Kinases, Assays and Inhibitors

Streamline Your Kinase Profiling Workflow by Integrating Bioluminescent Kinase Strips with a Multimode Detection System

Hicham Zegzouti, Ph.D. December 2015

[email protected] Promega Corporation Outline

Streamline Your Kinase Profiling Workflow 1. ADP-Glo Assay applications 2. Kinase Profiling Systems (Strips) 3. Automation using the strips 4. Signal generation and SMART data analysis 5. Profiling Data examples and validation

Gilson PIPETMAX

Promega Corporation 2 Kinase Research: parts of the equation

Kinase families (targets)

Kinase Assays (ADP-Glo)

Kinase Inhibitors (Drugs) Pharmaceuticals

Promega Corporation 3 Importance of Kinases in Drug discovery

US Marketed Small Molecule Kinase Inhibitors

• Kinases are the largest target in the drug discovery market • Diseases targeted: Oncology, Inflammation, Cardiovascular, Neurological (AD, PD), Diabetes… Two way paradigm Small and Large Screening Selectivity Potency Kinase Profiling Dose responses Looking for more? Promega Corporation 4 One assay platform - many applications

High-Throughput ADP-Glo™ is a Universal in vitro Screening Biochemical Assay for all types of Kinase Studies

Mode of action studies

ADP-Glo™ Assay Kinase inhibitor Platform profiling

Promega Corporation 5 Luminescent ADP detection assay requirements

ADP Detection

Bad

ATP Depletion

Promega Corporation 6 ADP-Glo™ Assay Format- 1:1:2

384-well plate 5µl kinase reaction + 5µl ADP-Glo™ Reagent 40 min. Incubation + 10µl Kinase Detection White Plates Format 1:1:2 (µl) Reagent 1,536-well 2.5/2.5/5 30-60 min. Incubation 384-well 5/5/10 10/10/20 96-well 25/25/50 50/50/100 Record Luminescence

Promega Corporation 7 Linearity of the ADP-Glo™ Assay

ADP conversion curves at different ADP/ATP concentrations

ADP-Glo™ can be used virtually with any ATP concentration

Promega Corporation 8 Comparison between ADP-Glo and Radioactivity Assay

Promega Corporation 9 Comparison between ADP-Glo and Radioactivity Assay

Vidugiriene J, et al (2009). Evaluating the utility of a Tai AW, Bojjireddy N, Balla T. (2011) A homogeneous and bioluminescent ADP-detecting assay for lipid kinases. Assay Drug nonisotopic assay for phosphatidylinositol 4-kinases. Anal Dev Technol. 7(6):585-97. Biochem. 417(1):97-102. Specific activity and response to known inhibitors determined by ADP-Glo™ correlated well with data from radiometric assay

Promega Corporation 10 Screening for Kinase inhibitors using luminescent ADP detection

Identification of true Kinase inhibitors vs false positives using Luminescence Kinase assay

Promega Corporation 11 Determination of inhibitor’s mechanism of action

PKA ATP Competitive inhibitor H-89

PKA ATP non Competitive inhibitor PKI Cellular Levels mM

Promega Corporation 12 ADP-Glo™ Assay in peer review A subset of ADP-Glo™ use citations for kinases ( a full list is available upon request)

A general framework for inhibitor resistance in protein kinases. Balzano et al. Chemistry & Biology (2011) 18(8):966-975 Evidence that Aurora B is implicated in spindle checkpoint signaling independently of error correction. Santaguida et al. The EMBO Journal (2011) 30, 1508-1519 Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for cdc7 kinase. Takagi et al. Combinatorial Chemistry & High Throughput Screening (2011) 14(8):669-687 A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases Tai et al. Anal Biochem. (2011) 417(1):97-102 Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1… Yang et al. Nucleic Acid Research 2012, 40(19):9621-9632 Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2… Ahmad et al., J Biomol Screen. 2012 Nov 7 Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells Antczak et al., ASSAY and Drug Development Technologies 2012, 10(1): 24-36. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability Luo et al., PNAS, 2012 vol. 109 (8), 2860-2865 .

Promega Corporation 13 Diverse kinase-substrate combinations with ADP-Glo

Protein Kinases

Non- Classical Kinases

ADP-Glo™ Kinase Assay detects the activity of any Kinase regardless of the substrate chemical structure Promega Corporation 14 Kinase Profiling Data Generated in Many Ways Existing profiling platforms - Fee-for-service model -

DiscoverX Reaction Biology Carna Biosciences Attractive model but comes with challenges Large panel kinase profiles may not be performed routinely

Promega Corporation 15 Issues Related to Kinase Profiling Current Challenges in creating selectivity profiles for Kinase inhibitors

 Addressing confidentiality issues related to outsourcing.

 In house profiling requires kinase assay developments and rigorous optimization which can be time consuming.

 The lack of available enzymes to do the profiling.

 Generating Selectivity profiles is costly and time consuming.

 Immediate accessibility of the data upon completion of Profiling.

There is a need for an easy and flexible solution that allows kinase selectivity profiles to be generated routinely without the time and cost challenges.

Promega Corporation 16 Addressing the kinome with large kinase panel KES: a complete kinase solution

Broad Human Kinome coverage with 174 Kinase Enzyme Systems

Promega Corporation 17 Profiling inhibitors against a subset Kinase panel with ADP-Glo Kinase Assay

EGFR EGFR

HER2/4

p38α

PI3K α/β/δ

Promega Corporation OK for small panels 18 Optimizing enzyme concentration for kinase inhibitor studies

Kinases need to be titrated to find the optimal amount to be used

ADP10%≈2.5ng

SB of 10 = 0.7ng

Determination of SB and % conversion values: kinase amount needed to generate a signal-to-background ratio of >=10 and optimal % ATP conversion.

Promega Corporation 19 Large kinase profiling data vs. routine small selectivity panels

Did we achieve our goal of ease of use and flexibility?

 Costly and time consuming

 Extensive assay development required.

Promega Corporation 20 Developing a simple kinase profiling solution Choosing the right kinases for flexible and targeted inhibitor profiling

Profiling with ADP-Glo platform made simple

• Ready to profile with simple protocol (No development time required) • Cost effective • Quick results with no compound sending out

Promega Corporation 21 Bioluminescent Kinase Selectivity Profiling Systems (Strips)

Promega Corporation 22 Kinase Selectivity Profiling Systems CMGC-1 example

Kinase Profiling strips manufactured by Signalchem to Promega specifications

2 strips

5µL Kinase Strip, 50X (some strips may contain 10µl) 2 strips

45µL Substrate/Co-Factor Strip

• 1.5mL • 25µL 5X Reaction DTT, 0.1M • Certificate of Analysis Buffer A Promega Corporation 23 Kinase profiling Systems: 3 types

Family based strips (14) General panel Custom panel

Kinase platform- CNRS Roscoff

TK-1 TK-2 TK-3 TK-4 CMGC-1 CMGC-2 TKL-1 STE-1 CAMK-1 CAMK-2 AGC-1 AGC-2 OTHER/CK-1 OTHER-2 General Panel ROS-1 ROS-2 ROS-3 AMPK EGFR ABL1 AXL1 c-MER ERK2 CDK1/CyclinA2 ALK2 ASK1 CHK1 AKT1 PKCa Aurora A CDC7/DBF4 FGFR1 CDK2/CyclinE1 AKT1 A1/B1/G1 PDGFRa TNIK FLT1 AMPK HER2 BRK EPHA1 FGFR1 GSK3b CDK2/CyclinE1 ALK4 HPK1 CHK2 p70S6Kb PKCb II Aurora B EIF2AK2 JAK3 GSK3b PKCa A1/B1/G2 PDGFRb AKT1 JNK1 AMPK HER4 BTK FAK FGFR2 JNK1 CDK3/CyclinE1 IRAK4 MINK1 MAPKAPK2 PDK1 PKCd CK2a1 IKKb LCK p38a ROCK1 A2/B1/G1 LCK IKKb JNK3 AMPK IGF1R CSK ITK FGFR4 JNK3 CDK5/p25 MLK2 MST1 MARK1 CAMK2a PKA PKCe DNA-PK NEK2 SYK Aurora A A1/B1/G2 EGFR TBK1 TGFbR2

InsR FYN A JAK3 FLT1 p38a CDK5/p35 RIPK2 NIK MELK CAMK2g PKC PKCg CK1α1 NEK3 MINK1 CAMK4 CK2a1 BRK ABL1 CHK2 Aurora KDR LCK PYK2 FMS p38b CDK6/Cyclin D3 TAK1-TAB1 PAK1/CDC42 PASK CAMK4 PRKG1 PKCi CK1epsilon PLK1 PAK1/CDC42 CHK1 IKKb p38a SRC A Aurora PDGFRa LYN B SYK MET p38d CDK9/Cyclin K TGFbR2 PAK3 PIM1 DAPK1 ROCK1 PKCtheta CK1g1 TBK1 IRAK4 DAPK1 CK1a1 RIPK2 JAK3 B PKCmu PDGFRb SRC TRKA RET p38g CLK3 ZAK TNIK STK33 RSK2 PKCz VRK2 ULK1 TAK1-TAB1 MAPKAPK2 CK1g1 (PKD1) MINK1 SYK CK2a1

Catalog COD Relevant kinases organized in: • Family based strips for intra-family inhibitor promiscuity analysis. • General panel for quick selectivity assessment. • Multiple strips are used for large selectivity profiling.

Promega Corporation 24 Simple profiling protocol for flexible and targeted inhibitor profiling

Kinase Profiling Strip Systems:

 Simple protocol: Two dilution steps

 One-time use design: Eliminating multiple freeze/thaw cycles ensures optimal kinase activity per experiment.

 Flexible kinase inhibitor profiling: Each strip has enough material to profile:

• Dose response for 1 compound against the 8 kinases at once.

• 10 compounds at a single dose

 Easy-to-automate inhibitor profiling.

Promega Corporation 25 Simple reaction assembly

Video available at: http://www.promega.com/resources/multimedia/cell-signaling/automation-of-the-kinase-selectivity-profiling-systems/

Promega Corporation 26 Helping customers with data analysis

• Automatically analyze results using the KSPS SMART Protocols found at promega.com/tools or built into the GloMax Discover.

• Separate tools are available for both dose-response and single dose data. Raw data is imported or copied into the tool and calculations are automatically generated.

Kinase Selectivity Profiling Systems (KPS) Single Dose SMART Protocol

Promega Corporation 27 Single Dose SMART Protocol Raw data is imported automatically in the template. Click to analyze.

Promega Corporation 28 Single Dose SMART Protocol Answer simple questions about the experiment: Strip kind, inhibitors, etc…

Promega Corporation 29 Single Dose SMART Protocol Data generated in a format that can be published

Promega Corporation 30 Dose Response SMART Protocol Raw data is imported into the template. Click to analyze. Answer simple questions about the experiment: Strip kind, inhibitors concentration…

Promega Corporation 31 Dose Response SMART Protocol

Accurate IC50 data generated in a format that can be published

Promega Corporation J.D. Parsons, A high-throughput method for fitting dose–response curves using Microsoft Excel, Analytical Biochemistry, 360 (2007) 309-311. 32 Flexible profiling: One Dose or Dose response

Kinase Gefitinib Dasatinib Tofacitinib SB203580 Roscovitine PF-477736 Tozasertib Enzastaurin

AXL1 108 92 105 94 102 54 107 101 EPHA1 84 1 102 79 97 63 77 100 Single dose Fak 95 84 118 106 109 86 91 115 ITK 102 101 99 94 100 33 24 99

Profiling ST-3 JAK3 99 113 0 87 94 36 88 103 TK family TK PYK2 133 134 122 106 134 120 80 116 (Up to 8/strip ) SYK 104 72 84 87 94 34 77 103 TRKa 95 82 60 89 87 9 1 89 Inhibitor Concentration = 1 µM Selectivity Promiscuity

Tofacitinib Titration PF-477736 Titration 150 150 AXL1 AXL1 EPHA1 EPHA1 Fak Fak 100 100 Dose response ITK ITK JAK3 JAK3 Profiling 50 IC50 PYK2 50 PYK2 (Up to 2/strip) JAK3:4.2nM SYK SYK

% Activity Enzyme TRKA: 1910nM TRKA % Activity Enzyme TRKA 0 0 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 [Tofacitinib], nM [PF-477736], nM

• Enabling flexible Kinase Profiling with the Strip Systems. • Verified selectivity profiles against 8 kinases at once. 33 Promega Corporation 33 Large selectivity panels created with the profiling strips 64 Kinases with 8 compounds at a single dose profiling ADP-Glo™ profiling systems

Strip Kinase #

Gefitinib

Dasatinib

SB203580

Tozasertib

PF-477736 Tofacitinib

Enzastaurin Roscovitine

EGFR 99 104 43 2 64 102 94 103 HER2 87 106 102 18 106 89 97 101 HER4 96 104 64 28 13 103 101 98 • Selective and promiscuous compounds IGF1R 102 105 98 103 100 102 96 97

ST-1 ST-1 InsR 98 100 95 97 94 97 101 88 TK Family TK KDR 84 93 4 86 83 83 91 34 PDGFRa 94 84 1 62 2 90 93 36 PDGFRb 85 88 0 85 1 77 92 40 ABL1 98 102 27 97 0 106 107 20 are identified. BRK 54 105 36 73 4 115 105 92 BTK 99 99 33 83 5 101 111 46 CSK 95 105 41 97 3 99 102 81

ST-2 ST-2 FYN A 90 93 8 74 0 67 97 30 TK Family TK LCK 90 88 22 74 2 89 92 27 LYN B 95 91 10 75 0 97 96 78 SRC 89 96 10 90 0 98 91 52 AXL1 94 101 54 108 92 105 102 107 EPHA1 79 100 63 84 1 102 97 77 Fak 106 115 86 95 84 118 109 91 • Inhibitor potency compared to different ITK 94 99 33 102 101 99 100 24

ST-3 ST-3 JAK3 87 103 36 99 113 0 94 88 TK Family TK PYK2 106 116 120 133 134 122 134 80 SYK 87 103 34 104 72 84 94 77 TRKa 89 89 9 95 82 60 87 1 CDK1/Cyclin A2 111 109 89 95 85 112 64 102 members of the same family or different CDK2/Cyclin A2 99 101 64 111 99 77 24 96 CDK3/Cyclin E1 89 90 92 99 116 99 44 106 CDK5/p25 97 98 75 89 104 100 26 99

ST-4 ST-4 CDK5/p35 101 105 75 107 99 96 25 100 CDK6/Cyclin D3 104 96 79 106 111 93 84 102 CMGCFamily families in the kinome. CDK9/Cyclin K 110 72 83 100 93 104 41 110 CLK3 90 98 87 106 92 98 98 92 ERK2 97 105 20 103 99 109 99 102 GSK3b 55 -3 86 86 103 105 96 102 JNK1 84 107 95 89 99 104 107 97 JNK3 30 106 86 81 95 93 106 88

ST-5 ST-5 p38a 3 102 99 64 24 105 101 103 p38b 10 103 103 94 54 100 101 105

CMGCFamily p38d 105 105 95 105 101 95 107 109 p38g 94 104 98 112 101 103 104 107 PKCa 96 5 77 88 107 106 103 102 PKCb II 105 4 75 91 101 100 105 95 PKCd 97 11 77 71 106 90 97 92 PKCe 94 9 66 99 97 97 106 100

ST-7 ST-7 PKCg 100 14 37 89 96 78 101 89

AGCFamily PKCi 93 97 84 101 97 99 98 99 PKCtheta 102 -3 66 97 95 87 100 102 PKCz 101 92 85 108 98 95 95 116 Inhibitors tested at 1µM CHK1 97 97 0 95 96 105 100 101 CHK2 100 106 8 80 98 107 109 90 MAPKAPK2 103 106 100 91 98 101 106 98 MARK1 93 96 6 97 97 78 103 97

ST-8 ST-8 MELK 98 109 43 101 106 86 94 14 Inhibitors Gefitinib Dasatinib Tofacitinib SB203580 Roscovitine PF-477736 Tozasertib Enzastaurin PASK 98 99 90 101 111 93 96 92

CAMKFamily PIM1 108 70 65 96 112 99 96 104 PKCmu (PKD1) 97 98 76 82 102 98 97 97 Aurora A 99 108 4 102 95 80 105 4 Kinase EGFR Abl/Src family JAK p38 CDK CHK Aurora PKC Aurora B 93 111 3 95 94 94 105 2 CK2a1 94 115 86 103 108 105 105 99

Family DNA-PK 102 91 85 106 104 99 113 115

CK ST-9 ST-9 CK1a1 86 103 93 104 98 102 95 107 CK1epsilon 58 95 96 100 105 96 86 107

Other/ CK1g1 92 99 97 102 105 94 88 104 VRK2 91 103 96 103 107 99 94 109 >60% Activity 20-60% Activity <20% Activity Promega34 Corporation 34 Validation of data created with ADP-Glo™ Profiling Systems

Comparison of ADP-Glo™ profiling to published radiometric data ADP-Glo™ profiling systems Radiometric data1

* Strip Kinase Kinase #

Gefitinib

Dasatinib

SB203580

Tozasertib

PF-477736 Tofacitinib Dasatinib

Enzastaurin Roscovitine Tofacitinib Tozasertib

Roscovitine

EGFR 99 104 43 2 64 102 94 103 21 96 97 78 EGFR HER2 87 106 102 18 106 89 97 101 80 109 103 102 HER2 HER4 96 104 64 28 13 103 101 98 4 100 98 93 HER4 IGF1R 102 105 98 103 100 102 96 97 97 101 98 44 IGF1R

ST-1 ST-1 InsR 98 100 95 97 94 97 101 88 97 97 94 52 InsR TK Family TK KDR 84 93 4 86 83 83 91 34 103 106 97 85 KDR PDGFRa 94 84 1 62 2 90 93 36 2 104 98 71 PDGFRa Data generated PDGFRb 85 88 0 85 1 77 92 40 2 95 91 72 PDGFRb ABL1 98 102 27 97 0 106 107 20 3 101 102 14 ABL1 BRK 54 105 36 73 4 115 105 92 5 101 103 95 BRK BTK 99 99 33 83 5 101 111 46 2 99 83 99 BTK CSK 95 105 41 97 3 99 102 81 7 101 89 78 CSK

ST-2 ST-2 FYN A 90 93 8 74 0 67 97 30 2 94 106 72 FYN A with ADP-Glo™ TK Family TK LCK 90 88 22 74 2 89 92 27 0 80 81 12 LCK LYN B 95 91 10 75 0 97 96 78 2 99 105 63 LYN B SRC 89 96 10 90 0 98 91 52 4 100 107 27 SRC AXL1 94 101 54 108 92 105 102 107 61 98 100 44 AXL1 EPHA1 79 100 63 84 1 102 97 77 3 98 97 78 EPHA1 platform is Fak 106 115 86 95 84 118 109 91 103 103 96 100 Fak ITK 94 99 33 102 101 99 100 24 102 99 105 99 ITK

ST-3 ST-3 JAK3 87 103 36 99 113 0 94 88 93 2 97 92 JAK3 TK Family TK PYK2 106 116 120 133 134 122 134 80 98 100 100 95 PYK2 SYK 87 103 34 104 72 84 94 77 65 107 103 99 SYK consistent with TRKa 89 89 9 95 82 60 87 1 97 94 98 7 TRKa CDK1/Cyclin A2 111 109 89 95 85 112 64 102 96 96 83 99 CDK1/Cyclin A2 CDK2/Cyclin A2 99 101 64 111 99 77 24 96 92 86 24 88 CDK2/Cyclin A2 CDK3/Cyclin E1 89 90 92 99 116 99 44 106 92 92 58 95 CDK3/Cyclin E1 CDK5/p25 97 98 75 89 104 100 26 99 102 100 31 94 CDK5/p25

ST-4 ST-4 CDK5/p35 101 105 75 107 99 96 25 100 100 99 31 100 CDK5/p35 published CDK6/Cyclin D3 104 96 79 106 111 93 84 102 103 111 96 121 CDK6/Cyclin D3

CMGCFamily CDK9/Cyclin K 110 72 83 100 93 104 41 110 84 100 79 93 CDK9/Cyclin K CLK3 90 98 87 106 92 98 98 92 95 106 105 110 CLK3 ERK2 97 105 20 103 99 109 99 102 93 89 88 91 ERK2 GSK3b 55 -3 86 86 103 105 96 102 97 100 96 104 GSK3b potencies of JNK1 84 107 95 89 99 104 107 97 106 108 98 95 JNK1 JNK3 30 106 86 81 95 93 106 88 100 100 101 107 JNK3

ST-5 ST-5 p38a 3 102 99 64 24 105 101 103 37 93 96 97 p38a p38b 10 103 103 94 54 100 101 105 77 103 101 93 p38b

CMGCFamily p38d 105 105 95 105 101 95 107 109 99 94 97 90 p38d radioactivity-based p38g 94 104 98 112 101 103 104 107 100 97 95 96 p38g PKCa 96 5 77 88 107 106 103 102 103 79 87 82 PKCa PKCb II 105 4 75 91 101 100 105 95 89 88 96 90 PKCb II PKCd 97 11 77 71 106 90 97 92 100 75 102 98 PKCd PKCe 94 9 66 99 97 97 106 100 102 99 98 98 PKCe

ST-7 ST-7 PKCg 100 14 37 89 96 78 101 89 86 91 96 94 PKCg Kinome profiling.

AGCFamily PKCi 93 97 84 101 97 99 98 99 101 103 92 99 PKCi PKCtheta 102 -3 66 97 95 87 100 102 97 89 90 86 PKCtheta PKCz 101 92 85 108 98 95 95 116 101 92 100 76 PKCz CHK1 97 97 0 95 96 105 100 101 96 94 94 77 CHK1 CHK2 100 106 8 80 98 107 109 90 103 104 97 100 CHK2 MAPKAPK2 103 106 100 91 98 101 106 98 100 97 101 101 MAPKAPK2 MARK1 93 96 6 97 97 78 103 97 107 119 115 114 MARK1

ST-8 ST-8 MELK 98 109 43 101 106 86 94 14 81 79 85 53 MELK PASK 98 99 90 101 111 93 96 92 110 105 93 99 PASK

CAMKFamily PIM1 108 70 65 96 112 99 96 104 102 102 105 99 PIM1 PKCmu (PKD1) 97 98 76 82 102 98 97 97 99 97 99 102 PKCmu (PKD1) Aurora A 99 108 4 102 95 80 105 4 93 84 89 5 Aurora A Aurora B 93 111 3 95 94 94 105 2 93 99 95 5 Aurora B CK2a1 94 115 86 103 108 105 105 99 105 104 106 114 CK2a1

Family DNA-PK 102 91 85 106 104 99 113 115 N/A N/A N/A N/A DNA-PK

CK ST-9 ST-9 CK1a1 86 103 93 104 98 102 95 107 98 100 91 97 CK1a1 CK1epsilon 58 95 96 100 105 96 86 107 90 101 81 99 CK1epsilon

Other/ CK1g1 92 99 97 102 105 94 88 104 97 101 94 99 CK1g1 VRK2 91 103 96 103 107 99 94 109 98 100 107 107 VRK2 1- Anastassiadis, T. et al;. Nat. Promega35 Corporation Biotechnol. 2011, 29, 1039 >60% Activity 20-60% Activity <20% Activity 35 SB203580 – kinase inhibition map against 112 kinases p38a Target

Off target IC50 (nM) <1nM 1 – 10nM 10 – 100nM 100nM - 1uM 1 - 10uM > 10uM

Promega Corporation 36 Gefitinib – kinase inhibition map against 112 kinases

IC50 (nM) <1nM 1 – 10nM 10 – 100nM 100nM - 1uM 1 - 10uM > 10uM

Promega Corporation 37 Comparison to binding assay results

EGFR

HER2 1 - InsR Kinase Kinase Kinase Kinase TK KDR PDGFRb 1 13 25 37 ABL1 EGFR SRC CDK5 TNIK BRK BTK

2 2 14 26 38 - CSK HER2 Fak CLK3 Aurora A

TK FYN A LCK LYN B 3 InsR 15 SYK 27 RSK2 39 CK1e SRC

FAK 3 - SYK

TK 4 16 28 40

TRKA KDR TRKA PIM1 CK1g1 4

- FGFR1 TK FGFR2 5 17 29 41

JNK1 PDGFRa FGFR1 AMPK NEK2 1

- JNK3 p38a

CMGC p38b 6 ABL1 18 FGFR2 30 CAMK2a p38g

CDK2/Cyclin E1 2 - CDK5/p25 7 19 31 CDK5/p35 BRK JNK1 CAMK2g

CMGC CLK3

- 1 C RSK2

AG 8 20 32

1 K M BTK JNK3 RIPK2

CA PIM1 2

- AMPK A1/B1/G1 CAMK2a 9 21 33

CAMK CAMK2g CSK p38a ASK1

1 -

TK RIPK2 L ASK1

MST1 10 22 34 1

- FYN A p38b MST1 PAK1/CDC42 STE PAK3 TNIK 11 23 35 PAK1/CDC42

Aurora A LCK p38g 1

- CK1epsilon K

Other/C CK1g1

2 12 24 36

- he Ot NEK2 r LYN B CDK2 PAK3 Promega Corporation 38 Comparison to binding results – Selectivity Scores

KSPS IC50 Ambit Kd <10µM 14 10 <100nM 2 2 Total Matching SELECTIVITY SCORES Kinases in <10µM 0.3415 0.2439 Data Sets = 41 <100nM 0.0488 0.0488 SB203580 Gefitinib KSPS IC50 Ambit Kd <10µM 17 7 <100nM 2 1 Total Matching SELECTIVITY SCORES Kinases in <10µM 0.4146 0.1707 Data Sets = 41 <100nM 0.0488 0.0243 Promega Corporation 39 Comparison to binding results - SB203580

Kinase IC50, µM Kd, µM Kinase IC50, µM Kd, µM Kinase IC50, µM Kd, µM

EGFR 5.614 2.9 SYK NC NC AMPK NC NC

HER2 1.656 NC TRKA NC NC CAMK2a NC NC

InsR NC NC FGFR1 7.271 NC CAMK2g NC NC

KDR 2.908 NC FGFR2 NC NC RIPK2 0.046 2.1

PDGFRb 4.585 NC JNK1 3.32 1.3 ASK1 NC NC

ABL1 NC NC JNK3 0.474 0.045 MST1 NC NC

BRK 1.306 9 p38a 0.016 0.017 PAK1/CDC42 NC NC

BTK NC NC p38b 0.113 0.25 PAK3 NC NC

CSK NC NC p38g NC 1.7 TNIK 1.391 1.5

FYN A NC NC CDK2 NC NC Aurora A NC NC

LCK 2.958 NC CDK5 NC NC CK1e 0.935 0.22

LYN B NC NC CLK3 NC NC CK1g1 NC NC

SRC NC NC RSK2 NC NC NEK2 NC NC

FAK NC NC PIM1 NC NC 40 Promega Corporation 40 Comparison to binding results - Gefitinib

Kinase IC50, µM Kd, µM Kinase IC50, µM Kd, µM Kinase IC50, µM Kd, µM

EGFR 0.0012 0.0018 SYK NC NC AMPK NC NC

HER2 0.168 1.1 TRKA 2.171 NC CAMK2a NC NC

InsR NC NC FGFR1 0.629 NC CAMK2g 5.777 NC

KDR NC NC FGFR2 NC NC RIPK2 0.062 0.8

PDGFRb 0.6648 NC JNK1 NC NC ASK1 NC NC

ABL1 8.244 NC JNK3 6.832 2.3 MST1 NC NC MeasuringBRK 3.107 BindingNC affinityp38a of a compound1.509 NC may notPAK1/CDC42 always translateNC intoNC levelBTK of inhibition4.605 NC of thep38b kinase activity3.492 andNC some leadsPAK3 can beNC discardedNC orCSK missedNC as a resultNC p38g NC NC TNIK 4.546 NC FYN A 3.556 NC CDK2 NC NC Aurora A NC NC

LCK 4.195 1.1 CDK5 NC NC CK1e NC 2

LYN B NC NC CLK3 NC NC CK1g1 NC NC

SRC 7.032 5 RSK2 NC NC NEK2 NC NC

PromegaFAK CorporationNC NC PIM1 NC NC 41 L Y 3 0 3 5 1 1 T i t r a t i o n P I 3 K p 1 1 0 a / p 8 5 a A Z D 6 4 8 2 T i t r a t i o n P I 3 K p 1 1 0 a / p 8 5 a

P I 3 K p 1 1 0 b / p 8 5 a 1 5 0 P I 3 K p 1 1 0 b / p 8 5 a 1 5 0

P I 3 K p 1 1 0 g P I 3 K p 1 1 0 g

t i

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t i

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3 - M e t h y l a d e n i n e T i t r a t i o n L Y 2 9 4 0 0 2 h y d r o c h l o r i d e T i t r a t i o n P I 3 K p 1 1 0 a / p 8 5 a Profiling kinases in strips to identify compound selectivityP Itowards3 K p 1 1 0 b / p 8 5 a 1 5 0 1 5 0

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3 - M e t h y l a d e n in e , n M L Y 2 9 4 0 0 2 h y d r o c h l o r i d e , n M Simultaneous profiling compounds against protein and lipid kinases.

Profiling PI3Kγ inhibitor Profiling PI3Kγ inhibitor

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Simultaneous Profiling of Protein and Lipid kinases identifies promiscuity of PI3K specific inhibitor towards other protein kinases such as CK2α1.

Promega Corporation 43 Potential Off-target effects of selective compounds

AS252424

PI3K IC50=20nM CK2 IC50=20nM

Simultaneous Profiling of Protein and Lipid kinases identifies promiscuity of PI3K specific inhibitor towards other protein kinases such as CK2α1.

Promega Corporation 44 Potential Off-target effects of selective compounds

Use of AS605240 compound

CK2 is highly inhibited by PI3Kγ compounds suggesting that their effects on inflammatory responses could be attributed to either CK2 or both PI3K and CK2 pathways?

Including lipid and protein kinases in profiling experiments may help prevent any misinterpretations related to the effects of compounds at the cellular level.

Promega Corporation 45 Technical Help: ADP-Glo™ Web Portal

ADP-Glo knowledge database contains:

ADP-Glo™ KES Application Notes: Serine- Threonine, Tyrosine, Lipid Kinases New! ADP-Glo Presentations, posters,… Protocols Promega Kinome Tree

App notes for Kinase Selectivity profiling using the strips (one for each Strip kind) New!

Check for updates at: www.promega.com/kinase

Promega Corporation 46 More reading: Publications

Book: H. Zegzouti and S. A. Goueli (eds.), Kinase Screening and Profiling: Methods and Protocols, Methods in Molecular Biology, vol. 1360, Springer New York 2016 New!

Chapter: H. Zegzouti, J. Hennek, S. Goueli, Using bioluminescent kinase profiling strips to identify inhibitor selectivity and promiscuity, Methods in Molecular Biology, 1360 (2016) 59-73. New! http://www.springer.com/us/book/9781493930722

Publication: J. Hennek, J. Alves, E. Yao, S. A. Goueli, H. Zegzouti (2015). Bioluminescent kinase strips: a novel approach to targeted and flexible kinase inhibitor profiling. Analytical Biochemistry, In Press. Coming Soon!

Promega Corporation 47 Advantages of Kinase profiling strips

Kinase Selectivity Profiling Strip Systems have the following advantages:

 Fast and simple reaction assembly: Two quick dilutions provide working stocks of kinases and substrate/co-factor solutions sufficient for 25 kinase reactions.

 One-time use design: Eliminating multiple freeze/thaw cycles ensures optimal kinase activity per experiment.

 Optimized kinase activity for inhibitor profiling: All kinases have been optimized to provide optimal ADP production when assayed at 10µM ATP.

 Formatted strips provide access to eight kinases at a time: Kinases from singular kinase families are grouped together for a more relevant selectivity profiles.

 Flexible kinase inhibitor profiling: Each strip has enough material to profile 10 compounds at a single dose or create a dose response for 1 compound against the 8 kinases at once.

Promega Corporation 48 Advantages of Kinase profiling strips

ADP-Glo™ Kinase Selectivity Profiling Systems generate data similar to the “gold standard” Radiometric assay

“Now even chemists can do kinase profiling?”

Promega Corporation 49 Summary

ADP-Glo™ Kinase Platform

1. ADP-Glo Kinase Assay 4. ADP-Glo Max Assay Kinase reactions up to 1mM ATP ATPase reactions up to 5mM ATP (ABC transporters,…)

2. Kinase Enzyme Systems 5. Lipid Kinase Systems ADP-Glo validated with Lipid 174 complete kinase assays substrates

Concentration Kinase ng/uL ERK2 8 GSK3b 3.12 Kinase profiling Systems JNK1 200 JNK3 25 p38a 820 well kinase strips for easy profiling p38b 2.5 p38d 30 p38g 6

Promega Corporation 50 Thank you

Questions? [email protected]

Promega Corporation 51 51