Screening and Profiling Kinase Inhibitors with a Luminescent ADP Detection Platform

Kinases have become one of the most important and widely studied families of enzymes in biochemical and medical research. Kinases are a class of phosphotransferases that use ATP to catalyze the transfer of phosphate to a wide variety of cellular substrates. Many cellular processes such as cell signaling, cell division and growth, development, differentiation, and cell death are orchestrated by kinases (1) (2). Under normal physiological conditions, the regulation of kinases is tightly regulated. However, under pathological conditions, kinase activities can be dysregulated, and the disruption of the intracellular signaling networks governed by these enzymes leads to many diseases including cancer and inflammation (3) (4) (5) (6). Consequently, kinases have become an important target for drug discovery, resulting in successful kinase inhibitor-based drugs, such as Gleevec® (imatinib) and Tarceva® (erlotinib) (7) (8).

Given the importance of kinase inhibitors as potential therapeutics, researchers have focused on developing technologies that monitor changes in kinase activities and their modulation by small-molecule inhibitors (9). Isotopic-based methods for measuring kinase activity have long been considered the gold standard. However, due to safety concerns and waste management issues, alternative approaches have been sought (9) (10) . The ideal kinase assay platform should be able to: 1) detect activity of many enzyme classes 2) use universal substrates 3) detect activity at low substrate conversion rates 4) have a large dynamic range and low data variability 5) be amenable to high-throughput applications, and 6) reduce false-positive results. With these goals in mind, we have developed a luminescence-based ADP detection assay, ADP-Glo™ Kinase Assay, that measures kinase activity by quantifying the amount of ADP produced in the kinase reaction (11). This assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase). Since the ADP-Glo™ Kinase Assay is universal and can be used with any kinase/substrate combination (11) (12) (13), we applied it to screening compounds for kinase inhibitor activity and creating inhibitor selectivity profiles for several kinase families spanning the human kinome.

The ADP-Glo™ Kinase Assay (Cat.# V9101, V9102, V9103) is performed in two steps. First, after the kinase reaction, an ATP-depletion reagent is added to terminate the kinase reaction and deplete any remaining ATP, leaving only ADP. Second, a detection reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be converted to light using a coupled luciferase/luciferin reaction (Figure 1). The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity (11). The assay can be performed over a wide range of ATP concentrations (low micromolar to millimolar). This allows detection of small concentrations of ADP in the presence of large amounts of ATP (Figure 2, Panel A), producing very high signal-to-background (SB) ratios (Figure 2, Panel B, and Table 1). The robustness of the ADP-Glo™ Kinase Assay and suitability for high-throughput applications is evidenced by high Z´-factor values reported in previous studies (13).

The ADP-Glo™ Kinase Assay is as sensitive as radioactivity-based methods and more sensitive than fluorescence-based technologies (11) (12) (13). In order to lower the background and further improve the sensitivity of the assay, we increased the purity of our ATP to have less ADP contamination. To assess the importance of ATP purity on ADP-Glo™ assay sensitivity, we compared the signal-to-background ratios generated in an ADP-Glo™ assay using Promega Ultra Pure ATP and ATP from other suppliers. Table 1 shows that the Promega ATP outperforms ATP from other sources (S, T and G) by greatly improving ADP-Glo™ assay sensitivity with SB ratios that are 2–3 times higher than those produced using other commercial preparations.

We wanted to ensure that screening chemical libraries with the ADP-Glo™ Kinase Assay would not generate a high rate of compound interference and, subsequently, false responses, since multiple enzymes are used as components of the ADP-Glo™ Kinase Assay. To address this issue, we performed two small-scale screenings of the Library of Pharmacologically Active Compounds (LOPAC, which contains 1,280 compounds) at 10µM each, using two kinases, epidermal growth factor receptor (EGFR; Cat.# V3831) and lymphocyte-specific protein tyrosine kinase (LCK; Cat.# V2691). For comparison, an additional LOPAC screen was performed with the ADP-Glo™ reagents using a mixture of 20% ADP and 80% ATP at 30µM total nucleotide concentration without EGFR or LCK present. The results show that only the specific inhibitory effects of the compounds on both kinases can be detected using this assay (Figure 3, Panels B and C), while no effect was observed on the performance of the ADP-Glo™ Kinase Assay in the presence of the LOPAC chemical compounds (Figure 3, Panel A). Using the ADP-Glo™ Kinase Assay to screen for kinase inhibitors generates specific compound-inhibitory data with less compound interference. These data also confirm what has been shown by others regarding the reproducibility and resistance to chemical interference of the combination of Ultra-Glo® Recombinant Luciferase used in the ADP-Glo™ assay and the formulations of the assay reagents (14) (15).

The universal product of all kinase reactions is ADP. This makes the ADP-Glo™ Kinase Assay applicable to a variety of kinase reactions regardless of the nature of their substrates (peptides, proteins, alcohols, lipids, and sugars) with no need for substrate modification. We have shown that the assay detects the activity of several kinases with high sensitivity and can be used to generate small selectivity profiles of compounds using kinases from different families (11) (16). To expand upon this application using a larger set of kinases, we optimized the ADP-Glo™ Kinase Assay protocol for 70 Kinase Enzyme Systems (KES) that represent all families of the human protein kinome. Individual KES available from Promega contain a purified kinase, substrate, reaction buffer, and reaction buffer components. The data described in Table 2 show that all kinases were detected with high sensitivity. This demonstrates that the ADP-Glo™ Kinase Assay is ideal for performing kinase assays using low amounts of enzymes and shorter enzyme reaction times (e.g., <1 hour). These features are critical for accurate determination of kinase inhibitor IC50 values.

and inhibitor mode of action studies (15) (17) (18). Here, we demonstrate that a single kinase assay platform can address all the needs of kinase research over a wide range of kinase families as demonstrated with the Promega Kinase Enzyme Systems. The ADP-Glo™ Kinase Assay can be used to detect the activity of kinases with high ATP Km values based on the assay’s ability to generate high signal strength at low ATP conversion rates and a tolerance for high ATP concentrations (up to 1mM). These assay features are ideal for primary and secondary compound screening as well as profiling of lead therapeutic candidates with kinase inhibitor activity.

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