Flexibility in the Development of LANCE® Ultra Assays for Tyrosine Kinases using General and Specific Substrates Francesco Lipari, Mireille Caron, Anja Rodenbrock, Anne Labonté, Mireille Legault, Christian Fafard, Véronique Brechler and Philippe Roby PerkinElmer Life and Analytical Sciences, Montreal (QC), Canada H3J 1R4 General substrates Cat.# TRF0100 TRF0101 TRF0120 TRF0121 TRF0122 AD0066 AD0068 various CR97-100 S4400 A2383 15575-020 6007290 6005250 2103-0010 2100-4160 2100-5060 2100-5090 2100-5110 EC50 = 55 pM 50000 0 -∞ -13 -12 -11 -10 -9 10 Enzyme time course ULight-IRS-1 ULight-JAK-1 300000 250000 150000 EC50 = 388 pM 100000 50000 0 -8 INSR kinase 100000 1 00 00 0 350000 200000 Interference by Tyr kinase in detection -∞ -13 -12 -11 -10 -9 -8 -7 50000 40nM 20nM 10nM 5nM 2.5nM 30000 20000 10000 250000 -6 50 100 150 200 250 0 nM 0.25 nM 150000 0.50 nM 100000 1.0 nM 2.0 nM 50000 0 0 [JAK3] 200000 0 log [c-Src Kinase] (M) log [c-Src Kinase] (M) [INSR] 40000 300 0 25 50 8 6 00 00 4 00 00 75 100 125 + ATP - no inhibition - ATP - no inhibition 2 00 00 -∞ -13 Time (min) Time (min) ATP titration and staurosporine curve 8 00 00 0 -12 11 -8 -7 -6 -5 -4 -3 100000 50000 0 - ∞ -8 -7 LANCE Signal (665 nm) 100000 80000 60000 IC50 = 19 nM 40000 20000 0 - ∞ -10 -9 -8 -7 -6 6 -4 -3 -5 -4 200000 150000 40000 50000 0 + 10 μM Staurosporine 0 5 10 15 Well number 20 25 -9 -8 -7 -6 -5 -3 Z' = 0.87 50000 + 10 μ M Staurosporine 5 10 15 20 Receptor Km app = 4.6 μ M 20000 10000 -∞ IC50 = 61 nM 30000 20000 10000 -10 -9 -8 -7 25 Well number Summary: Src kinase assays using ULight-polyGT and ULight-polyGAT as substrates have been developed. The enzyme is more efficient in phosphorylation of ULight-polyGT, but the apparent Km for ATP in the presence of the different substrates is similar. Excellent precision is obtained in a Z` experiment for both substrates. -7 -6 -5 -4 -6 -5 -4 IC50 = 34 pM 30000 -∞ -13 -12 -11 -10 -9 50000 40000 Z'=0.74 30000 20000 + 10 μM Staurosporine 10000 0 0 5 10 15 Well number 20 25 -7 no straurosporine Z' = 0.82 + 10 µM straurosporine 0 -6 + ATP : EC50 = 13 nM - ATP : EC50 > 30 nM -∞ -13 -12 -11 -10 -9 -8 -7 -6 log [JAK3 Kinase] (M) ALK AXL EGFR EPHB4 FGFR1 FLT1 HER2 IGF1R INSR KIT MET PDGFRA RET TIE2 BRK BTK FAK ITK JAK1 JAK2 JAK3 LCK LYNa SRC TYK2 ZAP70 Kinase assay signal/background (+ATP / -ATP) [Kinase] (nM) ULight -poly GT ULight -poly GAT ULight -CDK1 ULight -IRS-1 ULight -JAK-1 10 7 7 2 3 8 10 5 13 1 2 2 0,5 4 13 2 2 17 0,5 7 11 5 6 24 1 2 13 1 7 25 1 15 18 1 3 15 20 7 8 1 1 1 0,5 8 7 1 4 2 20 6 12 2 6 3 5 10 14 1 1 4 1 9 15 1 2 5 1 12 15 1 4 19 0,5 3 10 1 6 20 1 6 15 2 3 19 5 14 10 1 1 3 0,5 11 15 3 1 2 20 8 18 1 1 2 0,5 8 10 2 3 7 20 11 15 1 4 24 5 5 9 2 9 20 10 3 9 1 1 23 0,5 16 19 5 1 15 1 9 11 8 2 18 20 8 12 3 1 9 20 9 5 1 7 1 20 4 9 1 1 13 S/B > 4 Summary: ULight-polyGAT gives acceptable S/B values for all kinases tested (26/26) and ULightpolyGT works for 21/26 kinases. The other substrates provide acceptable S/B values with certain kinases only. 12 ULight-JAK-1 60000 -8 Log [Staurosporine] (M) 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -7 0 General protocol: same as other kinase assays. For kinase reaction the concentration of kinase was chosen to avoid interference. Used 200 μM ATP, 100 nM ULight-substrate, incubation 4 hours. 10000 Z’-factor determination no Staurosporine -8 20000 20000 Log [Staurosporine] (M) 70000 -3 40000 0 -∞ -8 Log [ATP] (M) ULight-IRS-1 150000 100000 30000 0 -2 40000 80000 no Staurosporine 0 -4 50000 -4 9 0 -5 60000 0 - ∞ -10 ULight-polyGAT Z' = 0.87 20000 0 Staurosporine Inhibition IC50 = 68 nM 100000 200000 no Staurosporine -6 log [Staurosporine] (M) ULight-polyGT 60000 -∝ 40000 log [ATP] (M) Z’-factor determination 80000 20000 0 -2 LANCE signal (665nm) In LANCE Ultra kinase assays, the phosphorylation of a ULight-labeled peptide substrate is detected with a specific anti-phospho-peptide antibody (Ab) labeled with europium chelate molecules (Eu). ULight is an innovative low-molecular weight red-shifted dye. The binding of the Eu-antibody (donor) to the phosphorylated ULight peptide (acceptor) substrate brings both the donor and acceptor dye molecules into close proximity. Upon irradiation at 320 or 340 nm, the excited europium chelate transfers its energy to the nearby ULight dye molecule that will in turn emit light at 665 nm. The intensity of light emission is proportional to the level of the ULight peptide phosphorylation. -5 40000 70000 log [Staurosporine] (M) Assay Principle -6 Km app = 134 μM 60000 log [ATP] (M) log [ATP] (M) Staurosporine Inhibition ATP Titration 150000 Cytoplasmic -9 200000 Km app = 1.5 µM LANCE signal (665 nM) -∞ 250000 LANCE signal (665nm) 0 ULight-JAK-1 50000 80000 LANCE signal (665 nM) 50000 300000 LANCE signal (665 nM) Km app = 1.6 µM 100000 ULight-IRS-1 350000 LANCE signal (665nm) LANCE Signal (665nm) LANCE Signal (665 nm) ATP Titration 150000 -9 40000 Kinase assays with ULight substrates Type Kinase 200000 -10 60000 Summary: JAK3 Tyr kinase interferes in phospho-Tyr detection by binding to the anti-phospho-Tyr antibody, whereas INSR kinase does not interfere. ATP titration and staurosporine curve ULight-polyGAT ULight-polyGT -11 log [INSR Kinase] (M) JAK3 kinase 80000 General protocol: 10 µL 50 nM biotin-phospho-Tyr-peptide plus 0-30 nM kinase ± 100 μM ATP 5 µL of 4X Eu-Antibody diluted in detection buffer (final 2 nM) 5 µL of 4X ULight -SA diluted in detection buffer (final 50 nM) Incubate 60 min at RT and read on EnVision instrument Peptide sequences: IRS-1 (CKKSRGDYMTMQIG), JAK-1 (CAGAGAIETDKEYYTVKD) General Protocol: 5 µL of 2X INSR or JAK3 kinase in kinase buffer 5 µL of 2X ULight -Substrate/ATP mix (final 50 nM ULight-JAK-1 or 200 nM ULight-IRS-1) Incubate up to 240 min at RT 5 µL of 4X EDTA in detection buffer (final 10 mM EDTA) Incubate 5 min at RT 5 µL of 4X Eu-Antibody (AD0068) in detection buffer (final 2 nM) Incubate 60 min at RT and read on EnVision instrument General Protocol: 5 µL of 2X Src kinase in kinase buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20) 5 µL of 2X ULight -Substrate/ATP mix in kinase buffer (final 50 nM substrate) Incubate 60 min at RT 5 µL of 4X EDTA in detection buffer (final 10 mM EDTA) Incubate 5 min at RT 5 µL of 4X Eu-Antibody (AD0066) in detection buffer (final 2 nM) Incubate 60 min at RT and read on EnVision instrument LANCE signal (665nm) 100000 Kinase profiling LANCE signal (665nm) 150000 LANCE signal (665nm) LANCE Signal (665 nm) LANCE Signal (665 nm) 200000 LANCE Signal (665nm) 3 Supplier PerkinElmer LAS, Inc. 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ULight-polyGAT ULight-polyGT 5 Materials 7 Enzyme titration Lance Signal (665 nm) 2 Item ULight™-poly GT (4:1) ULight-poly GAT (1:1:1) ULight-IRS-1(Tyr983) ULight-JAK-1(Tyr1023) ULight-CDK1(Tyr15) Eu-anti-phospho-Tyr (PY20) antibody Eu-anti-phospho-Tyr (PT66) antibody Kinases LANCE Detection Buffer 10X Staurosporine ATP 0.5 M EDTA pH 8 White OptiPlate™-384 TopSeal™-A EnVision® Multilabel Reader Mirror: LANCE/DELFIA Dual Excitation Filter: UV2(TRF) 320 nm Emission Filter: Eu 615 nm Emission Filter: LANCE 665 nm 4 Specific substrates LANCE signal (665 nm) Introduction LANCE Signal (665 nm) 1 Of the 518 known human kinases, 90 are tyrosine (Tyr) kinases. Protein Tyr kinases play a key role in signal transduction and normal cell growth. They are also involved in numerous proliferative diseases like cancer and atherosclerosis, in addition to a number of autoimmune diseases. For these reasons, Tyr kinases are often targets for new drug discovery in many laboratories involved in basic R&D, disease and therapeutics research, and HTS. PerkinElmer now offers LANCE Ultra, a new ultra-HTS compatible TR-FRET technology highly suited for kinase inhibitor screening. Five new Tyr kinase substrates will soon be available in the Ultra format. ULight-poly GT (4:1) and ULight-poly GAT (1:1:1) are general substrates that can be phosphorylated nonspecifically by Tyr kinases and can be used to quickly set up a kinase assay for an enzyme with unknown physiological substrate. Alternatively, specific ULightpeptide substrates corresponding to the phosphorylation site sequence from the physiological protein are available: ULight-IRS-1(Tyr983), ULight-JAK-1(Tyr1023), and ULight-CDK1(Tyr15). The phosphorylated substrates are detected using Eulabeled anti-phospho-Tyr antibodies. HTS-compatible assays have been developed for each substrate. Tyr kinase profiling of the five substrates illustrates the applicability of the substrates for a wide variety of Tyr kinases. 10 20 30 40 50 Well number Summary: Kinase assays using ULight-IRS-1 and ULight-JAK-1 as substrates have been developed using INSR and JAK3 kinases, respectively. Excellent precision is obtained in a Z` experiment for both substrates. Summary and Conclusions • Two types of ULight-labeled substrates for Tyr kinases were developed: general and specific. • The applicability of the substrates to the development of HTS assays is illustrated by demonstrating enzyme kinetic data, inhibition curves, and Z` experiments. The assays developed provide ideal parameters for HTS – only 3-4 additions per well, low quantities of substrate and antibody, and Z` values > 0.7. • Tyr kinases were assayed in detection assays to determine the interference of the enzyme on antibody binding. The kinase interferes in antibody-based kinase assays due to the presence of phospho-Tyr residues on the kinases. The concentration of the kinase in the reaction must be adjusted accordingly. • The substrates were tested with a variety of receptor and cytoplasmic Tyr kinases. All the kinases were active with ULight-polyGAT as substrate and 21/26 kinases were active with ULight-polyGT. The applicability of the general substrates to a wide variety of kinases illustrates their advantage in setting up Tyr kinase assays in a short period of time. The specific substrates showed activity only towards certain substrates. ULight-CDK1 prefers kinases form the Src family: LCK, LYNa, and SRC. ULight-IRS-1 contains the YMxM motif and select kinases of both receptor and non-receptor type were active with this peptide. Interestingly, ULightJAK-1 is active with a variety of kinases. 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