Streamlining ATPase and Kinase Assay Development Using Direct ADP Detection. Biology at Work

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1 Streamlining ATPase and Kinase Assay Development Using Direct ADP Detection

2 Summary Background on Transcreener ADP Assay, differentiators, competitive advantages Optimizing assays for kinases, a rule based approach ATPase case study: p97 An alternative to DIY assay development

3 Four assays, thousands of targets HTS Assays

4 Homogenous immunodetection of nucleotides with three far red fluorescent readouts.

5 Selective Nucleotide Detection NH 2 N N O O O N N 3 HO P O P O P O OH OH OH ADP vs. ATP H H OH O H H OH mp X µm Competitor ATP ADP 1 ATP ADP Bottom Top LogEC HillSlope span EC selectivity

6 Highly selective ADP detection enables initial velocity kinase assays PKA, 1 µm ATP mp mu 15 mu 7.5 mu 3.8 mu 1.9 mu Time, min ADP produced, µm ATP/ADP Standard 1 µm ATP ([ADP 2 Ab] determined from EC85 line) mu PKA 15 mu 7.5 mu mu 1.9 mu Time, min 2 15 mp % ATP Conversion

7 Transcreener ADP 2 Assay: Direct detection of ADP means less chance for interference

8 Competition: Luciferase Based ADP Detection Acceptor Kinase Acceptor OPO 3 Step 1 camp AC ATP ADP CK ATP Step 2 Creatine-P Luc Luciferin + O 2 Oxyluciferin+ AMP + PP i + CO 2 hν 1 Pretreatment Enzyme, 2 Coupling Enzymes PT Enzyme Inhibitors = False Negatives Coupling enzyme inhibitors = False Positives Two step detection Jabs, et al (1978) Clin. Biochem. 11, (5)

9 Competition: Fluorescent Peroxide Detection Acceptor Kinase Acceptor OPO 3 ATP ADP PEP 1. Pyruvate Kinase Pi Pyruvate 2. Pyruvate Oxidase Ac-PO 3 3 Coupling Enzymes 3. Peroxidase Fluor H 2 O 2 59nM Amplex Red Charter, et al (26) J Biomol Screen 26; 11; 39

10 Comparison of ADP Detection Assays

11 Transcreener ADP 2 FP Assay: Overnight Reagent and Signal Stability mp Day Reagent Stability Control -8 C -2 C 4 C RT 37 C mp hr Signal Stability 1hr 4hr 8hr 24hr ADP (µm) ADP (µm)

12 Transcreener ADP 2 Assay: Nanomolar Sensitivity % ATP Conversion.1 µm 1 µm 1 µm 1 µm 1 µm ZAP7,.1 µm ATP 8.% ATP Conversion Z' =.74 mp ng/ml ZAP7 no enzyme control Replicate Number

13 ADP 2 Antibody: Lot to Lot Reproducibility EC 85 calculated from lot µm ATP 33 µm ATP 333 µm ATP 25 1 mp = 6% Conversion 25 1 mp = 4% Conversion 25 1 mp = 5% Conversion mp mp mp % ATP Conversion % ATP Conversion % ATP Conversion

14 Concordance with Radioassay (PKA) Huss, et al, 27, J. Biomol. Screening

15 Transcreener ADP 2 Assay: Equivalence of different readouts TRF (nm) FI (nm) Slope 1.13 ± ± Similar IC 5 values are observed with the FP, TR-FRET, and FI assays IC 5 FI (nm) 4 4 TRF(nM) FI(nM) IC 5 FP (nm) IC5 TRF (nm) Choice of detection format may be influenced by instrument availability, library composition, or personal preference. All three assays yield Z values of.7-.9 at 1% ATP conversion, have deck and signal stability in excess of 8 hours, and use far red fluors to minimize compound interference.

16 ADP 2 Assay Universal: Any Kinase, Any substrate, Any ATP concentration Direct detection, far red fluors: less interference Sensitive: low substrate consumption, use less enzyme Single addition, mix and read format: easy automation Three fluorescent readouts, instrument-validated: flexibility, confidence > 8hr reagent and signal stability: easy automation 16

17 Rule #1: Set your instrument up correctly

18 Instrument Validation Program Specifications: Z >.7, 1% conversion of 1μM ATP

19 Rule #2: Use the optimal ADP Ab concentration

20 Dynamic Range can be Tuned for Robust Initial Velocity Detection at any ATP Concentration ADP antibody concentration sets the dynamic range of the assay EC 85 is optimal for initial velocity detection; ie, a good 1% conversion Ab binding profile can be affected by reaction components, especially proteins Ab titrations in the presence of ATP EC 85 vs. [ATP} mp EC [ADP 2 Antibody] (µg/ml).1 µm ATP 1 µm ATP 1 µm ATP 1 µm ATP 1 µm ATP

21 Dynamic Range can be Tuned for Robust Initial Velocity Detection at any ATP Concentration ATP/ADP Standard Curves ([ADP 2 Ab] determined from EC85 line) mp 2.1 µm ATP.33 µm ATP 1 µm ATP µm ATP 1 µm ATP 1 33 µm ATP 1 µm ATP µm ATP 1 µm ATP % ATP Conversion Z` at 1% Conversion Detection Format 1 µm ATP/ADP 1 µm ATP/ADP 1 µm ATP/ADP Transcreener FP Transcreener TR-FRET Transcreener FI

22 Application: Optimizing ATP for Minimal Enzyme Usage [ATP] um Enzyme/rxn Z' Factor % Conversion mu mu mu mu mu.71 9

23 Rule #3: Find the right enzyme concentration General considerations: ATP at K m or below, acceptor substrate saturating and non-limiting Enzyme at 5 nm or less to enable accurate dose response curves with potent inhibitors Reactions run under initial velocity conditions: < 2% ATP consumption, linear over time

24 Optimizing enzyme concentration: Streamlined approach mp % % ATP Conversion EC 4 EC 85 EC 6-85 Use of enzyme at 6-85% provides a good assay window for initial velocity detection and allows accurate IC 5 determinations directly from raw fluorescence data Polarization IC 5 (nm) PKA, mu/rxn R square.92 Slope 2.5 ± ADP IC 5 (nm) In this window,ic 5 values determined from raw fluorescence data are directly proportional to those determined from product (ADP) formation.

25 1) Scouting enzyme/time dependence in kinetic mode: Rock 1µM ATP 25.4 mp nm Rock 1 3 nm Rock 1 1 nm Rock 1.78 nm Rock 1 Standard Curve ADP produced, µm Time, min Time, min Conclusion: Reaction is linear for 2 hrs out to at least 1nM Rock 1 Reaction conditions: 1 um ATP, 1 um S6 Peptide (AKRRLSSLRA) at 3 degrees. Buffer: 5 mm Tris (ph 7.5), 5 mm MgCl 2,.1% Brij 35, 1 % DMSO. Detection components- 2 nm Tracer/ 3 µg/ml of Antibody

26 2) Determining optimal enzyme concentration in endpoint mode: Rock 1µM ATP, 12 min.3 mp EC 8 =2 nm ADP produced, µm [ROCK1], nm [ROCK1], nm Conclusion: EC 8 concentration of 2 nm provides >15 mp signal and is within the linear velocity region.

27 Kinase Enzyme Optimization 3.5 mp 2 1 EC 8 PKA DAPK1 ABL1 ROCK1 Standard Curve ADP produced, µm % Conversion [E], nm [E], nm

28 Equivalence of endpoint and kinetic data End Point Km Determination Kinetic Km Determination.6.8 V initial (µmol/min).4.2 Vmax Km Vinitial (µmol/min) V initial (µmol/min) Vmax Km Slope Kemptide, µm Kemptide, µm

29 p97 ATPase Disease implications: Human dementia Amyotrophic lateral sclerosis (ALS) Parkinson's disease Multiple cancers ATP ADP P97 Uses ATPase to pull polypeptide chain into the cytosol from the ER where it is then degraded by the proteosome

30 FP and TR-FRET reagent optimization for p97 5µM ATP 3 5 µμ ATP 25 mp 2 1 EC 85 = 3 µg/ml Em 665 /Em EC 85 = 46 nm tracer ADP 2 antibody, µg/ml ADP HiLyte 647 Tracer, nm

31 FP and TR-FRET standard 5µM ATP 3 5 µm ATP/ADP Standard Curve 2 5 µm ATP/ADP Standard Curve mp 2 1 Em 665 /Em % Conversion of 5 µm ATP % Conversion of 5 µm ATP ADP, µm % Conv mp Z` ADP, µm % Conv Z`

32 P97 ATPase: FP and TR-FRET assay comparison mp Human, -ATP Human, +ATP Mouse, -ATP Mouse, +ATP Em 665 /Em Human+ATP Human-ATP Mouse+ATP Mouse-ATP [P97], ng/ml [P97], ng/ml mp EC 8 = 4 ng/ml mp= 14 units EC 8 = 2 ng/ml mp= 16 units [P97], ng/ml Human+ATP Human-ATP Mouse+ATP Mouse-ATP The Transcreener ADP 2 FP Assay (>14 mp shift) was the better choice for p97 ATPase with the particular enzyme preparations used. Both the mouse and human p97 enzyme preparations can be used for screening, requiring only picograms of enzyme/well. < 1% of the ATP was consumed with both p97 assays, insuring initial velocity detection.

33 Summary Background on Transcreener ADP Assay, differentiators, competitive advantages Rules for ptimizing assays for kinases, ATPases 1. Set up instrument correctly 2. Optimize Ab concentration 3. Choose enzyme concentration An alternative to DIY assay development

34 DIY Assay Development: The Ugly Reality Source reagents Screen and/or profile inhibitors eventually Empirically test a matrix of enzymes, substrates, detection reagents and assay conditions

35 An Alternative Reality... Define project with BellBrook scientists. Screen and/or profile using fully optimized conditions. Transcreener Assay Development Report for GTPases 1-4 Km concentration Z 1% conversion Signal 1mP Take a week off... while we develop your assay.

36 Acknowledgements Tom Zielinski Andy Kopp Meera Kumar Tony Klink Matt Staeben NIH: NS5982, CA11535, GM69258

37 Examples of enzymes validated in Assays ADP Protein Kinases (many) Protein substrates Peptide substrates Autophosphorylation Carbohydrate Kinases Ketohexokinase Hexokinase phosphofructokinase Lipid Kinases Sphingosine kinase Shikimate kinase Pantothenate kinase PI3K 6 isoforms Various ATP-utilizing enzymes Acetyl CoA carboxylase Glutamine synthetase ATP Citrate Lyase Viral Helicase RNA Triphosphatase Hsp 7, 9 RecA GDP Gα proteins Cdc42 H-ras Rho A Arf 1 Fucosyltransferases 1,3 UDP α-1,3 Galactosyltransferase Glucosylceramide Synthase Hepatic UGTs AMP/GMP PDEs - several Ub, SUMO Ligases NAD Synthetase Acyl CoA Synthetase Sialyltransferases (CMP) EPIGEN HMTs EZH2 G9a DOT1L MLL4 SET7/9SET8 SUV39H1 PRMTs PRMT1 PRMT3 PRMT4 PRMT8 DNMTs DNMT1 DNMT3