Next Generation CSox-based Substrate Sensors for Continuous, Homogeneous and Quantitative Monitoring of Protein Kinase Activity

Peter Brescia1, Susan Cornell-Kennon2, Peter Banks1, Barbara Imperiali3 and Erik Schaefer2

1BioTek Instruments, Inc. Winooski, VT USA • 2AssayQuant Technologies, Inc. Marlborough, MA USA3Dept. of Biology & Chemistry, MIT Cambridge, MA USA

Abstract

Materials & Methods

PhosphoSens™ Chemistry & Workflow

BioTek Instrumentation & Settings

There are 518 human protein kinases, many of which have been shown to be dysregulated in disease states and therefore comprise greater than 30% of all drug development1-2. Harnessing chelation-enhanced fluorescence (CHEF) with the sulfonamido-oxine (Sox) chromophore in peptide or protein substrates has created a simple yet powerful method to measure the activity of protein kinases using a direct, single-step homogeneous, fluorescence-based assay for rapid and sensitive detection of serine/threonine and tyrosine kinase activities in a continuous (kinetic) format with both recombinant enzymes3-5 and with crude cell or tissue lysates6-11. This platform is ideal for elucidation of drug mechanism of action and is increasingly being applied earlier in the drug development process to address challenges and opportunities for next generation kinase inhibitors. Here we demonstrate the use of high-throughput peptide synthesis for degenerate Sox-based libraries to systematically identify improved substrates. Application of this approach to create robust assays for studying kinase activity and kinase inhibitor screening or characterization is presented.

A simple yet powerful kinase assay platform has been developed that allows continuous (kinetic) monitoring of kinase activity under optimal conditions including pH, selected metal ion cofactors, low to physiological (mM) ATP concentrations; and using optimized peptide substrates to enable both ATP competitive and ATP non-competitive (allosteric) kinase inhibitors to be selected and characterized. Assays can be performed using 96-, 384- and 1536-well plate formats and a wide range of sample types.

Figure 1. Chelation Enhanced Fluorescence (ChHF) mechanism for direct protein kinase activity sensing. Substrates are generated by standard solid-phase synthesis using Fmoc-Cys-Sox (+2 or -2 from the S/T or Y phosphorylation site), with flanking sequence on either side. Substrate phosphorylation and Mg(II) chelation creates the dramatic increase in fluorescence.

Figure 2. Fluorescence spectra of PhosphoSens Peptides. (A) Typical fluorescence changes upon phosphorylation of a peptide substrate for ERK1/2 MAPK: Ac-VPLL-pT-PG-[CSox]-RRG-COOH. (B) Excitation and emission spectra. The λExMax of chelated Mg(II) with the 8-hydroxyquinoline is 360 nm and the λEmMax is 485 nm.

Figure 3. Assay Workflow.

Materials were sourced and prepared per manufacturer recommendations. Briefly, the assay reaction was initiated by the addition of a master mix containing both a protein kinase and the CSox-peptide substrate to a well containing the final component needed to start the reaction. Typical final concentrations of each reaction component are as follows: 50 mM HEPES, pH 7.5, 1 mM ATP (or adjusted as needed), 1 mM DTT, 0.01% Brij-35, 0.5 mM EGTA, 10 mM MgCl2, 10 µM peptide substrate, 1-10 nM kinase (or adjusted as needed) and additional co-factors or additives (as needed).

The Sox-based peptide substrates (AssayQuant Technologies, Marlborough, MA) were resuspended to create a 1 mM (100x) stock. For substrate and ATP, a 10x working solution was prepared in ultrapure H2O just before use. A 10x DTT working solution was prepared from 1000x stock in ultrapure H2O just before use. Kinase reaction master mix was prepared per manufacturer instructions. Kinases were prepared as a 5x working concentration just before use in kinase dilution buffer and added to the wells to initiate reactions. Fluorescence measurements were taken kinetically every 2-5 minutes for up to 200 minutes at 30 ºC (assays can also be endpoint measurements by using 6M Gu-HCl).

Kinase titrations were prepared as an 11-point, 2-fold serial dilution using 1x kinase dilution buffer as well as a no-kinase control. Each dilution in the series represented 5x the final concentration of kinase in the reaction resulting in a concentration range that spans 3 log units (e.g., from 20 nM to 20 pM). Samples and standards were plated in triplicate with assays performed in Corning half-area 96-well, white, flat and round-bottom NBS microplates (Catalog No. 3642).

Figure 4. Synergy™ Neo2 Multi-Mode Microplate Reader is designed for speed and ultra high performance, incorporating BioTek’s patented Hybrid Technology™, with independent optical paths to ensure uncompromised performance. Continuously variable bandwidth quadruple monochromators, sensitive high- transmission filter-based optics and up to 4 PMTs provide ultra-fast measurements with excellent results. Advanced environment controls, including CO2/O2 control, incubation to 65 °C and variable shaking support live cell assays; cell-based detection is optimized with direct bottom illumination. Synergy Neo2 is controlled by integrated Gen5™ Microplate Reader and Imager Software.

A Synergy Neo2 was used for fluorescence intensity measurements in kinetic mode, reading from the top (excitation wavelength [λex] = 360 nm, emission wavelength [λem] = 485 nm).

Data Reduction

The background fluorescence was subtracted for each time point from the total fluorescence signal, both determined in triplicates, to obtain corrected relative fluorescence units (RFU). Corrected RFU values then were plotted vs. time.The slope of the initial linear portion of each curve was determined, which is the initial reaction rate (RFU/min., or converted to RFU/pmole kinase/min.). Reaction rates from linear or non-linear (kinases that exhibit a lag phase) fit of the data were generated using Gen5 software or by exporting the data to GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

Substrate Optimization

High-throughput methods were used to generate partially degenerate peptide libraries with the Sox fluorophore and then screened for increased reaction rates.

Reactions conditions were optimized and two rounds of selection were performed.

Fluorescent intensity data were collected every five minutes, and plotted as relative fluorescent units corrected for background (RFU, corrected) versus time.

Each line in the graph represents a different sequence (Figure 5).

Figure 5. Optimization of degenerate peptide sequences. Two rounds of selection were performed with partially degenerate peptide libraries for identification of optimized peptide substrates.

Enzyme TitrationKinases were serially diluted to achieve of range of concentrations (39 pM – 40 nM final) and then incubated with 10 µM AQT0099 substrate in a 50 mL reaction.

Fluorescent intensity data were collected every three minutes, and plotted as relative fluorescent units corrected for background (RFU, corrected) versus time.

Each line represents a different enzyme concentration (Figures 6A and 6C).

Velocity determinations were calculated for each reaction and plotted versus each enzyme concentration.

Figure 6. Representative data from a kinase-titration experiment. (A) EGFR WT tyrosine kinase with the AQT0099 peptide substrate, (B) Linearity across a range of EGFR WT concentrations, (C) EGFR(T790M/L858R) tyrosine kinase with the AQT0099 peptide substrate, and (D) Linearity across a range of EGFR(T790M/L858R) concentrations.

The data demonstrate linearity across a range of kinase concentrations from 10 nM to 160 pM for EGFR WT and from 1.3 nM to 39 pM for EGFR(T790M/L858R) (Figures 6B and 6D).

Kinase concentrations that provided a linear signal with time and resulted in <10% of the substrate being phosphorylated were used for subsequent experiments.

Z’ with Different [Enzyme] & Reaction Volumes

Sox-based tyrosine kinase assays were performed using the AQT0099 substrate (10 mM) and a range of EGFR concentrations (39 pM – 40 nM) in different assay volumes (25, 50, 75 µL) and assay precision was determined by calculation of Z’ values for each condition (Figure 7, Table 1).

A final reaction volume of 50 µL is recommended, however, the final choice is based on assay requirements.

Figure 7. Effect of assay volume on assay performance. Z’ values are plotted against EGFR concentrations at various assay volumes.

Table 1. Z’ Values for different final reaction volumes and EGFR concentrations. Bolded Z’ values are LLOQ’s (lowest concentration with CV < 20%).

Substrate Titration for Km & Vmax Determination

AQT0099 substrate was titrated using a 1.5-fold serial dilution (200 to 2.3 µM final concentration) in a final reaction volume of 50 µL and including a constant concentration of Wild-type or T790M/L858R EGFR.

Fluorescent intensity data were collected every three minutes and plotted as relative fluorescent units corrected for background (RFU, corrected) versus time. Background subtraction was achieved by subtracting RFU values from a no kinase control at each peptide concentration and time point.

Initial reaction velocities (slope of the line; RFU/second) were determined from the linear portion of each curve.

Figure 8. Representative data from a titration of the PhosphoSens Peptide Substrate AQT0099. Serially diluted substrate was incubated with 8 nM EGFR for 120 min at 30 ºC to determine reaction velocities (RFU Corrected/min) at each substrate concentration.

Figure 9. Serially diluted substrate was incubated with 1 nM EGFR(T790M/L868R) for 112 minutes at 30 ºC to determine reaction velocities (RFU Corrected/min) at each substrate concentration.

Conversion of Rates from RFU to µM/min

The same 1.5-fold serially diluted AQT0099 substrate concentrations (Figure 9) were incubated with 20 nM EGFR(T790M/L858R) to achieve complete phosphorylation (Figure 10).

A standard curve then was constructed by plotting the saturating RFU values for each curve versus the concentration of AQT0099 peptide substrate in the reaction (Figure 11).

The slope of the standard curve was determined and used to convert reaction rate velocities from Figures 8 and 9 to µM/second values by dividing the reaction velocities (RFU/second) by the slope of the phosphopeptide standard curve (RFU/µM) (Table 2).

Figure 10. Phosphopeptide standard curve: 20 nM EGFR (T790M/L858R) was used to completely phosphorylate all substrate and determine saturating RFU values at each substrate concentration to generate a phosphopeptide standard curve.

Figure 11. The slope for this curve was determined to be 621 ± 11 RFU Corrected/µM.

Table 2. Reaction Velocities (µM/min) were determined by dividing the reaction velocities from the kinetic progress curves (RFU Corrected/min) by the slope from the phosphopeptide standard curve (621 ± 11 RFU Corrected/µM).

The converted velocities then were plotted versus substrate concentration and the data fit to the Michaelis-Menten equation for determination of Km and Vmax (Figures 12A and 12B).

For EGFR the Km was determined to be 77 ± 1.9 µM and Vmax equal to 32 ± 0.004 µM/min and for EGFR (T790M/L858R) the Km was determined to be 92 ± 5.0 µM and Vmax equal to 0.55 ± 0.017 µM/min.

Figure 12. Data were fit with the Michaelis-Menten equation to determine the Km and Vmax for the EGFR (A) and EGFR (T790M/L858R) (B).

Inhibitor Characterization

Progress curves were generated for 3 nM EGFR or 1 nM EGFR (T790M/L858R) incubated with 20 µM AQT0099 in the presence of Tarceva (0 - 9.0 µM) for 150 minutes at 30 ºC to obtain RFU signals over time (Figure 13), which were then corrected by subtracting background fluorescence determined in control (no kinase) reactions.

Figure 13. Representative data from inhibitor dose-response titration.

Figure 14. Inhibition curves for EGRF and EGFR (T790M/L858R) in response to the potent kinase inhibitor Tarceva.

The IC50 value was determined from a plot of velocity (RFU Corrected/pmole/min) versus Tarceva concentration (each data point is the mean ± std dev) using a 4-parameter logistic curve fit (Figure 14).

For the EGFR, the IC50 value was determined to be 20 ± 0.29 nM, which is in the same range as an IC50 value of 40 nM reported by Kitagawa, et al. 2013, determined under similar conditions with 1 mM ATP.12

The IC50 value determined for the EGFR (T790M/L858R) was 7.6 ± 0.19 µM or 380-fold less sensitive to Tarceva, illustrating the resistance to the drug that develops in patients.

This platform is ideal for elucidating drug mechanism of action and is increasingly being applied earlier in the drug development process to address challenges and opportunities for next generation kinase inhibitors including determining Ki, kinact, IC50 & residence times, etc.13-27

Conclusions

1. The PhosphoSens CSox technology demonstrates improved performance over other methods to address the challenges of next generation protein kinase inhibitor discovery and characterization.

2. The ability to systematically develop Sox-based substrates with improved kinetic performance using degenerative CSox-peptide libraries was demonstrated for the EGFR. Using this approach, we have developed both highly-generic and highly-selective sensors.

3. Generic sensors allow screening of large number of purified recombinant kinases while highly-selective sensors enable quantitative kinase profiling in crude unfractionated cell and tissue lysates.

4. AssayQuant Technologies currently has validated kinase assays to individual Tyrosine and Serine/Threonine kinases.

5. Combined with the HTS capable Synergy Neo2, the platform allows for the investigation of kinase activation or profiling and elucidation of drug mechanism of action and potency, using a wide range of sample types.

INTELLECTUAL PROPERTY & LICENSING: Use of the sulfonamido-oxine (Sox) fluorophore to report kinase activity via chelation-enhanced fluorescence (CHEF) is covered by patents 10/681,427, 10/682,427, 07872278.2 (issued 2016) and 62,331,903 (pending) exclusively licensed by MIT to AssayQuant Technologies, Inc. PhosphoSens™ is a trademark of AssayQuant Technologies, Inc. REFERENCES: 1Fabbro, D. 2015, 25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations, Mol Pharmacol, 87, 766-775. • 2Roskoski, R., Jr. 2015, A historical overview of protein kinases and their targeted small molecule inhibitors, Pharmacol Res, 100, 1-23. • 3Shults, M. D.; Imperiali, B. 2003, Versatile fluorescence probes of protein kinase activity, J Am Chem Soc, 125, 14248-14249. • 4Shults et al. 2006, Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases, Anal Biochem, 352, 198-207. • 5Luković et al. 2008, Recognition-domain focused chemosensors: versatile and efficient reporters of protein kinase activity, J Am Chem Soc, 130, 12821-12827. • 6Shults et al. 2005, A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates, Nat Methods, 2, 277-283. • 7Stains et al. 2012, Interrogating signaling nodes involved in cellular transformations using kinase activity probes, Chem Biol, 19, 210-217. • 8Li et al. 2009, Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates, Anal Biochem, 384, 56-67. • 9Luković et al. 2009, Monitoring protein kinases in cellular media with highly selective chimeric reporters, Angew Chem Int Ed Engl, 48, 6828-6831. • 10Stains et al. 2011, A p38alpha-selective chemosensor for use in unfractionated cell lysates, ACS Chem Biol, 6, 101-105. • 11Peterson et al. 2014, Selective mitogen activated protein kinase activity sensors through the application of directionally programmable D domain motifs, Biochemistry, 53, 5771-5778. • 12Kitagawa et al. 2013, Activity-based kinase profiling of approved tyrosine kinase inhibitors. Genes Cells 18(2):110-22. • 13Desai et al. 2013, Rapid discovery of a novel series of Abl kinase inhibitors by application of an integrated microfluidic synthesis and screening platform, J Med Chem, 56, 3033-3047. • 14Akl et al. 2014, Olive phenolics as c-Met inhibitors: (-)-Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models. PLoS One 9: e97622. • 15Devkota et al. 2014, High-throughput screens for eEF-2 kinase. J Biomol Screen 19: 445-452. • 16Dumble et al. 2014, Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor. PLoS One 9: e100880. • 17Edupuganti et al. 2014, Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors. Bioorg Med Chem 22: 4910-4916. • 18He et al. 2014, Cyclometalated iridium(III)-beta-carboline complexes as potent autophagy-inducing agents. Chem Commun (Camb) 50: 5611-5614. • 19Paris et al. 2014, The spleen tyrosine kinase (Syk) regulates Alzheimer amyloid-beta production and Tau hyperphosphorylation. J Biol Chem 289: 33927-33944. • 20Sartini et al. 2014, Structure-based optimization of tyrosine kinase inhibitor CLM3. Design, synthesis, functional evaluation, and molecular modeling studies. J Med Chem 57: 1225-1235. • 21Schwartz et al. 2014. Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance. Proc Natl Acad Sci U S A 111: 173-178. • 22Silva et al. 2014, A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity. Anal Biochem 450: 63-69. • 23Tjin Tham Sjin et al. 2014, In vitro and in vivo characterization of irreversible mutant-selective EGFR inhibitors that are wild-type sparing. Mol Cancer Ther 13: 1468-1479. • 24Hagel et al. 2015, First Selective Small Molecule Inhibitor of FGFR4 for the Treatment of Hepatocellular Carcinomas with an Activated FGFR4 Signaling Pathway. Cancer Discov 5: 424-437. • 25Kuzmič et al. 2015, An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations. Anal Biochem 484: 82-90. • 26Cheng et al. 2016, Nitidine chloride suppresses epithelial-to-mesenchymal transition in osteosarcoma cell migration and invasion through Akt/GSK-3β/Snail signaling pathway. Oncol Rep. • 27Cheng et al. 2016, Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants. J Med Chem. 59: 2005-24.

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