Optimization of Luminescent Assay for Screening of Cyclin-dependent Kinase 2 Inhibitors Suthar and Patel: Luminescent Assay for Cdk2 Cyclin-dependent Kinases Are Most Extensively Studied Targets for Cancer Chemotherapy since the Tumor Cells Exhibit False Checkpoints and Can Proliferate Even If the G

found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes. Protein kinases are the validated drug targets for cancer and extensive efforts to develop kinase inhibitor for variety of cancers has lead to more than 40 inhibitors in clinical trials [1,2]. Current drive in cancer research has been towards the development of drugs which target the cell cycle progression. Among the key class of protein targets that are known to be cell cycle regulators are cyclin-dependent kinases (CDKs) [3]. Cyclins and CDKs are most extensively studied targets for cancer chemotherapy because the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised [4]. CDKs are involved in the molecular mechanisms at the point, which can overcome the barrier of checkpoints in cancerous cells [5]. Inappropriate activation of CDKs occurs through subunits mainly cyclin A, D and E [6]. So for tumor therapy CDKs are more important target at cell cycle level over signal transduction level [7]. Importance of the CDKs in cell cycle has driven interest in development of selective and potent inhibitors for overall blockade of cell cycle to achieve growth arrest [8]. Small molecule inhibitor (SMI) as CDK inhibitors seems to appear as attractive anti-cancer agents because of their selectivity, potency and cell permeability [9]. There are a number of assay technologies available for measuring kinase activity. Bioluminescent, fluorescent, and radiolabeled assays are examples of different types of technologies available for high-throughput applications, each offering their own advantages and disadvantages [10]. Bioluminescent methods are automation friendly and have low background, and the luminescent output is not affected by fluorescent compound interference. Availability of different assay biochemistry for bioluminescent methods at cost effective rate is a big advantage for screening large number of compounds [11]. Fluorescence based methods are

Protein kinases are the validated drug targets for cancer and extensive efforts to develop kinase inhibitor for variety of cancers has lead to more than 40 inhibitors in clinical trials [1,2] .Current drive in cancer research has been towards the development of drugs which target the cell cycle progression.Among the key class of protein targets that are known to be cell cycle regulators are cyclin-dependent kinases (CDKs) [3] .Cyclins and CDKs are most extensively studied targets for cancer chemotherapy because the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised [4] .CDKs are involved in the molecular mechanisms at the point, which can overcome the barrier of checkpoints in cancerous cells [5] .Inappropriate activation of CDKs occurs through subunits mainly cyclin A, D and E [6] .So for tumor therapy CDKs are more important target at cell cycle level over signal transduction level [7] .
Importance of the CDKs in cell cycle has driven interest in development of selective and potent inhibitors for overall blockade of cell cycle to achieve growth arrest [8] .Small molecule inhibitor (SMI) as CDK inhibitors seems to appear as attractive anticancer agents because of their selectivity, potency and cell permeability [9] .
There are a number of assay technologies available for measuring kinase activity.Bioluminescent, fluorescent, and radiolabeled assays are examples of different types of technologies available for high-throughput applications, each offering their own advantages and disadvantages [10] .Bioluminescent methods are automation friendly and have low background, and the luminescent output is not affected by fluorescent compound interference.Availability of different assay biochemistry for bioluminescent methods at cost effective rate is a big advantage for screening large number of compounds [11] .Fluorescence based methods are cost effective, but inherent fl uorescent properties of compounds can interfere in measurement and produce false positive results.Higher assay backgrounds with fl uorescent assays also limit the dynamic range and sensitivity of the assay [12] .Radiolabeled methods are very sensitive, but the use of radioactive labels requires a regulatory compliance and associated costs for disposal [13] .
The aim of present investigation was to optimize the assay for CDK2/cylin A and CDK2/cyclin E with use of bioluminescence-based biochemical reaction for quantification of residual ATP, hence extent of phosphorylation and inhibition of phosphorylation.

Kinase concentration and reaction time optimization:
Two-fold serial dilutions of CDK2/cyclin (0.976, 1.953, 3.906, 7.812, 15.62, 31.25,62.5, 125, 250, 500 ng/well of CDK2/cyclin A or CDK2/cyclin E) were prepared in duplicate across the plate, using fixed amount of ATP (100 μM) and histone H1 (10 mg/ml) running from well 10 to well 1 in decreasing concentration.Assay dilution buffer (25 mM β-glycerophosphate, 20 mM MOPS pH 7.0, 5 mM EGTA, 1 mM DTT and 1 mM NaVO 3 ) was added to all wells to bring the contents to volume of 25 μl.Content was mixed using shaker and incubated for different time interval (5, 10, 15 and 20 min) for both CDK2/cyclin A and CDK2/ cyclin E) at room temperature.To each well, 25 μl Kinase-Glo® plus reagent was added (equal to the volume of the kinase reaction mixture) at different time intervals (5, 10, 15 and 20 min).Content of plate was mixed and incubated at room temperature for 1 min to stabilize luminescence signal prior to measurement with luminescence plate reader (Glo-Runner, Turner Biosystem, USA) (fi gs. 1 and 2).

Substrate (histone h1) concentration optimization:
Substrate was common for both CDK2 complexes (CDK2/cyclin A and CDK2/cyclin E), was histone H1.Optimal kinase amount (250 ng/well CDK2/cyclin A and 125 ng/well CDK2/cyclin E) and reaction time (20 min for CDK2/cyclin A and CDK2/cyclin E)  from the previous experiment were used to determine optimal substrate concentration.Two-fold serial dilutions of kinase substrate (histone H1) were made across the plate using 250 ng/well CDK2/cyclin A or 125 ng/well CDK2/cyclin E with 100 μM ATP.For a control, same titration was performed without CDK2.The contents of plate were mixed and incubated at room temperature for 20 min for CDK2/cyclin A and CDK2/cyclin E. To each well, 25 μl of Kinase-Glo® plus reagent was added.Luminescence was recorded after 1 min of incubation, for stabilization (fi g. 3).

Enzyme Dil ution Buffer and Water effect on IC 50 of CYC202:
Three-fold serial dilution of CYC202 (R-ro scovitine/ seleciclib) ranging from 100 μM to 0.005 μM were made across the plate using 250 ng/well CDK2 / cyclin A or 125 ng/well CDK2/cyclin E. To each well 2.5 mg/ml histone H1 was added.Reaction was started by addition of 100 μM ATP.Contents were mixed and incubated for 20 min for both CDK2/ cyclin A and CDK2/cyclin E. To each well, 25 μl of Kinase-Glo® plus reagent was added.The plate was mixed and incubated at room temperature 1min to stabilize the luminescent signal.Luminescence was recorded in a plate reader (fi gs. 4 and 5).

Z'-factor analysis:
For Z' determin ation three plates were prepared with enzyme (250 ng/well CDK2 /cyclin A or 125 ng/ well CDK2/cyclin E) and three plates were prepared without enzyme to obtain the average of signal and average of background.Following equation was used to calculate the Z' of assay.Total 48 wells with enzyme (250 ng/well CDK2/cyclin A and 125 ng/well CDK2/cyclin E) and 48 wells without enzyme were prepared.Substrate histone H1 (2.5mg/ ml) was added to each well.Five microlitres of assay dilution buffer was added to each well.Five microlitres of ATP (100 μM) was added to each well to initiate the kinase reaction.Plates were incubated according to the predetermined reaction times (20 min for CDK2/cyclin A and CDK2/cyclin E).Twenty    1).

RESULTS AND DISCUSSION
For CDK2/cyclin A assay, SD signal = 42392.7,SD background = 452.3,M signal = 2651028 and M background = 1836.1.Therefore, Z' for CDK2/Cyclin A assay was found as 0.802007.As per the Eqn. 1, Z' of assay was calculated and that was 0.802 which was considered good.For CDK2/cyclin E assay, SD signal = 46551.09,SD background = 467, M signal = 628198.3and M background = 1894.4.Therefore, Z' for CDK2/cyclin A assay was found as 0.774 as per Eqn. 1.With Z' factor higher than 0.5 both assays are suitable for screening of inhibitors at different ATP concentrations using the Kinase-Glo ® Assay technology.This can help to determine selectivity and potency of CDK2 inhibitors.
Optimal reaction time was 20 min for both CDK2/cyclin A and CDK2/cyclin E. For both enzymes CDK2/cyclin A and CDK2/cyclin E optimal substrate concentration was 2.5 mg/ml histone H1.Difference in IC 50 of CYC202 was observed for water and EDB as a diluent.Difference in IC 50 of CYC202 was not negligible in case of CDK2/cyclin A optimized assay, however, difference was considerably higher for CDK2/cyclin E assay (Table

TABLE 1 : IC 50 OF CYC202 WITH EDB AND WATER AS A DILUENT (IN μM) IC 50 (μM) with Water
Difference in IC 50 of CYC202 was not negligible in case of CDK2/Cyclin A optimized assay, difference was considerably higher for CDK2/cyclin E assay.