Sci USA 2006; 103(31 ): 11473-8.]. The screen was carried out at a 5-concentration titration ranging from 92 nM to 57.5 µM. To eliminate false positive compounds that affect the assay detection reagents, a counter-screen assay that consisted of the glCK reaction product ATP and the ATPLite detection reagent without the glCK enzyme was screened in parallel. From assay calibration against known ATP concentration titrations, we found that the glCK assay generated 20 µM ATP, which was applied to the counter-screen. The parallel screens of both the primary and counter-screen assays revealed concentration-response curves for all the primary hits, and provided a rich source of information for potency, efficacy, and false positive hits. Using the hit selection criteria of having IC₅₀ < 10 µM and > 70% maximal inhibition in the glCK assay with <30% maximal inhibition in the counter-screen assay, a total of 37 primary hits were selected with a hit rate was 0.9 %.

These 37 hits were cherry picked and retested in the glCK and counter-assay in a 1:3 titration for 12 concentrations for the measurements of IC₅₀ values. A total of 30 compounds out of 37 primary hits were confirmed (Table 2). Of the confirmed hits, three compounds showed sub-micromolar potencies (Fig. 4). It is interesting to note that several structural scaffolds were repeatedly found to be active. A class of chelator compounds that included disulfiram, thiram and sodium diethyldithiocarbamate inhibited glCK with IC₅₀ values of 0.5 µM, 4.1 µM and 16.3 µM respectively (Table 2). In addition a second class of molecules containing a polyphenol scaffold was also found among the hits (Table 2). Polyphenols belong to a well-known class of general kinase inhibitors, but their activity on carbamate kinase has not been reported elsewhere. Many of the polyphenolic inhibitors identified in this screen are dopamine mimetics, and have previously reported activity as dopamine receptors modulators. Since Giardia infection is limited to the intestine, this potential off-target activity could theoretically be eliminated by synthesizing analogs with less systematic absorption to increase local drug concentration in gastrointestinal tract.

Table 2

Confirmed Hits in the Luminescence Assay

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.

Of the 30 hits confirmed by the luminescence assay, 13 compounds were readily available for purchase and were tested with a secondary assay employing a different coupling enzymes and readout (UV spectroscopy). Of these, 4 compounds demonstrated good correlation between the two assays, with less than 2-fold difference in IC₅₀ values, 5 compounds showed less than 10-fold difference in IC₅₀ values, and one compound had a 17-fold difference between the two IC₅₀ values (Table 3). The latter three compounds were not confirmed in the absorbance assay, with IC₅₀ values evaluated as greater than 200 μM, the maximum concentration used in the assays (Table 3). The differences in inhibitor potency might be attributed in part to significant differences between the assays. For example, the substrate concentration was 150 µM in the luminescence assay versus 40 µM in the absorbance assay, and enzyme was concentration 0.5 nM in the luminescence assay versus 5 nM in the absorbance assay. The higher enzyme concentration in the secondary assay was necessary to detect the absorbance signal. Considering these differences, 9 out of 13 compounds showed IC₅₀ values with less than 10-fold difference between the two assays. Two of the top compounds, disulfiram and succimer showed good confirmation in potencies and have submicromolar IC₅₀ values in both assays (Table 3).

Table 3

Secondary Assay Results