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Quality Data. Quality Support.

Kinase HotSpotSM Over 451 kinases, assayed with gold-standard 33P radiolabeled technology. Radio-isotope quality data at fluorescent prices. Custom panels, assay conditions available. The largest selection of functional kinase assays from any service provider.

Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR: “Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. “Nature Biotech 2011; 29(11):1039-1045.

Methyltransferase HotSpotSM Methyl-Transferase assayed with gold-standard 3H radiolabeled technology. 28 HMT’s and DNMT’s available for screening and profiling. Exclusive assay services available in an important new target category.

Proprietary Methyltransferase ProteinsA growing list of HTS-validated active human recombinant HMT’s, including previously unavailable protein complexes exclusive to RBC. Check www.reactionbiology.com for a current list.

HDAC Spectrum Profile against every HDAC and SIRT isoform in this RBC exclusive panel. Class IIa specific substrate is available. HAT’s also available.

Protease RBC’s 70+ protease panel available for profiling and screening.

Deubiquitinase12 DUB enzymes available for profiling and HTS.

RBC offers a suite of services for early stage drug discovery that can allow researchers to expand their discovery horizons while controlling, even reducing, budgetary expenditures. RBC specializes in high quality enzymatic assay services with flexible conditions and fast turnaround.

All Assay services include: n Dedicated IC50 controls from known inhibitors for every targetn Custom assay conditions availablen Single dose or IC50 determinationn Complete confidentialityn High level scientific support to help interpret dataAnd More: n MOA studiesn Enamine compound library screens n Custom assay development n Cell based assays for HTS and Confirmationn CYP/hERG

For more information please visit: http://www.reactionbiology.com/

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Quality Data. Quality Support.Miniaturized 33P-Based Kinase Profiling and Screening Platform - 457 Kinases

The HotSpotSM

Advantage

Services Available:

n Kinome-wide profiling for: — selectivity determination — off-target interactions — and profiling-based drug discovery.

n Larger scale HTS

n Large scale single dose, duplicate profiling

n IC50 profiling — 5 or 10 dose with curve fitting

n ATP — competitive evaluation

n Compound Inhibition Mode

n Ki determination

n Custom and focused kinase panels

n Custom assay development

n Substrate determination

n Research collaborations

GOLD STANDARD33P-based functional assay avoids the false positives and negatives generated by other assay formats

HIGH QUALITY DATAZ’ > 0.7

HIGH REPRODUCIBILITYR2 > 0.95

LOW COSTHotSpotSM is priced competitively vs.fluorescence based assay services

FLEXIBLE CONDITIONSIC50, qHTS, HTS, variable ATPconcentration, customer-tailored conditions

NO SPECIAL SUBSTRATE LABELINGUsing both protein and peptide as substratesNo assay interfering labelingFreedom to use your specific substrate

NO COMPOUND INTERMEDIATE DILUTIONSReduces assay to assay variability

LOW COMPOUND CONSUMPTIONOnly nanoliters needed for assays

FULLY VALIDATEDAll assays come with DMSO as positive control, EDTA as negative control and IC50 standard kinase inhibitors

Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR: “Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. “Nature Biotech 2011; 29(11):1039-1045.

For more information please visit: http://www.reactionbiology.com/

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Quality Data. Quality Support.

Kinase HotSpotSM: 457 KinasesThe Largest Collection of Kinases for Profiling & Screening

CLK1CLK2CLK3CLK4c-MERc-MET COT1/MAP3K8CSKc-SrcCTK/MATKDAPK1DAPK2DCAMKL1DCAMKL2DDR1DDR2DLK/MAP3K12DMPKDMPK2/CDC42BPGDRAK1/STK17ADYRK1/DYRK1ADYRK1BDYRK2DYRK3DYRK4EGFREPHA1EPHA2EPHA3EPHA4EPHA5EPHA6EPHA7EPHA8EPHB1EPHB2EPHB3EPHB4ERBB2/HER2ERBB4/HER4ERK1/MAPK3ERK2/MAPK1ERK5/MAPK7 ERK7/MAPK15FAK/PTK2FERFES/FPS FGFR1 FGFR2 FGFR3 FGFR4 FGR FLT1/VEGFR1 FLT3 FLT4/VEGFR3 FMS FRK/PTK5 FYN GCK/MAP4K2 GLK/MAP4K3GRK1 GRK2 GRK3 GRK4 GRK5 GRK6 GRK7 GSK3a

GSK3bHaspinHCK HGK/MAP4K4

HIPK1HIPK2 HIPK3 HIPK4 HPK1/MAP4K1 IGF1R IKKa/CHUK IKKb/IKBKB IKKe/IKBKE IR IRAK1 IRAK4 IRR/INSRR ITK JAK1 JAK2 JAK3 JNK1 JNK2 JNK3 KDR/VEGFR2 KHS/MAP4K5 LATS1 LATS2 LCK LCK2/ICK LIMK1 LIMK2LKB1 LOK/STK10 LRRK2 LYN LYN B MAPKAPK2 MAPKAPK3 MAPKAPK5MARK1 MARK2/PAR-1Ba MARK3 MARK4 MEK1 MEK2 MEK3MEKK1 MEKK2 MEKK3 MELK MINK/MINK1 MKK4 MKK6 MLCK/MYLK MLCK2/MYLK2 MLK1/MAP3K9 MLK2/MAP3K10 MLK3/MAP3K11 MNK1MNK2 MRCKa/CDC42BPA MRCKb/CDC42BPB MSK1/RPS6KA5 MSK2/RPS6KA4 MSSK1/STK23 MST1/STK4 MST2/STK3

MST3/STK24MST4MUSKMYLK3 MYO3B NEK1 NEK2 NEK3NEK4 NEK5NEK6NEK7NEK9NEK11NLKOSR1/OXSR1P38a/MAPK14 P38b/MAPK11P38d/MAPK13P38g/MAPK12p70S6K/RPS6KB1p70S6Kb/RPS6KB2PAK1PAK2PAK3PAK4 PAK5PAK6 PASKPBK/TOPK PDGFRa PDGFRb PDK1/PDPK1 PHKg1 PHKg2 PIM1 PIM2 PIM3 PKA PKAcb PKAcg PKCa PKCb1 PKCb2 PKCd PKCepsilon PKCeta PKCgPKCiota PKCmu/PRKD1 PKCnu/PRKD3 PKCtheta PKCzeta PKD2/PRKD2 PKG1a PKG1b PKG2/PRKG2 PKN1/PRK1 PKN2/PRK2 PKN3/PRK3 PLK1 PLK2 PLK3 PLK4/SAK PRKX PYK2 RAF1RET RIPK2

RIPK3RIPK5ROCK1 ROCK2RON/MST1R ROS/ROS1 RSK1 RSK2 RSK3RSK4 SGK1 SGK2 SGK3/SGKLSIK1SIK2SIK3SLK/STK2 SNARK/NUAK2 SRMS SRPK1 SRPK2 SSTK/TSSK6 STK16 STK22D/TSSK1 STK25/YSK1 STK32B/YANK2 STK32C/YANK3STK33 STK38/NDR1 STK38L/NDR2 STK39/STLK3 SYK TAK1 TAOK1 TAOK2 TAOK3/JIK TBK1 TEC TESK1 TGFBR2 TIE2/TEK TLK1 TLK2 TNIK TNK1 TRKA TRKB TRKC TSSK2 TSSK3/STK22C TTBK1 TTBK2 TXK TYK1/LTK TYK2 TYRO3/SKY ULK1 ULK2 ULK3 VRK1 VRK2 WEE1 WNK1 WNK2 WNK3 YES/YES1 ZAK/MLTK ZAP70 ZIPK/DAPK3

Mutant KinasesABL1 (E255K) ABL1 (F317I) ABL1 (G250E) ABL1 (H396P) ABL1 (M351T) ABL1 (Q252H) ABL1 (T315I) ABL1 (Y253F) ALK (C1156Y) ALK (L1196M) ALK (F1174L) ALK (R1275Q) AXL (R499C)BRAF (V599E) BTK (E41K) CHK2 (I157T) c-Kit (A829P) c-Kit (D816H) c-Kit (D816V) c-Kit (D820E) c-Kit (T670I) c-Kit (V559D) c-Kit (V559D/V654A) c-Kit (V559D/T670I) c-Kit (V560G) c-Kit (V654A) c-MER (A708S)c-MET (D1228H) c-MET (D1228N) c-MET (F1200I) c-MET (M1250T) c-MET (Y1230A) c-MET (Y1230C) c-MET (Y1230D) c-MET (Y1230H) c-MET (Y1235D)c-Src (T341M) EGFR (G719C) EGFR (G719S) EGFR (L858R) EGFR (L861Q) EGFR (T790M) EGFR (T790M, L858R)EGFR (d746-750/T790M) EGFR (d746-750) EGFR (d747-749/A750P) EGFR (d747-752/P753S) EGFR (d752-759) FGFR1 (V561M) FGFR2 (N549H) FGFR3 (G697C) FGFR3 (K650E) FGFR3 (K650M) FGFR4 (N535K) FGFR4 (V550E) FGFR4 (V550L) FLT3 (D835Y) FLT3 (ITD)JAK2 (V617F)LRRK2 (G2019S)LRRK2 (I2020T)LRRK2 (R1441C)MEK1 (P124L)p38a (T106M)PDGFRa (D842V)PDGFRa (T674I)PDGFRa (V561D)PKD2 (G870E)RET (E762Q)

RET (G691S)RET (M918T)RET (R749T)RET (R813Q)RET (V804L)RET (V804M)RET (Y791F)TIE2 (R849W)TIE2 (Y897S)TIE2 (Y1108F)Atypical KinasesAMPK(A1/B1/G1)AMPK(A1/B1/G2)AMPK(A1/B1/G3)AMPK(A1/B2/G1)AMPK(A2/B1/G1)AMPK(A2/B2/G1)AMPK(A2/B2/G2)DNA-PKEEF2KEIF2AK1/HRIEIF2AK2EIF2AK3/PERKEIF2AK4/GCN2mTOR/FRAP1PDK1/PDHK1PDK2/PDHK2PDK3/PDHK3PDK4/PDHK4TRPM7/CHAK1

Lipid KinasesPI3K (p110a(E542K)/p85a)PI3K (p110a(E545K)/p85a)PI3K (p110a(H1047R)/p85a)P13K (p110a/p65a)PI3Ka (p110a/p85a)PI3Kb (p110b/p85a)PI3Kd (p110d/p85a)PI3Kg (p120g)PI3KC2aPI3KC3PI4KaPI4KbP14K2APIP5K1APIP5K1CSPHK1SPHK2

For more information please visit: http://www.reactionbiology.com/

Wild Type KinasesABL1 ABL2/ARGACK1AKT1AKT2 AKT3ALKALK1/ACVRL1 ALK2/ACVR1 ALK3/BMPR1A ALK4/ACVR1B ALK5/TGFBR1 ALK6/BMPR1B ARAF ARK5/NUAK1 ASK1/MAP3K5 Aurora A Aurora B Aurora C AXL BLK BMPR2 BMX/ETK BRAF BRK BRSK1 BRSK2 BTK CAMK1a CAMK1b CAMK1d CAMK1g CAMK2a CAMK2b CAMK2d CAMK2g CAMK4 CAMKK1 CAMKK2 CDC7/DBF4 CDK1/cyclin A CDK2/cyclin A1 CDK1/cyclin B CDK1/cyclin E CDK2/cyclin A CDK2/cyclin E CDK3/cyclin E CDK4/cyclin D1 CDK4/cyclin D3 CDK5/p25 CDK5/p35 CDK6/cyclin D1 CDK6/cyclin D3 CDK7/cyclin H CDK9/cyclin K CDK9/cyclin T1 CDK16/cyclin Y CHK1CHK2CK1a1CK1dCK1epsilonCK1g1CK1g2CK1g3CK2aCK2a2c-Kit

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Quality Data. Quality Support.

Methyltransferase HotSpotSM Miniaturized 3H-Based Histone and DNA Methyltransferase

Profiling and Screening Platform

GOLD STANDARD WITHHIGH QUALITY DATA ANDHIGH REPRODUCIBILITYn Exclusive 3H-AdoMet (SAM) based functional assay avoids the false positives and negatives generated by other assay formats.

FLEXIBLE CONDITIONSn IC50, qHTS, HTS, variable SAM concentration, customer- tailored conditions.

NO SPECIAL SUBSTRATE LABELINGn Can use nucleosome, histone protein and peptide as substrates for HMTs.n Use Plasmid or DNA fragment as DNMT substrate.n No assay interfering labeling.

NO COMPOUND INTERMEDIATE DILUTIONSn Avoid compounds crashing out of solution.

LOW COMPOUND CONSUMPTIONn Only nanoliters needed for assays.

FULLY VALIDATEDn All assays come with DMSO as positive control, No-Enzyme as negative control and IC50 standard inhibitors.

“DNA Is Not Destiny -The new science of epigenetics rewrites the rules of disease, heredity, and identity.” – Discover Magazine

Services Available:n Larger scale HTSn Large scale single dose, duplicate profilingn IC50 profiling — 5 or 10 dose with curve fittingn Custom assay developmentn Substrate determinationn Research collaboration

Targets Available

n DNMT1n DNMT3an DNMT3bn DNMT3b/3l comp.n DOT1n EZH1 complexn EZH2 complexn G9an G9a/GLP complexn GLPn MLL 1 complex n MLL 2 complexn MLL 3 complexn MLL 4 complexn NSD1

The HotSpotSM

Advantage

n NSD2n PRMT1n PRMT3n PRMT4n PRMT5n PRMT6n PRMT8n SET1B complexn SETD2n SET7/9n SET8n SETMARn SMYD2n SUV39H1n SUV39H2

For more information please visit: http://www.reactionbiology.com/

McCabe MT et al, “EZH2 inhibition as a therapeutic strategy for lymphoma

with EZH2-activating mutations”.Nature, 2012 492, 108-112

Largest MT panel available for screening

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Proprietary Active Human Recombinant HMT’s & DNMT’s HMT Proteins

n High purityn Validated by Kinetic Assays n Batch-to-batch consistency guaranteed n Highly active enzymes suitable for HTS. n Exclusive complex combinations availablen Bulk discounts available by quote.n Initial in-house work can be followed up with RBC’s Methyltransferase HotSpotSM service using identical proteins.

Reaction Biology Corporation (RBC) offers an expanding line of active, human recombinant HMT’s that are compatible with HTS assay conditions.

n Each RBC HMT is fully evaluated with a gold-standard radioisotope filtration binding assay and FlashPlate assay, using an array of potential substrates. n In addition to a simple study of enzyme activity progression curve, Michaelis-Menten kinetic parameters are also determined with at least one optimized substrate. n Each protein is tested with a group of commercially available inhibitors to evaluate their potencies as references.n Each RBC HMT has reproducible batch-to- batch consistency.

In RBC’s experience, many recombinant HMT preparations can require high enzyme concentrations, saturating substrate levels and lengthy sample processing (e.g. SDS-PAGE/autoradiography; Antibody generation or substrate labeling) to simply demonstrate methyltransferase activity. In response to this problem, RBC has created optimized proteins essential for HMT drug discovery work. RBC is committed to producing HMT’s that our customers can use for valid and economical screening and profiling assays.

Proteins Available:

n ASH1L n DNMT1n DNMT3an DNMT3b /3L Comp.n DMNT3b n Dot1L n EZH1 Complex n EZH2 Complex n G9a n G9a-GLPn GLPn GST-GARn Histone H2An Histone H2Bn Histone H3.3n MLL1 Complex n MLL2 Complex n MLL3 Complex n MLL4 Complex n NSD1 n NSD2n NSD3

n Nuclear Extract (HeLa)n Nucleosomes (HeLa Mono/Di & HeLa Oligo)n PRDM8n PRDM9n PRMT1n PRMT3n PRMT4n PRMT5/MEP50n PRMT6n PRMT8 n SET 1A Complex n SET 1B Complexn SET8 n SET 7/9n SETD2 (GST)n SETD2 (His) n SMYD2 n SUV39H1n SUV39H2n SUV420H1n WRAD2 Complex

For more information please visit: http://www.reactionbiology.com/

McCabe MT et al, “EZH2 inhibition as a therapeutic strategy for lymphoma with

EZH2-activating mutations”. Nature, 2012 492, 108-112

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Quality Data. Quality Support.A Full Panel of HDAC, HAT and SIRT Targets

Services Available:

n Profiling for: — HDAC — HAT — SIRTn Larger scale HTS

n Large scale single dose, duplicate profiling

n IC50 profiling — 5 or 10 dose with curve fitting

n Radioisotope filtration assays for HAT

n Compound Inhibition Mode

n Ki determination

n Specific Substrate for Class2a HDAC’s

n Research collaborations

HDAC’s

n HDAC1 n HDAC2n HDAC3 n HDAC4 n HDAC5n HDAC6 n HDAC7 n HDAC8n HDAC9 n HDAC10 n HDAC11n Nuclear Extract n SIRT1n SIRT2 n SIRT3n SIRT5

HAT’s (Histone Acetyltransferase)

n p300 n CBPn hGCN5 n KAT5 n MYST2 n MYST4n pCAF

For more information please visit: http://www.reactionbiology.com/

McCabe MT et al, “EZH2 inhibition as a therapeutic strategy for

lymphoma with EZH2-activating mutations”.Nature, Nature. 2012 Oct 10. doi: 10.1038

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Quality Data. Quality Support.Profiling and Screening Platform71 Targets and Growing

Kallikrein 12 Kallikrein 13 Kallikrein 14 MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10 MMP12 MMP13 MMP14 PapainPlasma Kallikrein Plasmin Proteinase A Proteinase K TACE Thrombin alpha Tissue Plasminogen Activator (tPA) Trypsin Tryptase beta2 Tryptase gamma1 Urokinase Urokinase

Activated Protein C ADAM-9 ADAM-10 Angiotensin-converting enzyme (ACE 1) Angiotensin-converting enzyme (ACE 2) BACE 1 Calpain 1 Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10 Caspase 11 Caspase 14 Cathepsin B Cathepsin C Cathepsin D Cathepsin E Cathepsin G

Cathepsin H Cathepsin K Cathepsin L Cathepsin S Cathepsin V Cathepsin X/Z Chymase Chymotrypsin Complement activated C1s Dipeptidyl peptidase IV (DPP-IV) DPP-VIII DPP-IX Elastase Factor VIIa Factor Xa Factor XIa Granzyme B Hepatitis C virus NS3/4A protease

HIV Protease Kallikrein Kallikrein 5 Kallikrein 8

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sQuality Data. Quality Support.Cellular Assays: 56 Cell Lines for

Compound Profiling Targets Available:

n Kinases o pAKT, pERK, pSTAT, etc.n HMTs o H3K27me3, H3K4me2, H3K79me2, H3K36me2, H3K9me2, H3K27me2n HATs o H3K27ac and H3K9acn HDACs o Acetylated alpha-tubulin, acetylated histone

Reaction Biology Corporation (RBC) is offering expanding types of cell-based assays for our clients. We carry 56 normal and cancer cell lines in house, and still growing. We provide service for compound profiling (6, 8 or 10 dose with curve fitting). In addition, we are able to perform research collaborations and custom assay development for our clients as well.

Services Available:n Cell Proliferation Assays o CellTiter-Glo® Luminescent Cell Viability Assay, MTT Cell Proliferation Assay

n Cell Apoptosis Assays o Apo-ONE® caspase 3/7 assay, Mitochondrial Apoptosis Detection ( JC-1)

n Cell Phosphorylation Assays o ELISA, Western Blot

n Cell Based Histone Methylation Assays o AlphaLISA, ELISA (DELFIA), Western Blot

n Cell Based Histone Acetylation Assays o AlphaLISA, ELISA (DELFIA), Western Blot

n Cell Based Histone Deacetylation Assays o ELISA (DELFIA), Western Blot

n Custom Tailored Cell Based Assays o Cell Invasion, Cell Migration

Cell Lines:

n A-253n A-431n A-A549n Cal 27n Detroit 562 n DU 145 n FaDu n HCT 116n HDMECn HeLan Hep G2n HGF-1 n H-9 n H-Hn HPLF n HOK n Hs 578Bstn Hs 578Tn HT-1080 n HuT 78 n HuT 102 n IMR-32 n K-562 n MDA-MB-231

n MIA PcCa-2n MJ (G11)n MRC-5n MSTO-211Hn NCI-H441n NIH/3T3n NK-92n OVCAR-3 n PC-3n PC-12n PLC-PRF-5 n SK-Hep-1n SK-OV-3n SNU-16 n SNU-182n SU-DHL-6n SW480n SZ-4n THLE-3n U2OSn U87MG

For more information please visit: http://www.reactionbiology.com/

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Quality Data. Quality Support.The Perils and Promise of Off-Target Interactions

The HotSpotSM

Advantage

Off-Target Primary Hits Name Intended Primary

Target Hit

SC-68376 p38 MAPK CK1δ AG 1024 IGF-1R RIPK2

Akt inhibitor X AKT PIM3

SB 202474 Inactive Haspin

DMBI PDGFR FLT3

ATM kinase inhibitor ATM PIM3

As reported in Nature Biotechnology November 2011, RBC and Fox Chase Cancer Center screened 178 kinase inhibitors against 300 wild-type kinases. The research led to conclusions with important implications for kinase-targeted drug discovery:n Kinase inhibitors can be unexpectedly promiscuous. l 6ofthetop17specifickinaseinhibitorsinhibited other kinases more potently than the ones they were intended to target. l Most off-target hits fall outside of the kinase family of the intended target.

n Inhibitorpromiscuityarguesforprofilingearlierinthelead identificationprocess. l On-target SAR optimization is not enough. An “optimized” compound lead candidate can cause issues with off-target activity that may invalidate months or years of work. l Measuring promiscuity can help choose between competing scaffolds.

n The surprising promiscuity of kinase inhibitors makes target-agnostic drug discovery a viable option. l Although drug research is most often carried out by choosingaspecifictarget,themostpromisingresults can be in unexpected areas. l Byprofilingacompoundlibrarysubsetwithout preference to target, large value can be tapped by findingeffectiveinhibitorsinavarietyofkinases,and only choosing the target after the inhibitor has been found.

n Kinase inhibitors can be more easily repurposed than thought. l Compounds that have had animal or clinical validation create compelling value opportunities if they can be repurposed to an alternate target.

Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR: “Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor

selectivity. “Nature Biotech 2011; 29(11):1039-1045.

Kinase selectivity. A ranked bar chart of selectivity scores (S(50%)) for all tested kinases. This score corresponds to the fraction of all tested inhibitors that inhibit catalytic activity by >50%. Each bar represents the selectivity score of an individual kinase. Insets identify the 14 kinases that were not inhibited by any compound (left) and the seven most frequently inhib-ited kinases (right).

Individual Kinases

For more information please visit: http://www.reactionbiology.com/