Target-Specific Brochure

Target-Specific Assays • Kinase Assays • Epigenetic Assays • RAS Assays • Targeted Protein Degradation Assays • Protease Assays • Phosphatase Assays • Ion Channels • and more Reaction Biology has provided their service for numerous projects with us, especially on multiple kinase assays which have guided our way through drug development. I was very impressed by their level of expertise and professionalism. The insightful advice they provide has been instrumental to our successes. The reproducibility, quality, and reporting of the results have been outstanding throughout the many years we have been working together.

Dr Laurent Meijer Chair & Chief Scientific Officer at Perha Pharmaceuticals & ManRos Therapeutics

Reaction Biology offers a variety of target-specific assays for drug discovery. Most of our assays are high-throughput compatible and can be customized to your specific requirements.

Kinase Assays Kinase Screening - FreeChoice ...... p. 14 Kinase Screening - Panels ...... p. 15 KinaseFinder ...... p. 17 Kinase SubstrateFinder ...... p. 18 NanoBRET Intracellular Kinase Assay ...... p. 19 Cellular Phosphorylation Assay ...... p. 20 In Vivo Kinase Activity Models ...... p. 22

Target-Specific Assays Target-Specific

3 4 Target-Specific Assays Customized KinaseDrug Discovery Additional Assays Nuclear Receptor Assays GPCR Assays Ion ChannelAssays Metabolic Pathway Assays Phosphodiesterase (PDE)Assays Phosphatase Assays Protease Assays PARP Assays Ubiquitin-Proteasome Assays Targeted Protein DegradationAssays Assays RAS-related eader DomainAssays Epigenetic Assays Cell-Based EpigeneticAssays Histone DeacetylaseAssays Histone AcetyltransferaseAssays Demethylase Assays R Methyltransferase Assays

...... p.51 ...... p.50 ...... p.49 ...... p.46 ...... p.43 ...... p.42 ...... p.41 ...... p.40 ...... p.39 ...... p.38 ...... p.37 ...... p.36 ...... p.35 ...... p.34 ...... p.33 ...... p.32 ...... p.31 ...... p.29 ...... p.25 kinase inhibitorswhicharenowusedinthecliniconadailybasis. Throughout thehistoryofourcompany, ourscientistswerededicatedtousetheirexpertiseadvance related diseases. comprising enzymatic,biophysicalandcell-basedassaysaswellinvivomodelsthattargetkinase- Reaction Biologyoffersthemostusedandvaluedkinasediscovery platformintheindustry hit toleadandoptimization. research backgroundswhoareavailabletoassist you ateach phaseofthedrugdiscovery process: hitidentification, Reactionyears. Ourstaffincludesexpert serving clientsforover20 Biology hasbeen Expertise existing assaysanddevelopnewmakingustheCRO ofchoiceforeveryproject. Wedrug discoveryservices.We customizedprojectsaswellintegrated providefee-for-service, cancustom-tai Flexibility immediate technicalsupportandadviceforeveryresearch projectweundertake.Callusanytime. Weto provid understandtheneedsofsmallandlargeresearchorganizationsalike.Ourscientistsarededicated Customer service with otherkinaseassayformats. directly measurestheactivity of thekinasewithhighreproducibilityandavoids false negative andpositives common radioactivity-based assays for enzymatickinaseinhibitorscreening. The Reaction Biologyuses state-of-the-art Quality ofassays platform inindustry. AlmosthalfofthoseinterviewednamedReaction BiologytheirfavouriteCRO. HTStec KinaseProfiling Trends Survey has reportedthattheReaction Biologykinasescreenningisthemostused Our clientshaveidentifiedusastheirfavoriteCRO Your trustedCROforkinasedrugdiscovery scientistsfromavarietyof assay ing lor

5 Target-Specific Assays 6 Target-Specific Assays Cell-based assays: Biochemical assays: Explore ourportfolioof730+kinaseassaysofferedwithavarietyassayformats: need. custom-tailored assaydevelopment,Reaction Biologyisguaranteedtoprovidethekinaseassayyou With thelargest portfolioofkinaseassaysavailablefordrugdiscoveryand20yearsexpertisein Kinase assaysatReactionBiologyscreeningfacilities ACVR1B ACVR1 (R206H) ACVR1 (Q207D) ACVR1 (G356D) ACVR1 (G328V) ACVR1 ABL2 ABL1 (Y253H) ABL1 (Y253F) ABL1 (V299L) ABL1 (T315I) ABL1 (Q252H) ABL1 (M351T) ABL1 (H396P) ABL1 (G250E) ABL1 (F317L) ABL1 (F317I) ABL1 (E255V) ABL1 (E255K) ABL1 AAK1 Kinase • • • • •

Target engagementviaNanoBRETassayformat Kinase activitytestingviaCellPhosphorylationassayformat HTRF-based activityassay(performedinUS) activityassays(ADP-GloLuminescence-based performedinUSandGermany) Radiometric activityassays(HotSpotperformedinUS, HS, PQ,NB HS, NB HS, NB NB NB HS, PQ, NB HS, PQ, NB HS HS, PQ, NB HS HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ HS, NB HS, PQ,NB HS HS, PQ,NB HS, PQ,NB NB Assay Format ALK (G1202R) ALK (F1174S) ALK (F1174L)-NPM1 ALK (F1174L)-EML4 ALK (F1174L) ALK (C1156Y) ALK AKT3 (G171R) AKT3 (E17K) AKT3 (aa106-479) AKT3 AKT2 (E17K) AKT2 (aa107-481) AKT2 AKT1 (E17K) AKT1 (aa106-480) AKT1 ADK ACVRL1 ACVR2B ACVR2A Kinase HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ, CPA HS, NB HS, NB PQ HS, PQ HS, NB PQ HS, PQ, NB HS, NB PQ HS, PQ,NB, CPA NB HS, PQ,NB PQ PQ Assay Format 33 PanQinase performedinGermany) AURKB (G160L) AURKB AURKA ATM ARAF (Y301D/Y302D) ALK-TPM3 ALK-TPM1 ALK-TFG (Tex4Aex20) ALK-TFG ALK-NPM1 ALK-KLC1 ALK-KIF5B ALK-EML4 ALK (T1151M) ALK (T1151-L1152insT) ALK (S1206R) ALK (R1275Q) ALK (L1196M) ALK (L1152R) ALK (G1269S) ALK (G1269A) Kinase HS HS, PQ,NB, CPA HS, PQ,NB HTRF HS, PQ HS HS HS HS HS, PQ HS HS PQ HS HS HS HS, PQ HS, PQ HS HS HS Assay Format CAMK1D CAMK1 BUB1B BTK (Y485F) BTK (T474I) BTK (P190K) BTK (E41K) BTK (C481S) BTK BRSK2 BRSK1 BRAF-SRGAP3 (Kex16Bex9) BRAF-KIAA1549 (Kex15Bex9) BRAF-KIAA1549 BRAF-FAM131B BRAF (V600K) BRAF (V600E) BRAF (V600D) BRAF (V600A) BRAF (T599_V600insT) BRAF (R506_K507insVLR) BRAF (L597V) BRAF (K601E) BRAF (G469A) BRAF (G464V) BRAF (d485-489/P490Y) BRAF BMX BMPR2 BMPR1B BMPR1A BMP2K BLK BCR-ABL1 AXL (R199C) AXL AURKC Kinase HS, PQ,NB HS, NB PQ HS HS HS, NB HS, NB HS, NB HS, PQ, NB HS, PQ, NB HS, PQ, NB HS HS HS HS HS HS, PQ,NB, CPA HS HS HS HS HS HS HS HS HS HS, PQ HS, PQ,NB HS HS, PQ HS, PQ,NB NB HS, PQ,NB CPA HS HS, PQ,NB, CPA HS, PQ,NB Assay Format CDK3-CCNC CDK2-CCNO CDK2-CCNE2 CDK2-CCNE1 CDK2-CCND1 CDK2-CCNA2 CDK2-CCNA1 CDK20-CCNT1 CDK20-CCNH CDK1-CCNE1 CDK1-CCNB1 CDK1-CCNA2 CDK19-CCNC CDK18-CCNY CDK17-CDK5R1 CDK17-CCNY CDK16-CCNY CDK15-CCNY CDK14-CCNY CDK13-CCNK CDK12-CCNK CDK12 (R722C)-CCNK CDK11A-CCNL2 CDK11A-CCNK CDK10-CCNQ CDK10-CCNL2 CDC7-DBF4 CDC42BPG CDC42BPB CDC42BPA CAMKK2 CAMKK1 CAMK4 CAMK2G CAMK2D CAMK2B CAMK2A CAMK1G Kinase PQ HS HS HS, PQ,NB PQ HS, PQ HS, NB PQ PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ HS, PQ, NB HS, PQ, NB PQ HS, NB HS, PQ,NB NB HS, NB HS, PQ,NB HS, PQ,NB HS, PQ NB NB PQ NB HS, PQ HS HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ,NB HS, NB Assay Format CSNK1A1L CSNK1A1 CSK CSF1R COQ8B CLK4 CLK3 CLK2 CLK1 CIT CILK1 CHUK CHEK2 (I157T) CHEK2 CHEK1 CEP43-FGFR1 CDKL5 CDKL3 CDKL2 CDKL1 CDK9-CCNT2 CDK9-CCNT1 CDK9-CCNK CDK8-CCNC CDK7-CCNH-MNAT1 CDK7-CCNH CDK7 CDK6-CCND3 CDK6-CCND2 CDK6-CCND1 CDK5-CDK5R2 (p25) CDK5-CDK5R1 CDK5-CDK5R1 CDK4-CCND3 CDK4-CCND2 CDK4-CCND1 CDK3-CCNE2 CDK3-CCNE1 Kinase HS, NB HS, PQ HS, PQ,NB HS, PQ,NB NB HS, PQ, NB HS, PQ HS, PQ, NB HS, PQ, NB HS HS, NB HS, PQ HS HS, PQ, NB HS, PQ, NB HS NB NB NB NB HS HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ NB NB HS, PQ,NB HS, PQ HS, PQ,NB NB HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ,NB HS HS, PQ,NB Assay Format

7 Target-Specific Assays 8 Target-Specific Assays EGFR (d746-750) EGFR (d746) EGFR (C797S/L858R) EGFR (C797S) EGFR (C797A) L858R) EGFR (C775S/T790M/ EGFR (A767_S768insTLA) Y764insFQEA) EGFR (A763_ Y764insFHEA) EGFR (A763_ EGFR EEF2K DYRK4 DYRK3 DYRK2 DYRK1B DYRK1A DSTYK DMPK DDR2 (T654M) DDR2 (N456S) DDR2 DDR1 DCLK3 DCLK2 DCLK1 DAPK3 DAPK2 DAPK1 CSNK2A2 CSNK2A1 CSNK1G3 CSNK1G2 CSNK1G1 CSNK1E (R178C) CSNK1E CSNK1D Kinase HS, PQ HS HS, PQ HS, PQ HS HS HS HS HS HS, PQ, CPA HS, PQ HS, PQ HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ,NB HS, NB NB HS, PQ HS HS, PQ HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ HS HS, PQ,NB HS, PQ,NB Assay Format EGFR (R999A) EGFR (N771_P772insH) EGFR (L861Q) EGFR (L858R) L858R) EGFR (L792H/C797S/ EGFR (L792H) EGFR (L792F/L858R) EGFR (L792F) EGFR (L747S) EGFR (L718Q) C797S/L858R) EGFR (K728A/T790M/ EGFR (K728A) EGFR (K716Q/L718Q) C797S/L858R) EGFR (K716A/T790M/ L858R) EGFR (K716A/C797S/ EGFR (K716A) EGFR (G719S) EGFR (G719D) EGFR (G719C) EGFR (D770GY) N771insNPG/T790M) EGFR (D770_ N771insNPG) EGFR (D770_ EGFR (D761Y) EGFR (d752-759) EGFR (d747-752/P753S) EGFR (d747-749/A750P) EGFR (d747-749) C797S/L858R) EGFR (d746-750/T790M/ C797S) EGFR (d746-750/T790M/ EGFR (d746-750/T790M) EGFR (d746-750/C797S) EGFR (d746-750/C797A) T790M/L858R) EGFR (d746-750/C775S/ Kinase HS HS HS, PQ,CPA HS, PQ,CPA HS HS HS HS HS HS, PQ HS HS HS HS HS HS HS, PQ,CPA HS HS, PQ HS HS HS HS HS, PQ,CPA HS, PQ HS, PQ,CPA HS HS HS, PQ,CPA HS HS, PQ,CPA HS HS, PQ Assay Format G778insCG) ERBB2 (V777_ ERBB2 (R896C) ERBB2 (P780-Y781insGSP) ERBB2 (P1170A) ERBB2 (D769Y) ERBB2 (D769H) G776insYVMA) ERBB2 (A775_ ERBB2 (775YVMA776) ERBB2 EPHB4 EPHB3 EPHB2 EPHB1 EPHA8 EPHA7 EPHA6 EPHA5 EPHA4 EPHA3 EPHA2 EPHA1 EIF2AK4 EIF2AK3 EIF2AK2 EIF2AK1 EGFR (V769_D770insGE) EGFR (T790M/L858R) L858R) EGFR (T790M/L792H/ C797S/L858R) EGFR (T790M/L792H/ L858R) EGFR (T790M/L792F/ C797S/L858R) EGFR (T790M/L792F/ L858R) EGFR (T790M/C797S/ EGFR (T790M/C797S) EGFR (T790M) Kinase HS HS HS HS HS HS HS PQ HS, PQ, CPA HS, PQ, NB, CPA HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, NB HS, PQ HS, PQ HS, PQ HS HS, PQ,CPA HS HS HS HS HS, PQ,CPA HS HS, PQ,CPA Assay Format FLT3 (D835Y) FLT3 (D835V) FLT3 (D835H) FLT3 FLT1 FGR FGFR4 (V550M) FGFR4 (V550L) FGFR4 (V550E) FGFR4 (N535K) FGFR4 FGFR3 (V555M) FGFR3 (K650Q) FGFR3 (K650M) FGFR3 (K650E) FGFR3 (G697C) FGFR3 FGFR2 (V564F) FGFR2 (R612T) FGFR2 (N549H) FGFR2 (K659N) FGFR2 (K641R) FGFR2 (K526E) FGFR2 (E565G) FGFR2 (C491S) FGFR2 (C491F) FGFR2 (C491A) FGFR2 FGFR1 (V561M) FGFR1 FES FER ERN2 ERN1 (R728A) ERN1 (R727A) ERN1 ERBB4 ERBB2 (V777L) Kinase HS, PQ,NB, CPA NB NB HS, PQ,NB, CPA HS, PQ,NB HS, PQ, NB HS HS HS HS HS, PQ, NB HS HS HS, PQ HS, PQ HS, PQ,NB HS, PQ,NB HS HS HS HS HS HS HS HS HS HS HS, PQ,NB, CPA HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, NB HS HS HS, NB HS, PQ,CPA HS Assay Format IRAK4 IRAK3 IRAK1 INSRR INSR IKBKE IKBKB IGF1R HIPK4 HIPK3 HIPK2 HIPK1 HCK GSK3B GSK3A GSG2 GRK7 GRK6 GRK5 GRK4 GRK3 GRK2 GRK1 GAK FYN (Y531F) FYN FRK FLT4 V592insVDFREYEYD) FLT3 (Y591_ FLT3 (R834Q) FLT3 (R595_E596insEY) FLT3 (N841I) FLT3 (K663Q) FLT3 (ITD)-W51 FLT3 (ITD)-NPOS FLT3 (ITD) FLT3 (F594_R595insREY) FLT3 (F594_R595insR) Kinase HS, PQ,NB NB HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ, NB, CPA HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ HS, PQ, NB HS, PQ, NB HS, PQ, NB HS, PQ, CPA HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS NB HS, PQ,NB HS, PQ,NB, CPA HS, PQ,NB HS, PQ,CPA HS NB HS NB NB HS HS HS, PQ,CPA HS HS Assay Format KSR1 (L639F) KSR1 (A635F) KSR1 KIT (Y823D) KIT (V654A) KIT (V560G/N822K) KIT (V560G/D816V) KIT (V560G) KIT (V559D/V654A) KIT (V559D/T670I) KIT (V559D) KIT (V559A) KIT (T670I) KIT (L576P) KIT (K642E) KIT (D820Y) KIT (D820E) KIT (D816Y) KIT (D816V) KIT (D816I) KIT (D816H) KIT (D816F) KIT (D816E) KIT (d557-558) KIT (A829P) KIT KDR JAK3 JAK2 (V617F) JAK2 (JH1) JAK2 (JH1&2) JAK2 JAK1 (S729C) JAK1 (aa866-1154) JAK1 (aa850-1154) JAK1 ITK untagged IRAK4 (aa104-460) Kinase HS HS HS HS HS, PQ HS HS HS, PQ HS, PQ, NB HS, PQ, NB HS, PQ, NB HS HS, PQ NB HS HS HS HS HS, PQ,NB HS HS, NB HS HS, PQ HS HS, PQ,NB HS, PQ,NB, CPA HS, PQ,CPA HS, PQ,NB HS, NB NB HS HS, PQ,NB PQ HS PQ PQ HS, PQ,NB PQ Assay Format

9 Target-Specific Assays 10 Target-Specific Assays MAP3K4 MAP3K3 MAP3K21 MAP3K20 MAP3K2 MAP3K19 MAP3K14 MAP3K13 MAP3K12 MAP3K11 MAP3K10 MAP3K1 MAP2K7 MAP2K6 (S207D/T211D) MAP2K6 MAP2K5 MAP2K4 MAP2K3 MAP2K2 MAP2K1 (S218E/S222E) MAP2K1 (P124L) MAP2K1 (F53L) MAP2K1 MAK LYN (d23-43) LYN LTK LRRK2 (R1441C) LRRK2 (I2020T) LRRK2 (G2019S) LRRK2 LIMK2 LIMK1 LCK LATS2 LATS1 KSR2 (R676S) KSR2 Kinase NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, NB HS, PQ NB HS, NB HS, PQ, NB HS, PQ, NB HS, PQ HS, PQ PQ HS, NB HS, PQ HS, PQ HS HS, PQ PQ HS PQ HS, PQ HS HS HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, NB HS, NB HS HS Assay Format MATK MASTL MAST4 MAST3 MARK4 MARK3 MARK2 MARK1 MAPKAPK5 MAPKAPK3 MAPKAPK2 MAPK9 MAPK8 MAPK7 (CD) MAPK7 (aa5-397) MAPK7 MAPK6 MAPK4 MAPK3 MAPK15 MAPK14 (T106M) MAPK14 MAPK13 MAPK12 MAPK11 MAPK10 MAPK1 MAP4K5 MAP4K4 MAP4K3 MAP4K2 MAP4K1 MAP3K9 MAP3K8 MAP3K7-TAB1 MAP3K7 MAP3K6 MAP3K5 Kinase HS, PQ HS, PQ NB HS, NB HS, PQ,NB HS, PQ, NB HS, PQ, NB HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ, NB HS, PQ, NB HS PQ HS NB NB HS, PQ,NB HS, PQ HS, NB HS, PQ,NB HS, PQ HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ HS, NB HS, PQ,NB HS, NB HS, PQ,NB HS, PQ PQ HS HS HS, PQ Assay Format MOK MKNK2 MKNK1 MINK1 MET-TFG MET-KIF5B MET (Y1235D) MET (Y1230S) MET (Y1230H) MET (Y1230D) MET (Y1230C) MET (Y1230A) MET (V1092I) MET (T992I) MET (T1173I) MET (P991S) MET (M1250T) MET (M1250I) MET (L1195V) MET (L1195F) MET (K1244R) MET (H1094Y) MET (H1094L) MET (G1163R) MET (F1200l) MET (F1200I) MET (D1288H) MET (D1228Y) MET (D1228V) MET (D1228N) MET (D1228H) MET (D1228G) MET (D1228A) MET MERTK (A708S) MERTK MELK (T460M) MELK Kinase NB HS, PQ,NB HS, PQ,CPA HS, PQ HS HS HS, PQ, NB HS HS, PQ, NB, CPA HS, PQ, NB, CPA HS, PQ, NB, CPA HS, PQ, NB, CPA HS, NB HS, NB HS, NB HS, NB HS, PQ,NB, CPA HS HS, PQ HS HS HS HS HS, PQ CPA HS, PQ,NB CPA HS HS HS, PQ,NB, CPA HS, PQ,NB HS HS HS, PQ,NB, CPA HS, NB HS, PQ,NB HS, NB HS, PQ,NB Assay Format NTRK2 NTRK1-TPR NTRK1-TPM3 NTRK1-TFG NTRK1 (L657M) NTRK1 (G667S) NTRK1 (G667C) NTRK1 (G595R/L657M) NTRK1 (G595R/G667S) NTRK1 (G595R/G667C) NTRK1 (G595R/G667A) NTRK1 (G595R/A608D) NTRK1 (G595R) NTRK1 (F589L) NTRK1 (A608D) NTRK1 NRK NLK NIM1K NEK9 NEK8 NEK7 NEK6 NEK5 NEK4 NEK3 NEK2 NEK11 NEK1 MYO3B MYO3A MYLK4 MYLK3 MYLK2 MYLK MUSK MTOR MST1R Kinase HS, PQ,NB HS HS HS HS HS HS, PQ, NB HS HS HS HS HS HS HS HS HS, PQ,NB NB HS, PQ,NB HS, NB HS, PQ,NB HS HS, PQ HS, PQ,NB HS, NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS HS HS, NB HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ HS, PQ,NB, CPA Assay Format PI4KB PI4KA PI4K2B PI4K2A PHKG2 PHKG1 PEAK1 PDPK1 PDK4 PDK3 PDK2 PDK1 PDGFRB-TPM3 PDGFRB PDGFRA-FIP1L1 PDGFRA (V561D) PDGFRA (T674I) PDGFRA (D842V) PDGFRA PBK PASK PAK6 PAK5 PAK4 PAK3 PAK2 PAK1 OXSR1 NUAK2 NUAK1 NTRK3 (L686M) NTRK3 (G696A) NTRK3 (G623R/L686M) NTRK3 (G623R) NTRK3 (G623E) NTRK3 Kinase ADP-Glo (Ger) ADP-Glo (US), ADP-Glo (US) ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (US), HS, PQ, NB HS, PQ, NB HS HS HS HS HS HS, PQ HS HS, PQ,CPA HS HS, PQ,NB HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ HS, PQ HS HS, PQ,NB HS, PQ,NB HS HS HS HS HS HS, PQ Assay Format PIP5K1C PIP5K1B PIP5K1A PIP4K2C PIM3 PIM2 PIM1 PIKFYVE PIK3CG (L1049R)-PIK3R1 PIK3CG PIK3CD-PIK3R1 PIK3CB-PIK3R1 PIK3CB (E633K)-PIK3R1 PIK3CB (E1051K)-PIK3R1 PIK3CB (D1067Y)-PIK3R1 PIK3CB (D1067V)-PIK3R1 PIK3CB (D1067A)-PIK3R1 PIK3CA-PIK3R1/p65a PIK3CA-PIK3R1 PIK3CA (Q546K)-PIK3R1 PIK3CA (M1043I)-PIK3R1 PIK3CA (I800L)-PIK3R1 PIK3CA (H1047Y)-PIK3R1 PIK3CA (H1047R)-PIK3R1 PIK3CA (H1047L)-PIK3R1 PIK3CA (E545K)-PIK3R1 PIK3CA (E545A)-PIK3R1 PIK3CA (E542K)-PIK3R1 PIK3CA (C420R)-PIK3R1 PIK3C3 PIK3C2G PIK3C2B PIK3C2A Kinase ADP-Glo (Ger) ADP-Glo (US), ADP-Glo (Ger),NB ADP-Glo (Ger) ADP-Glo (US), NB HS, PQ, NB, CPA HS, PQ, CPA HS, PQ, CPA NB ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (US), Glo (Ger),NB ADP-Glo (US),ADP- Glo (Ger),NB ADP-Glo (US),ADP- ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (US) Glo (Ger),NB ADP-Glo (US),ADP- NB NB NB NB ADP-Glo (US),NB NB ADP-Glo (US),NB NB ADP-Glo (US),NB NB Glo (Ger),NB ADP-Glo (US),ADP- ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (Ger) ADP-Glo (US), Assay Format

11 Target-Specific Assays 12 Target-Specific Assays PRKCI PRKCH PRKCG PRKCE PRKCD PRKCB (2) PRKCB (1) PRKCA PRKACG PRKACB PRKACA-DNAJB1 PRKACA (L206R) PRKACA PRKAA2-PRKAB2-PRKAG3 PRKAA2-PRKAB2-PRKAG2 PRKAA2-PRKAB2-PRKAG1 PRKAA2-PRKAB1-PRKAG3 PRKAA2-PRKAB1-PRKAG2 PRKAA2-PRKAB1-PRKAG1 PRKAA2 PRKAA1-PRKAB2-PRKAG3 PRKAA1-PRKAB2-PRKAG2 PRKAA1-PRKAB2-PRKAG1 PRKAA1-PRKAB1-PRKAG3 PRKAA1-PRKAB1-PRKAG2 PRKAA1-PRKAB1-PRKAG1 PRKAA1 (aa1-312) PRKAA1 PNCK PLK4 PLK3 PLK2 PLK1 PKN3 PKN2 PKN1-TECR PKN1 PKMYT1 Kinase HS, PQ HS, PQ HS, PQ HS, PQ,NB HS, PQ HS, PQ HS, PQ HS, PQ HS HS, NB HS HS HS, PQ, NB HS HS HS HS HS HS NB HS HS HS HS HS HS PQ PQ, NB HS HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ HS, PQ HS HS, PQ HS, PQ,NB Assay Format RET (V804E) RET (V778I) RET (S904F) RET (S904A) RET (S891A) RET (R912P) RET (R813Q) RET (R749T) RET (M918T) RET (L790F) RET (L730M) RET (L730I) RET (G810S) RET (G810R) RET (G810C) RET (G691S) RET (E762Q) RET (A883F) RET RAF1 (Y340D/Y341D) RAF1 (Y340/Y341D) RAF1 (R391W) PTK6 PTK2B PTK2 (aa411-686) PTK2 PRKX PRKG2 PRKG1 (B) PRKG1 (A) PRKDC PRKD3 PRKD2 (G870E) PRKD2 PRKD1 PRKCZ (aa184-592) PRKCZ PRKCQ Kinase HS, PQ HS HS HS HS, PQ HS HS, PQ HS, PQ HS, PQ, NB HS PQ PQ HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ HS HS, PQ,NB HS PQ HS HS, PQ,NB HS, PQ,NB PQ HS, PQ,NB, CPA HS, PQ,NB HS, PQ,NB HS HS, PQ HS, PQ HS, PQ HS HS, PQ HS, PQ HS, PQ HS, PQ HS, PQ Assay Format SGK2 SGK1 SBK3 SBK1 RPS6KB2 RPS6KB1 RPS6KA6 RPS6KA5 RPS6KA4 RPS6KA3 (L608F) RPS6KA3 (I416V) RPS6KA3 RPS6KA2 RPS6KA1 ROS1-TPM3 ROS1-GOPC ROS1 (G2101C) ROS1 (G2101A) ROS1 (G2032R) ROS1 ROCK2 ROCK1 RIPK4 RIPK3 RIPK2 RIPK1 RIOK2 RET-PRKARA1A RET-NCOA4 RET-KIF5B RET-CCDC6 RET-BCR RET (Y806H) RET (Y791F) RET (V804M)-KIF5B RET (V804M) RET (V804L)-KIF5B RET (V804L) Kinase HS, PQ,NB HS, PQ,NB NB HS HS, PQ HS, PQ, CPA HS, PQ, NB HS, PQ HS, PQ, NB HS HS HS, PQ, NB HS, PQ, NB HS, PQ, NB HS HS HS HS HS HS, PQ HS, PQ,CPA HS, PQ,CPA HS, PQ HS HS, PQ,NB NB NB HS HS HS HS, PQ HS HS, PQ HS, PQ HS HS, PQ,NB HS HS, PQ,NB Assay Format CPA PQ NB HS STK35 STK33 STK32C STK32B STK32A STK3 STK26 STK25 STK24 STK17B STK17A STK16 STK11-CAB39-STRADA STK11 STK10 SRPK3 SRPK2 SRPK1 SRMS SRC (Y530F) SRC (T341M) SRC SPHK2 SPHK1 SNRK SLK SIK3 SIK2 SIK1 SGK3 Kinase

: : : :

Cell Phosphor Radiometric NanoBRET IntracellularKinase Assay Radiometric HotSpotKinase ActivityAssay NB HS, PQ, NB HS HS, NB NB HS, PQ, NB HS, PQ, NB HS, PQ HS, PQ,NB NB HS, PQ HS, PQ,NB PQ HS, NB HS, NB HS, PQ HS, PQ HS, PQ HS, PQ,NB HS HS HS, PQ,NB, CPA ADP-Glo (US) ADP-Glo (US) HS, NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ Assay Format 33 ylation Assay PanQinase KinaseActivityAssay TNNI3K TNK2 TNK1 TNIK TLK2 TLK1 TIE1 TGFBR2 TGFBR1 TESK2 TESK1 TEK (Y897S) TEK (Y897C) TEK (Y1108F) TEK (R849W) TEK (P883A) TEK (A1124V) TEK TEC TBK1 TAOK3 TAOK2 TAOK1 SYK (aa356-635) SYK STK4 STK39 STK38L STK38 STK36 Kinase NB HS, PQ, NB HS, PQ, NB HS HS, PQ, NB HS, PQ, NB NB HS, PQ,NB HS, PQ HS HS, NB HS, PQ,NB HS, NB HS, PQ,NB HS, PQ,NB HS, NB HS, NB HS, PQ,NB, CPA HS, PQ,NB HS, PQ,NB HS, PQ HS, PQ HS PQ HS, PQ HS, PQ,NB HS, PQ HS, PQ,NB HS, PQ,NB NB Assay Format ZAP70 (Y319F) ZAP70 YES1 (T348I) YES1 WNK3 WNK2 WNK1 WEE2 WEE1 VRK2 VRK1 ULK3 ULK2 ULK1 TYRO3 TYK2 (JH2) TYK2 (JH1) TYK2 TXK TTK TTBK2 TTBK1 (aa1-480) TTBK1 TSSK6 TSSK3 TSSK2 TSSK1B TRPM7 Kinase HS HS, PQ HS HS, PQ, NB HS, PQ HS, PQ HS, PQ NB HS, PQ,NB HS, PQ HS, PQ HS, NB HS, PQ,NB HS, NB HS, PQ,NB NB NB HS, PQ,NB HS, PQ,NB HS, PQ,NB HS, PQ PQ HS, PQ HS HS HS, PQ HS, PQ,NB HS Assay Format

13 Target-Specific Assays 14 Target-Specific Assays Kinase Assays Kinase Profiling Trends Survey. and profilingservices.ItisthemostusedkinasescreeningserviceinindustryaccordingtoHTStec With over730+ kinases,Reaction Biologyoffersthelargestselectionofkinasesavailableforscreening Kinase Screening–FreeChoice (HotSpot We offertworadiometricassayformatswhich differonlyinthewayofsubstrateretentionviaafiltermembrane with otherassayformats. 33 Compound screeningonproteinkinasesisperformedwith highlysensitiveradiometricassays.Phosphatefrom Lipid kinasesarescreenedwith ADP-Glo Platformfrom Promega. facility). Assay formats P-labelled ATP istransferredontoasubstrateanddirectlymeasuredavoidingfalsepositivesnegatives common • • • • • •

TM A referencecompoundisincludedineverystudyfornoadditionalcost Deliverables: singleconcentration%inhibition;IC Customized assaydevelopmentpossible High-throughput compatible allosteric inhibitors Any classofinhibitorcanbetestedincludingATP aswell competitiveandnon-competitive Get thehighestqualitydatapossiblewithgoldstandardradiometricassayformat assay, usedintheUSfacility)orona flashplatesurface( 50 and/orKivalues 33 PanQinase TM assay, usedinthe German Kinase Mapper Kinase PanelScreening inhibited morethan75%areshowninblue. more than90%arehighlightedinredcircles,those Wild Type Kinase Panel. Kinasesthatwereinhibited Shown isanexamplewithSorafenibprofiledthe graphic presentationofkinasescreeningresults. A kinasemappertoolcanbeusedbycustomersfor two weeks. panels arerunonceortwicepermonthallowingustoofferscreeningwithaturnaroundtimeofonly Screen againstthelargestselectionofkinasesandmostwidelyusedpanelinindustry. Ourkinase Lipid KinasePanel Atypical KinasePanel Mutant KinasePanel Wild Type KinasePanel Panels runatUSfacility • • • •

Deliverable: %ofinhibition(singlepoint)orIC Panels runwithHotSpot Visualize yourresultswiththekinasemapper Highest reproducibility TM 17 24 301 377 # ofkinases assayincludeafreecontrolcompound‘sIC ERK1/MAPK3 ERK2/MAPK1 CDKL4 CDKL1

50 CLK3

CLK2

HIPK2

Lipid KinasePanel Mutant KinasePanel Wild Type KinasePanel IC50 Wild Type KinasePanel Panels runatGermanfacility

DYRK3

WNK1

p38g/MAPK12 NRBP1

p38bMAPK11 NRBP2

p38a/MAPK14 CDK4/cyclin D3 BubR1

CDK6/cyclin D3 Bub1

CDK3/cyclin E valuedetermination

PLK3

HIPK1

Nek5

DYRK2 Nek1

p38d/MAPK13 Aurora A Aurora

CDK2/cyclin ERK5/MAPK7

CDK4/cyclin D1 WNK3 CDK2/cyclin A CLK4 PLK2 CDK6/cyclin D1 CDK2/cyclin E

CDK2/cyclin O

CDK3 AAK1 CDKL3

TBK1 MYLK4 DYRK4

MYLK3 PLK1 JNK1

A1 IKKb/IKBKB

IKKa/CHUK

WNK2 Nek3

CLK1

HIPK3

CDK2 JNK3 PLK4/SAK

CDK4 DYRK1/DYRK1A SgK069

DAPK1 CDK6 110 CDK1/cyclin A SgK CDKL2 BIKE

MLCK2/MYLK2 JNK2

CDK1/cyclin B Nek4

DAPK2 CDK1/cyclin E WNK4 TSSK2 IKKe/IKBKE NLK

TSSK1 CDKL5

CDK1 GAK

ZIPK/DAPK3 CDK18 MSSK1/STK23 SSTK/TSSK6 PRK Nek11

GSK3 DYRK1B

SIK CDK5/p25 ERK3

MLCK/MYLK STK16

QIK CDK5/p35 CDK5 ANP

DRAK1/STK17A SRPK2

CDK7/cyclin H HIPK4 PERK/PEK

β Nek2

CDK17 Wee1B ANP

TSSK3/STK22C Wee1 β/NPR2

GSK3 B Aurora

α/NPR1 PRP4

MARK1 SBK1

ERK4

MARK2 Nek9

DRAK2 C Aurora

TTN α CAMKK1

GCN2

HSER

CDK9/cyclinCDK9/cyclin K CDK16 PM

TSSK4 CDK9/cyclin PINK1 PEAK1

ERK7/MAPK15 γ

MYT1

CDK7 Nek8

TLK2 CHED GUCY2D

SgK223

KLCK2/ICK CAMKK2 F

HRI

ULK2 ASTK QSK TLK1

Obscn~b MARK3 T1 Nek6

T2 SRPK1

KIS

BRSK2 SgK196

TTK TXK

CDK9 SgK493 GUCY2F

RSK2 BRSK1 SPEG~b MAK TBCK

ULK1 TEC

RSK3 -b PKCmu MARK4 SgK071 BTK Nek7 CK2

-b PKCnu AMPK CK2

Obscn PFT

T Trb2 PBK/ Tyk2-b

CCRK ULK3 BMX/ETK

AMPK Trb1 Sgk424 SgK396 α2

SNARK PRPK

MSK2 α1 Lyn B

AIRE2 ERN2/IRE2 TK Sgk307 A ITK

MSK1 -b α2 SPEG

Nek10 Lyn

OPK CTK/M

-b ULK4 α1 ERN1/IRE1 Pim2 YES/YES1 CDK8 CSK HCK

ARK5 Pim3 MOK CDK12 CLIK1L

Haspin Abl1

Slob RSK1 NIM1 Fused

CAMK2a Pim1 Lck MELK SCYL3

CDK13 Abl2/Arg

-b SNRK RNAseL CAMK2b RSK4 SCYL2 c-Src

-b HUNK CDK11 BLK

CDK14 FES/FPS CAMK2g Trio Trb3 PIK3R4 MNK1 Trad

MAPKAPK2 Fer Fyn PITSLRE EphA5 CAMK2d MNK2 SCYL1 EphA3 CDK10 Jak1-b Fgr CDK20 MAPKAPK3 EphA4 P FRK/PTK5 EphB1 PKD2 ASK Jak3-b

CAMK1d LKB1 EphA6 PhKg2 MAPKAPK5 CDC7/DBF4 Sgk495 CAMK1a CHK1 SRMS EphA7 EphB3 PhKg1 Jak2-b EphB2 CASK STK33 Brk EphA8 EphB4 RTK106 EphA2 FAK/PTK2 CAMK1g Su CAMK1b EphA1 PYK2 CAMK4 Syk DCAMKL2 VACAMKL EphB6 DCAMKL3 PSKH2 DCAMKL1 EphA10 ZAP70 PSKH1 CHK2 IR IGF1R TYK1/LTK ROS/ROS1 ALK IRR/INSRR

CCK4/PTK7 DDR2 DDR1 MuSK TrkA TrkB

50 ANK1

Y TrkC

STK32b ROR1 ROR2

MASTL

ANK3 Y RON/MST1R

RYK Met

SgK494 MAST2 Tyro3/Sky

AXL Ret

NDR2 Tie2 Mer

MAST3 FGFR1

NDR1 TIE2/TEK foreveryassay

FGFR4 FGFR2

MAST4

LA FGFR3 TS1

MAST1 LA

TS2

ROCK2 Lmr3 PDGFR

PKG1b

PKG2

ROCK1 Ack1 α PDGFR

PKG1a STK22D/TSSK1 DMPK β Lmr1 Tnk1 FLT3

Kit FMS FLT1/VEGFR1 DMPK2 NIK

GCN2~b FL

PKN2/PRK2 Lmr2 T4/VEGFR3KDR/VEGFR2

MRCKa MLKL PKN1/PRK1 ERRB4/Her4

MRCKb Tpl2/COT

TTBK2

VRK3

TTBK1 IRAK4 PKN3/PRK3 PKCzeta LIMK1

PRKX

MSK2/RPS6KA4 LIMK2 HER3

PKCiota

MSK1/RPS6KA5 PRKY

PKAcg

PKCd RIPK5 Jak2

BMPR2 IRAK2 PKCtheta EGFR

MEKK1 Jak3 VRK2 ERBB2/Her2 VRK1

MISR2

PKCeta

PKAcb HH498 14 96 340 340 # ofkinases

PKA

p70S6Kb/RPS6KB2 RIPK3 PKCepsilon

PDK1/PDPK1 p70S6K/RPS6KB1 TGFBR2 T

AK1 TESK1 Tyk2 PKCg TESK2 Jak1

OSR1/OXSR1

STLK3

STRAD/STLKS STLK6 PKCb1 ILK

PKCb2 RIPK2 PKCa

SGK2 MAP3K4

RSK3 IRAK3

Akt3 ZAK/M

RHOK/GRK1 YSK4 L

TK IRAK1

SGK3

MKK7

GRK2

P ALK1 GRK3

SGK1

AK2 MLK2/MAP3K10 RSK4 LRRK1

T ACTR2B AOK3/JIK ALK2 ANKRD3 ACTR2 LRRK2

MEKK6 ANKK1

GRK7 MKK6

CK1g2 DLK/MAP3K12 AK3

Akt1 HPK1/MAP4K1 LZK CK1epsilon MLK4

Akt2

CK1d ALK3

MKK4 P

AK1 ARaf

RSK2 GCK KSR1

ALK6 MLK3/MAP3K11 RSK1

GRK6 KSR2

P MLK1/MAP2KP LOK/STK10

GLK/MAP4K

SLK/STK2

MEK5 AK6

RSKL2

RSKL1 ALK7

AOK1

T AOK2/

GRK4

MYO3B GRK5 KHS/MAP4K5 KHS2

MST4

CK1g3

AO1 CK1g1 RAF1

MYO3A MAP3K8 BRaf

CK1a1

TNIK

Ck1a1L NRK/ZC4

ALK4

ALK5 MEK3 ASK1/MAP3K5

MAP3K7

MINK/MINK1 MEK2 HGK/MAP4K4 MEKK2

MST3/STK24 MEKK3

MEK1 STK25/YSK1

P MST2/STK3 MST1/STK4

AK5 AK4

15 Target-Specific Assays Kinase Assays 16 Target-Specific Assays Kinase Assays 33 Example ofIC will beavoided. individual kinase.False positivesor falsenegativesthatmayoccurwhentestingwithasuboptimalconcentration Sorafenib activitywasdeterminedwith6concentrationson320wildtypeproteinkinasesforIC Using theIC determination. PanQinase

IC50 values, replicate #1 50 TM valuedeterminationsetupyieldsatrueoftheinhibitioncompoundforevery 50 assayformat value determination forsorafenibwiththeWildType valuedetermination KinasePanelbyusingthe IC 50 values,replicate#2 protein kinases. experiments withponatinibon320wildtype Selectivity profilesfromtwoindependent High Reproducibility 50 value

the characterizationofphysiologicalpathways. The KinaseFinder canidentifykinasesthatphosphorylate asubstrateofinterest.Thisserviceisidealfor KinaseFinder D H G B A C E F 12 • • • • Tyr kinases custom panel Wild Type KinasePanel Ser/Thr &Tyr kinases Ser/Thr kinases Type ofKinaseFinder

34 56 Deliverable: Absoluteactivitymeasurementofeachkinaseonyoursubstrate substrate Option tofollowupwithanSDS-PAGE andautoradiogramtovisualizethephosphorylated mutations Option tocomparepeptideswithphospho-site Potential substratescanbepeptidesorproteins 78 91 01 2 custom substrate

plate togetherwith The targetsubstrateisincubated withdifferentkinasesineverywellofamulti-well Assay procedure transfer of of phosphorylatedsubstrate is performedviascintillationcounting 33 selected bycustomer P whentheproteinorpeptide isrecognizedassubstrate.Quantification # ofkinases iaeP-ATP Kinase 33 + 376 339 245 94 P-ATP that servesasphospatedonor. Thekinasewillcatalysethe Assay format 33 33 33 PanQinase PanQinase PanQinase HotSpot HotSpot TM TM TM TM TM Kinase 1 Kinase 3 Kinase 2 Kinase 4

17 Target-Specific Assays Kinase Assays 18 Target-Specific Assays Kinase Assays peptide libraries. substrate panelscomprisevariousproteins,whereasthephysiologicalincludebiotinylated The KinaseSubstrateFinder canidentifysuitablesubstratesforaspecifickinaseofinterest.Thegeneric Kinase SubstrateFinder Kinase activity (cps) • • • autophos. 2000 4000 6000

Ser/Thr &Tyr PhysiologicSubstratePanel Tyr PhysiologicSubstratePanel Ser/Thr &Tyr GenericSubstratePanel Ser/Thr GenericSubstratePanel Tyr GenericSubstratePanel Type ofKinaseSubstrateFinder 0 Deliverable: absoluteactivitymeasurementofkinasewitheachsubstrate Testing ofphysiologicsubstrateswithradiometricassayusing Testing ofgenericsubstrateswithATP consumptionassayADP-Glo (Promega)

S1 concentration 1 no enzyme concentration 3 concentration 2

Substrate S6

S10

S11

S12

S13

background # ofsubstrates S14 720 145 58 39 19 a MELKassay. Substrate 14istobemostsuitableforestablishmentof without substrate. substrate andautophosphorylationoftheMELKkinase controlsofeach substrates. Controlsareno-enzyme MELK kinaseactivitywasmeasuredwithavarietyof Example ofMELKassaydevelopment 33 P-ATP NanoBRET system. kinaseandafluorescent tracer.between aluciferase-tagged Quantitationandspecificityarekeyattributesofthe The assayisacompoundcompetitionthatrelieson bioluminescenceresonanceenergytransfer(BRET) Assay principle HEK293 cellstransientlyexpressingNanoLuc DDR1 inhibitionbyDasatinib reader. was measuredonanEnVision 2104multilabelmicroplate reference compoundDasatinib for1hour. The BRETsignal fusion vectorweretreatedwiththeTracer K-4 and NanoBRET IntracellularTarget EngagementKinaseAssay membrane integrity. the quantitativedeterminationofkinaseinhibitoroccupancyinlivecells,withoutdisruptioncellular Reaction BiologyofferstargetengagementassaysusingPromega’s NanoBRETtechnologythatenables • • •

Deliverable: apparentbindingaffinityofinhibitor(IC High-throughput compatible Intact cellswithphysiologicalATP andpHvalues. concentration,proteincomplex,co-factors ® - DDR1 50

Normalized BRET response [%] ) 100 120 20 40 60 80 0 -10 compound [log M] -8 -6 -4

19 Target-Specific Assays Kinase Assays 20 Target-Specific Assays Kinase Assays Cellular Phosphorylation Assay Cellular Phosphorylation The assaysareperformedwith 8compoundconcentrationsinduplicateforIC50value determination. antibodiesoncapturedVEGF-R2.substrate phosphorylationis quantified byELISAwithpan-phospho-tyrosine minutes toallowfortargetbinding. Aftera3-minutestimulation withligandVEGF-A, cellsarelysedandthe Human endothelialcellsare knowntoexpressVEGF-R2. Thecellsincubatewiththe testcompoundfor90 Example ofVEGF-R2signaling compound activityinaphysiologicalenvironmentonsubstrate. as aresultoftreatmentwithyourinhibitorinintactcells.Theassayshavebeendesignedtoaddress The CellularPhosphorylationAssayquantifieschangesinthephosphorylationstateofasubstrate vivo includingpharmacodynamicprofiling. Most ofthesecellscanbeusedtostudytumorgrowthin administration. active, orkinaseactivityistriggeredbyligand of interestwhichiseitheroverexpressedorconstitutively Assays areperformedwithcellsexpressingthekinase Assays basedonendogenouskinases • • •

Deliverable: %inhibitionofkinaseactivityandIC Assay canbeperformedwithbloodcontainingdrugforplasma-inhibitorystudy Physiological kinase,substrateandATP concentrations one day inhibitor add 90 min stimulus add 3 min lysis 50 receptor determination kinase ligand ELISA VEGF

phospho-substrate [%] 100 50 0 -9 -8 compound [logM] IC 50 -7 -6 -5 quantified via ELISA. transmembrane domain.The cellswereincubatedwiththreeEGF-R-specific inhibitorsandtheir potencywas Rat1 fibroblastsexpress theintracellulardomainofEGF-Rrelevant mutationsanda containingdisease- Example ofEGF-Rmutant analysis EGF-R Δ746750/C797S EGF-R L858R EGF-R Δ746-750/T790M/C797S EGF-R T790M/L858R EGF-R T790M/C797S/L858R can bequantifiedviaELISA. that receptors causesconstitutiveauto-phosphorylation artificial transmembranedomain.Dimerizationofthe intracellular domainofEGF-R mutantsfusedtoan Rat 1fibroblastsweretransfectedtostablyexpressthe Assays basedonexogeneouskinases EGF-R WildType lapatinib osimertinib osimertinib

brigatinib

21 Target-Specific Assays Kinase Assays 22 Target-Specific Assays Kinase Assays for theinvestigationofinhibitorsinvivosetting. exogenous receptorkinaseunderthecontrolofaninduciblepromotor. Thesemodelsmake Reaction Biologyofferstwomodelsbasedonfibroblastcellswhichwereengineeredtoexpressan such asanoverexpressedorconstitutivelyexpressedkinase. Genetically engineeredtumormodelsarewellsuitedforinvestigationofasingledrivergrowth In VivoKinaseTumor Models regression. with theanti-ErbB2antibody Herceptinresultingintumor mice. Atanaveragetumorsizeof400mm NIH3T3-ErbB2-Rrep cellswereimplantedsubcutaneously into Example: ErbB2InhibitionwithHerceptin inhibited inthepresenceofdoxycycline. is expressedintheabsenceofdoxycyclineandexpression is ErbB2 underthecontrolofaTet-inducible promoter. ErbB2 NIH3T3 cellswerestablytransfectedtoexpresshuman Example: ErbB2Model human ErbB2receptor human IGFreceptor Kinase • • •

Assess compoundefficacyandevaluatemechanismsofdrugresistance Implantation ofengineeredcellsforcomparabletumorgrowth Target ahumankinaseinmicewithintactimmunesystem 3 , miceweretreated Cell line NIH3T3 MEF

tumor volume [mm 3] 1000 1500 2000 500 - doxy 0 0 day aftertumorimplantation vehicle Herceptin 5 Therapy startedatday17 10 + doxy 15 excellent tools 20

25 ATP andSubstrateCompetition Assay activation ofatargetkinase byanupstreamkinaseinasocalledcascadeassay. The KinaseActivationAssay issuitableforthediscoveryofallostericcompounds thatinhibitthe Kinase ActivationAssay association anddissociation. a sensorchipandtheanalyteflowstotarget.Target bindingismonitoredinreal-timeforboth: changes inthemolecularmassofatargetafterbinding oftheanalyte.Thetargetisimmobilizedto SPR iscommonlyusedtodeterminethekineticsoftarget-analyte bindingkinetics.Theassaydetects Kinetics, Bindingaffinity compound‘s therapeuticefficacy. your compoundincludingbindingaffinity, residencetime,on-andoff-rates,thatarecrucialtoyour Using avarietyofbiochemicalandbiophysicalmethods,wecandeterminethekineticbehavior Mechanism ofActionAnalysis we determinetheIC To determinewhetheracompound‘smechanismofactionisATP competitiveorsubstratecompetitive, construct selection,proteinproduction,substraterequirementsandassayconditionoptimization. of thecustom-tailoredassayforyourdrugdiscoveryproject.We willbehappytoprovideguidancein Our experienceofestablishingmorethan730kinaseassaysisthebasisforsuccessfuldevelopment Custom AssayDevelopment validated kinaseassays.Contactustotalkaboutthebestapproachforasuccessfulscreeningproject. Bring your own compoundlibrary or use one of our libraries for high-throughput screening High-Throughput Screening Customized KinaseDrugDiscovery 50 orKivaluesatvariousATP andsubstrateconcentrations. with our well of

23 Target-Specific Assays Kinase Assays 24 Target-Specific Assays Epigenetic Assays E acetylation andphosphorylation. The targetfamiliesinclude proteinsthatregulatepost-translational processes suchasmethylation, cell-based assays. assay development,high-throughputscreening, SARsupport,mechanism ofactionanalysesand Reaction Biologyoffersextensiveepigeneticdrugdiscoveryservicesincludingproteinproduction, pigentic TYPE OFMARK Add marks WRITERS A Phosphate group Acetyl group Methyl group ssa ys histone RNA, DNAorhistone modiﬁcations Detect marks READERS DNA Remove marks ERASERS Reader DomainAssays reader domaininhibitors. More than100assayshavebeenestablishedforscreening, leadoptimizationorselectivityprofilingfor Reaction Biologyoffersbothbiochemicalandbiophysicalassaystostudy epigeneticreaderdomains. • • • ITC

TSA Visualize yourbromodomainprofilingresultswiththemappertool. Extensive coverageofthebromodomainfamily. All readerdomainproteinsareproducedatourfacilityandavailableforpurchase. MST domains bromo Screen Alpha SPR HTRF FP • • • • Biochemical assayformatstoquantifycompoundbinding: bromodomain targets availableatReactionBiologyfor Assay formats • • • the molecularlevel on- andoff-ratesparametersofagent-target interactionon Biophysical assayformatsfordeterminationofbindingaffinity,

Thermal shiftassay(TSA) Isothermal titrationcalorimetry(ITC) Microscale thermophoresis(MST) Surface plasmonresonance(SPR) Fluorescence polarization(FP) HTRF AlphaScreen

25 Target-Specific Assays Epigenetic Assays 26 Target-Specific Assays Epigenetic Assays dissociation rates?5.Wheredoesitbind? bind? 2.Isthecompoundspecifictomytarget?3.Howstrongisbinding?4.Whatareassociationand Using SPRwecandescribeseveralparametersoftheinhibitor-bromodomain interaction:1.Whichofmycompounds binding whichismeasuredwithanopticalreadout. compound flowsoverthesensorchipandbindstotargetincreasingmolecularmassofproteinupon enzymatic targetssuchasbromodomains.Thetargetproteinsareimmobilizedonthesurfaceofasensorchip. SPR measuresbiomolecularinteractionsinrealtimeforscreeningoftargetsthatareenzymesaswellnon- plasmonresonance(SPR)assay Surface (acetylated peptide) Substrate BRD4 withindividualbromodomains, SPRrevealedthatRVX-208 is about10-timesmoreselectiveforBD2overBD1. Inhibitors thatselectivelybind tooneofthedomainsmayaffectdifferentbiologicaloutcomes. Byusingrecombinant Human bromodomainBRD4 containstandembromodomains(BD1andBD2)thathave uniquebiologicalfunctions. Example ofBRD4domain interactionwithRVX-208asdetectedbySPR RelativeRelative response Response [RU] [RU] 15 25 35 45 55 -5 5 -50 bromodomain BD1: KD=2.27µM 50 150 Ac K 6xHis 250 Time [S] Ni Time [s] acceptor bead 350 450 Biotin oxygen singlet 550 650

Relative Response [RU] Relative response [RU] 15 25 35 45 55 -5 5 donor bead Streptavidin-coated Excite -50 BD2: KD=0.22µM 50 150 250 Time [S] screening andprofiling. offers AlphaScreenassaysformanyreaderdomains resulting inareducedfluorescentsignal.Reaction Biology of aninhibitorinterfereswithsubstrate/proteinbinding resulting inanenhancedfluorescentemission.Thepresence of thecomplexresultsinasequencechemicalreactions protein bringsthebeadsintocloseproximity. Laser excitation beads. Abindinginteractionbetweenthesubstrateand and abromodomainproteinarecapturedonAlphaScreen For bromodomainscreening, anacetylatedpeptide AlphaScreen Time [s] 350 450 550

assay 650

Relative Response [RU][RU] 1,00E-08 10 20 30 40 50 60 0 1,00E-07 Concentration [M] Concentration [M] 1,00E-06 1,00E-05 substrate BD1 BD2 1,00E-04 BRD7-GST BRD4-Tndm-His BRD4-Tndm-GST BRD4-2-His BRD4-2-GST BRD4-2 BRD4-1-His BRD4-1-GST BRD4-1 BRD4 Full length BRD3-Tndm-His BRD3-Tndm-GST BRD3-2-His BRD3-2-GST BRD3-1-His BRD3-1-GST BRD2-Tndm-His BRD2-2-His BRD2-2-GST BRD2-1-His BRD2-1-GST BRD1-His BRD1-GST BPTF-[PHD-BRD]-His BPTF-[BRD]-His BAZ2B-His BAZ2A-His BAZ2A-GST BAZ1B-His BAZ1A-GST ATAD2B-His ATAD2B-GST ATAD2-His ASH1L-[BRD]-GST Reader Domain TS TS, AS TS TS, AS TS SPR TS, AS TS SPR TS, AS TS, AS TS TS, AS TS TS, AS TS TS, AS TS, AS TS TS, AS TS TS, AS TS TS, AS TS, AS TS, AS AS TS TS, AS TS TS, AS TS TS, AS TS Assays HP1beta-[CHR]-His HP1beta-[CHR]-GST HP1alpha-GST HP1alpha-[CHR]-His EP300-His EP300-GST EED CREBBP-His CREBBP-GST CHD7-[CHR]-GST GST CHD4-[PHD-CHR]- CHD4-[CHR]-GST CHD2-[CHR]-His CHD2-[CHR]-GST CHD1-[CHR]-His CHD1-[CHR]-GST CECR2-His CECR2-GST CDg1-[CHR]-GST CBX7-[CHR]-GST BRWD3-2-GST BRWD1-2-His BRWD1-2-GST BRPF3-His BRPF3-GST BRPF1b-His BRPF1b-GST BRPF1a BRDT-Tndm-His BRDT-2-His BRDT-1-His BRD9-His BRD9-GST Reader Domain TS, AS TS TS TS TS TS HTRF TS, AS TS TS TS TS TS TS TS TS TS, AS TS TS TS TS TS, AS TS TS, AS TS TS, AS TS TS TS, AS TS TS, AS TS, AS TS Assays TAF1-1-GST SP140L-His SP140L-GST SP140-His SP140-GST SP110c-GST SP100-His SP100-GST SMARCA4-His SMARCA2b-His SMARCA2a-His bromodomain SMARCA2a PHIP-Tndm PHIP-2 PB1-6 PB1-5 PB1-4 PB1-3 PB1-2 PB1-1 MPP8-[CHR]-GST L3MBTL1-His L3MBTL1 KAT5-3-[CHR]-His KAT5-3-[CHR]-GST KAT5-2-[CHR]-His KAT2B KAT2A HP1gamma-His HP1gamma-GST HP1beta-Strep HP1beta-His HP1beta-GST Reader Domain TS TS, AS TS TS, AS TS TS TS TS TS, AS TS, AS TS HTRF TS TS TS TS TS TS TS TS TS AS TS TS TS TS TS TS TS TS TS TS, AS TS Assays length UHRF1-His Full UHRF1-[TDR]-His His UHRF1-[TDR-PHD]- UHRF1-[TDR-PHD] UHRF1-[SRA] UHRF1-[PHD]-His UHRF1-[PHD] UHRF1 Full length TRIM66 TRIM33b-His TRIM33a TRIM28 TRIM24 TAF1L-Tndm-GST TAF1L-2 TAF1L-1 TAF1-2-His TAF1-2-GST Reader Domain AS TS, AS AS TS TS AS TS TS TS TS TS TS TS TS TS TS AS TS Assays AlphaScreen AS... Resonance Plasmon Surface SPR ..... Fluorescence Time-Resolved Homogenous HTRF ..... Assay Shift Thermal TS....

27 Target-Specific Assays Epigenetic Assays 28 Target-Specific Assays Epigenetic Assays Example oftheselectivity profile ofbromosporine the bindingaffinityofprotein/bromosporine interaction. difference inmeltingtemperatures oftargetproteinsboundtobromosporineversusDMSO controlisproportionalto The bindingofbromosporine to77bromodomainproteinswascharacterizedusing the BromoMELT assaykit.The identifying ligands, buffer conditions, co-factors anddrugsaffectingproteinstability.identifying ligands,bufferconditions,co-factors melting temperatureuponthebindingofaligand.Protein meltingmeasurementsareusefulfor BromoMELT isathermalshiftassayforbromodomaintargetsthatmeasuresthechangeinprotein BromoMELT • • • •

High-throughput compatible Any inhibitorcanbecharacterizedwithinhours Includes 77proteinsrepresenting63bromodomains machine Available asserviceorkittoeasilyperformtheassayinyourownlabusingaqPCR binding affinityoftheinteraction. protein plusligand(purpleline)isproportionaltothe melting temperaturesofproteinonly(blueline)and of thetargetprotein.Thedifferencebetween temperature atwhichthereis50%denaturation The thermalshiftassaydeterminesthemelting Assay principle S-adenosyl-L-homocysteine (SAH)duringthetransferofradioactivemethylgrouptohistonesubstrate. S-adenosyl-L-homocysteine (SAM)asthemethyldonorthatisconvertedto Methyltransferases usetritium-labeledS-adenosyl-L-methionine Assay principle Methyltransferase Assays histone HA2substrate(HotSpot assay, left)orH4-biotinsubstrate (FlashPlateassay, right). inhibitionofPRMT5/MEP50activity byinhibitorsincomparisontoSAHoneither Concentration-dependent Example: InhibitionofPRMT5/MEP50 activity methyltransferases. Reaction Biologyoffersradiometricactivityassaysandrecombinantproteins forover30 • • • •

Deliverable: %inhibition(singleormultipleconcentration)IC Substrates canbenucleosomes,histones,peptidesorothersubstrates Detection ofinhibitorswithvaryingbindingmodes Direct measurementofenzymeactivityviaradiometricassay + SAM PMT SAH + 50 values

29 Target-Specific Assays Epigenetic Assays 30 Target-Specific Assays Epigenetic Assays of inhibitors to epigenetic targets including enzymes and non-enzymatic proteins. of inhibitorstoepigenetictargetsincludingenzymesandnon-enzymatic Biophysical assayssuchassurfaceplasmonresonance(SPR)canbeusedtodeterminethebindingaffinities SAM analoguessuchasMTA andSAH. inhibitorthatbindstoitstargetPRMT5/MEP50onlyinthepresenceofSAM EPZ015666 isasubstrate-competitive Example ofaco-factoranalysisbySPR MLL1 Complex METTL21A GLP G9a EZH2 Complex Complex EZH2 (Y641F) EZH1 Complex DOT1L DNMT3b/DNMT3L DNMT3b DNMT3a DNMT1 COMT (V108M) COMT ASH1L Methyltransferase Relative response [RU] 0 1.5 3 5.5 7 9.5 .5 .5 .5 - 50 0100150 KD =nobinding no co-factor no co KD =nobinding time [s] - factor available Protein √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ MEP50 PRMT5 (C449S)/ PRMT4 PRMT3 PRMT1 PRDM9 NSD3 NSD2 (T1150A) NSD2 (E1099K) NSD2 NSD1 NRMT2 NRMT1 MLL4 Complex MLL3 Complex MLL2 Complex Methyltransferase Relative response [RU] 7 9.5 0 1.5 3 5.5 .5 .5 .5 - 50 0100150 presence ofMTA KD =2.1x10 presence of KD =2.1*10 time [s] - MTA -5 5 M M available Protein √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ SUV420H1-tv2 SUV39H2 SUV39H1 SMYD3 SMYD2 SETDB1 SETD2 SET8 SET7 SET1B PRMT8 PRMT7 PRMT6 PRMT5/MEP50 Methyltransferase Relative response [RU] 7 9.5 0 1.5 3 5.5 .5 .5 .5 - 50 0100150 presence ofSAH KD =6.6x10 presence KD =6.6*10 time [s] of SAH -7 - 7 M M available Protein √ √ √ √ √ √ √ √ √ √ √ √ √ √ or Demethylase Assays(KDM) species tocatalyzehydroxylationreactions. dioxygenases thatuseareactiveFe(IV)-oxoferryl the process.JmjCareFe(II)/2-oxoglutarate-dependent that catalyzedemethylationofKme2orKme1producingperoxide(H monoamineoxidases demethylases (LSD)histonedemethylasesubfamilies.LSDsareflavin-dependent containing(JmjCs)andlysine-specific Reaction BiologyoffersassaysforbothJumonjiC-domain determination ofareference inhibitior. H LSDs activitywasdetectedby quantificationof Example ofLSD1inhibition • • •

2 O test concentrations)orIC Deliverable: %inhibition(singleormultiple Customized conditionsareavailable the activityofdemethylases Three assayformatsareavailabletomeasure 2 usingAmplexRed reagentforIC activity [%] 100 120 20 40 60 80 0 -9 -8 compound [logM] -7 -6 50 determination -5 Tranylcypromine 50 -4 value -3 reference inhibitors. antibody forIC technology withKDM5Cand substrate-specific (homogeneous timeresolved fluorescence) KDM5C activitywasdetected usingHTRF Example ofKDM5Cinhibition LSD1 KDM6B KDM5C KDM5B KDM5A KDM4D KDM4A Demethylase activity [%] 100 120 20 40 60 80 0 -9 2 O 50 2 valuedeterminationoftwo compound [logM] ) andformaldehyde(H -8 Assay format Amplex Red AlphaLISA AlphaLISA -7 HTRF HTRF HTRF HTRF -6 -5 2,4 PDCA GSK-J1 -4 available Protein √ √ √ √ √ √ √ 2 -3 CO) in

31 Target-Specific Assays Epigenetic Assays 32 Target-Specific Assays Epigenetic Assays cofactor. Reaction BiologyoffersradiometricactivityassaysforHAT Aas enzymesusingtritiatedacetyl-Coenzyme Histone AcetyltransferaseAssays(HAT) activity. inhibitors againstCREP-binding proteinCBP Full concentration-response ofthreereference Example ofCBPinhibition Assay principle reflect theenzymeactivity. A astheacetyldonor. Thetritium-acetylgroupistransferedontohistonesubstratethatmeasureddirectlyto Histone acetyltransferasesacetylatelysinesonhistonesandotherproteinsusingtritium-labelledacetyl-Coenzyme • • •

activity [%] Deliverable: %inhibition(singleormultipletestconcentrations)IC Customized conditionsareavailable Direct measurementofenzymeactivityviaradiometricassay 100 120 20 40 60 80 0 -9 -8 compound [logM] -7 + -6 -5 - CoenzymeA -4 Garcinol Curcumin Anacardic Acid -3 HAT pCAF p300 MYST4 MYST2 KAT5 hGCN5 CPB HAT 50 determination + available Coenzyme A Protein √ √ √ √ √ √ √ Histone Deacetylase (HDAC) and Sirtuin Assays Histone Deacetylase(HDAC)andSirtuin fluorecence. designed foreachenzyme.Thedeacetylatedfluorigenicsubstrateissusceptibletocleavagebyaproteaseyield The assayisperformedwithpurifiedhumanproteinandafluorigenicacetylatedpeptidesubstratespecifically assaysforbothZn Reaction Biologyoffersfluorescence-based inhibitors againstHDAC1 activity. Full concentration-response oftworeference Example ofHDAC1inhibition Each assayisoptimizedbasedonitsspecificsubstrate: dependent sirtuins. Assay principle

activity [%] • • 100 120

20 40 60 80 0 -10 - Sirt5:Ac-L - HD - HD - Deliverable: %inhibition(singleormultipletestconcentrations)IC Customized conditionsandkineticstudiesareavailable HDAC 1,2,3,6,10,andSirt3: -9 AC 4,5,7,9,11:Trifluoroacetyl lysine AC 8:p53residues379-382(RHKAcKAc), compound [logM] -8 ys(Succinyl)-AMC -7 -6 -5 Trichostatin A SAHA -4 -3

p53residues379-382(RHKKAc) pan-SIRT inhibitor,pan-SIRT againstSIRT5 activity. Full concentration-response ofNicotinamide,a Example ofSIRT5 inhibition activity [%] 100 120 20 40 60 80 0 -9 2+ -dependent HDACs-dependent andNAD -8 compound [logM] -7 -6 50 determination -5 Nicotinamide -4 -3 + -

33 Target-Specific Assays Epigenetic Assays 34 Target-Specific Assays Epigenetic Assays Cell-based EpigeneticAssays determination. infrared imaging. Theresultsareplottedinacurvefor EC50 were subjectedtoWestern Blottingandquantificationvia Lysates ofU266B1ce Example fordetectionofHDAC activityincancercells include ELISA,Western assays. Blot,NanoBRETandHDAC-Glo methylation changesofsubstratesinaphysiologicalenvironmentusingintactcells.Thedetectionoptions Cell-based assaysarevaluabletoolsforevaluatinginhibitorpotencytoaffectacetylationand/or • • •

Deliverable: IC ELISA andWestern Blotdetectthe Evaluate compoundactivityinintactcells. Compound bindingtoBromodomain Compound bindingtoHDAC Bromodomain-Histone Interaction Histone Methylation Histone Deacetylation Histone &Tubulin Deacetylation Readout lls treatedwithTrichostatin A (TSA) 50 valuesofepigeneticenzymeinhibition cells seed one day endogenous substratesfordirectactivitymeasurement inhibitor add NanoBRET Target EngagementAssay NanoBRET Target EngagementAssay NanoBRET Target EngagementAssay Western Blot;AlphaLISA HDAC-Glo Western Blot;ELISA Assay format 16hrs lysis H3 ELISA me2 R EGFR signalingincludingkinases,phosphatasesand transcriptionfactors. inhibitors againsttheERK/MAPKandPI3Ksignaling pathwayaswellupstreampathwayssuch In additiontoRASspecificassaysweoffermorethan 80Ras Pathway relatedassaysfortesting Reaction Biologyprovidesavarietyofservicestodiscovernewinhibitors targetingtheRASpathway. Most ofourassaysareavailablewithwildtypeandmutatedRASvariants. facilitates enhancedRas signalingincancercells. associated withpoordiseaseprognosis.MutatedRas islocked intheactivatedGTP boundstateand The smallGTPase, Ras, isaknownoncogenethatmutatedinlargepercentageofcancersand SPR DirectBindingAssay Thermal ShiftDirectBindingAssay Protein-Protein InteractionofRAS andcRAF Protein-Protein InteractionofRAS andSOS1 Nucleotide ExchangeAssay Available AssayFormats as P a thw a y A ssa ys of compoundsbindingRas andRas mutantsorSOS. Surface PlasmonResonance (SPR)determines thekinetics measurement ofcompoundselectivitytoRas mutantpanel. Compound bindingaffinitymeasurementsuitedfor quantification ofnucleotideexchangereaction. cRAF bindingtoRas. Thisassayisalsosuitedfor HTRF-based assay for testingofcompoundsthatdisrupt SOS1 bindingtoRas. HTRF basedassayfortestingofcompoundsthatdisrupt labeled GDPtoGTP Measuring ofSOS1/2mediatedexchangefluorescently Description

35 Target-Specific Assays Ras Pathway Assays 36 Target-Specific Assays Protein Degradation Assays T program. program. out to reach usPlease to your discuss Targeted options for supporting discovery Protein drug Degradation for development work the collaboration of new Targeted or FTE-based Protein molecules. Degradation We fee-for-service, are currently shared-cost talking to biotech companies to and pharma perform cycles neededforthegenerationofpotentandoptimizedproteindegradationmolecules. the bindingmodeofternarycomplextosignificantlyreducenumbercompoundsandscreening the INDstage.TheAIpoweredTargeted Protein Degradationdiscoveryplatformwillenablepredictionof the firstCROsinsynthezisingproteindegradationmoleculesandadvancedtwoPROTAC moleculesinto we havecreatedtheTargeted Protein Degradationdrugdiscoveryplatform.Medinoahhasbeenoneof Together withMedinoah,amedicinalchemistryprovider, andPMRBioinfo,anAIcomputationalcompany, Rethinking PROTAC: AnAIbasedplatformtosupportyourdrugdiscoveryproject argetd Workflow of discovery of of new discovery TargetedWorkflow Protein molecules platform: Degradation Drug-to-Ligase engagement P Primary screeningfor Drug-to-Target and Drug Synthesis Drug Design rotein D egrada Drug-to-Ligase engagement Degrader Discovery Cellular screening for Targeted Protein Drug-to-Target and tion A ssa Biophysical analysisofthe ternary complex formation ys Cellular Screening for in vivo tumormodels Phenotypical testing Target Degradation in cellularand protein interactions.Reaction Biologyoffersfluorescent-based assays forscreeningofDUBinhibitors. molecules. Ubiquitinationofproteinsaffectsproteindegradation,cellularlocation,activitiesandprotein- Deubiquitinase enzymes(DUBs)areagroupofproteasesthatcleaveubiquitinfromproteinsandother U deubiquitinase, AMC is released and it’s fluorescencecanbemeasured. deubiquitinase, AMCisreleasedandit’s methylcoumarin (AMC),thatisquenchedwhenubiquitinylated. Uponincubationwitha Fluorescent Ubiquitin-AMC isasubstratecontainingthefluorophore,7-amido-4- Assay procedure

activity [%] biquitin • • • 100 120

20 40 60 80 0 -9 Deliverable: %inhibition(singlepoint)orIC Custom assaydevelopment quantificationofdeubiquitinaseand20Sproteasomeactivity Fluorescence-based ﬂuorophore -8 compound [logM] -7 -P Ub -6 roteasome -5 quenching UCH-L1 I2 Ub Aldehyde DUB -4 -3 P Ub inhibitors. IC inhibitionofdeubiquitinaseA20bytwo Concentration-dependent Example ofA20/TNFAIP3 inhibition concentrations. signal ﬂuorescence a thw 50 profiling, Kidetermination 50 a value determinationisbasedon10compound y A ssa ys USP8 USP5 USP2 USP14 USP10 UCHL3 UCHL1 SENP2 SENP1 NEDP1 BAP1 Ataxin3 A20 20S Proteasome Available Targets

37 Target-Specific Assays Ubiquitin-Proteasome Assays 38 Target-Specific Assays PARP Assays P epigenetic regulation,forexample,bypolyADP-ribosylation ofhistonesubstrates. onto targetnuclearproteinsforminglongbranchedPoly ADP-ribose chains.PARPs playarolein Poly (ADP-ribose) polymerase(PARP) isafamilyofproteinsthattransferADP-ribose unitsfromNAD+ quantified viascintillationcounting. Adenylate-NAD Assay procedure ARP A activity [%] 100 120 • • • • 40 60 80 20

0 Deliverable: %inhibition(singlepoint)orIC Custom assaydevelopment High-throughput compatible Gold standardassayformat:radiometricactivity -10 + compound [logM] serves as co-factor fortransferof servesasco-factor ssa -8 + ys PJ34 Veliparib (ABT-888) Olaparib (AZD2281) -6 - ADPRibose -4 N O NH 2 32 P-labelled ADP-ribose unitsontohistoneswhichwillbe compound concentrations. inhibitors. IC inhibition ofPARP2Dose-dependent bythreereference Example ofPARP2 inhibition PARP 50 profiling 50 valuedeterminationisbasedon10 - ADPRibose + N O NH 2 Assay formats quencher suppressingfluorescence unlesssubstrateiscleaved. Lower image:The peptidesubstratescontainafluorophoreand protease releasingafluorescent product. Upper image:Fluorogenicpeptidesubstrateiscleavedby target P proteases areavailableforcustomizedorders. proteases, serinemetalloproteases,aspartyldipeptidasesandothers.Over80 Reaction Biologyoffersa65-memberproteasepanelforroutineprofiling. Membersincludecysteine rotease • • • •

Deliverable: %inhibition(singlepoint)orIC Custom-assay development High-throughput compatible activityassay Fluorescence-based A ssa ys 50 valuedetermination CathepsinG Cathepsin E Cathepsin D Cathepsin C Cathepsin B Caspase 9 Caspase 8 Caspase 7 Caspase 6 Caspase 5 Caspase 4 Caspase 3 Caspase 2 Caspase 14 Caspase 11 Caspase 10 Caspase 1 Calpain1 BACE1 ADAM10 ACE2 ACE1 MMP 1 Matriptase 2 Kallikrein 7 Kallikrein 5 Kallikrein 14 Kallikrein 13 Kallikrein 12 Kallikrein 1 HIV protease Factor XIa Factor Xa Factor VIIa Elastase DPPVIII DPPIX DDP-IV Chymotrypsin Chymase Cathepsin V Cathepsin S Cathepsin L Cathepsin H Proteases Urokinase Tryptase g1 Tryptase b2 Trypsin Thrombin TACE Proteinase K Proteinase A Plasmin Plasma Kallikrein Papain Neprilysin MMP 9 MMP 8 MMP 7 MMP 3 MMP 2 MMP 14 MMP 13 MMP 12 MMP 10

39 Target-Specific Assays Protease Assays 40 Target-Specific Assays Phosphatase Assays P basic hydrolysisreaction. than kinases,phosphatasesarelessspecificfortheirsubstrates.Allcatalyzethesame Protein phosphatasesplayimportantrolesincellsignallingprocessesinterplaywithkinases.Different fluorescent afterdephosphorylation. range ofproteinphosphatases.Thereactionproduct DiFMUP is The fluorinatedMUPderivateissuitableassubstrateforalarge DiFMUP-based assayprinciple O H hospha activity [%] 2 • • • • 100 120 20 40 60 80

0 -9 + Deliverable: %inhibition(singlepoint)orIC Custom-assay development High-throughput compatible activityassay Fluorescence-based HO O -8 non-ﬂuorescent OH P compound [logM] O -7 F t ase F -6 O A -5 O ssa -4 PHPS1 PTP1B Inhibitor NSC-87877 phosphatase -3 ys Full concentration-response of3referenceinhibitors. Example ofPTPRC/CD45inhibition F O ﬂuorescent 50 valuedetermination F O O + HPO 4 PTPRC_CD45 PTPN7_LC-PTP PTPN6_SHP1 PTPN2_TC-PTP PTPN12_PTP-PEST PTPN11_SHP2-FL PTPN11_SHP2 PTPN1_PTP1B PP2A alpha/PPP2R1AComplex PP1B PP1A DUSP22_MKPX Phosphatases fluorescence polarizationsignal. tracer fromanantibodyselectiveforAMPandGMPresultinginreductionofthe PDE convertscAMPorcGMPtoAMPGMPwhichdisplaceafluorescent Assay principle conditions includingpulmonaryhypertension,acuterefratorycardicfailure,erectiledysfunction,etc. cyclic nuleotides‘ signalingpathways.PDEinhibitorshavefoundutilityinthetreatmentofavariety Phosphodiesterases (PDEs)catalyzethehydrolysisofcyclicAMPandGMP, therebyregulatingthese Full concentration-response forthreereferenceinhibitorsofPDE10A. activity [%] P 100 120 hosphodiesteras 20 40 60 80 0 -10 • • • •

Deliverable: %inhibition(singlepoint)orIC Custom-assay development High-throughput compatible (BellBrook labs) Activity ofenzymesismeasuredwiththeTranscreeneer AMP2/GMP2FPPDEassayplatform -9 compound [logM] -8 -7 -6 -5 -4 TC-E 5005 IBMX Methoxymethyl-IBMX

( PD E) A 50 values ssa ys PDE9A PDE8A PDE7A PDE5A PDE4D PDE4C PDE4B PDE4A PDE3B PDE3A PDE2A PDE1C PDE1B PDE1A PDE10A PDEs

41 Target-Specific Assays Phosphodiesterase Assays 42 Target-Specific Assays Metabolic Pathway Assays analogous coupledreaction asdescripbedaboveforIDHs. provides compoundscreening againstNQOsbymonitoringenzymeactivityinan NQOs areinvolvedindetoxificationandbiosyntheticpathways. Reaction Biology NAD(P)H dehydrogenase[quinone]1-NQO IDHs arealsoavailableforpurchase. resorufin inasecondaryreaction. intheconversionofresazurintofluoresecent the initialreactionisaco-factor measuring enzymeactivityinacoupledsystemwhereinNADPHproduced factor NADPH. Reaction BiologyprovidescompoundscreeningagainstIDHby that catalyzetheconversionofisocitratetoa-ketoglutarate(aKG)andco- Isocitrate dehydrogenases1and2(IDH1IDH2)arekeymetabolicenzymes Isocitrate dehydrogenase-IDH detecting theproductionofADP. drug target.Reaction BiologyprovidescompoundscreeningagainstACC by ACC isacrucialmetabolicenzymeandattractive tomalonyl-CoA. of acetyl-CoA enzymethatcatalyzes ACCthe ATP-dependent is a biotin-dependent carboxylation Acetyl-CoA Carboxylase(ACC) M et abolic P a thw a y A ssa ys NQO2 NQO1 NQO IDH2 WT IDH2 R172Q IDH2 R140Q IDH2 R140K IDH1 Y139D IDH1 WT IDH1 R132H IDH1 R132C IDH1 R100Q IDH1 R100A IDH1 G97D IDH ACC2 ACC1 Carboxylase Q Patchassay gigaohm seals. patch clampdatabasedontrue provide whole-cell 48 or16cellsinparallel,respectively. Bothsystems clamp platformsthatallowforthetestingofupto QPatch HTXandQPatch 16areautomatedpatch Reaction Biologyofferscell-basedionchanneltestingfordrugdiscovery andevaluationofdrugsafety. I on • • • •

Deliverable: IC andligand-gatedionchannels Investigation ofvoltage-gated clamp,QPatchThree formats:twoelectrode-voltage andmanualpatchclamp evaluationofcompoundsafety panelisavailableforpre-clinical Cardiac-safety C hannel 50 valueofinhibitionionchannelactivity A ssa ys hCav1.2 hNav1.5 hERG Ionchannels with recording in the whole-cell format. with recordinginthewhole-cell setup channels inaplanarpatch-clamp Schematic ofacellexpressingsodium Assay principle Q Patch, ManualPatch Clamp Q Patch, ManualPatch Clamp Q Patch, ManualPatch Clamp

43 Target-Specific Assays Ion Channel Assays 44 Target-Specific Assays Ion Channel Assays compounds andtodeterminetheeffectsofonbiophysicalpropertiesachannel. canbeusedtoassessmechanismofaction activity ofpreliminaryactivesfromscreens,manualpatch-clamping Manual patchclampingisthegoldstandardforinvestigationofionchannelactivity. In addition toconfirmingthe Manual PatchClampAssay Quinidine wasperfusedfor5minutes.10µM shows closetocompleteinhibitionofhERG. on theQPatch potassium channel.Eachconcentrationof inCHOcellsstablyexpressinghERGvoltage-dependent hERG potassiumchannelinhibitioninthepresenceofvarious concentrationsofQuinidine.Recordings weremade Example ofhERGinhibitionbyQuinidine

Current [pA] - 200 200 400 600 0 0 1000 pre 2000 - drug 3000 Time [ms] 4000 drug 5000 post 6000 - drug 7000 8000 - 0 20 40 - - - 80 60 40 20

Clamp Voltage [mV] 10 µ 3.3 µ 1.11 µ 370 123 4 saline 1 nMQuinidine M Quinidine nM Quinidine nM Quinidine M Quinidine M Quinidine necessary forreceptoractivation. allosteric modulator(PAM) glutamateandglycine thatare simultaneouslywith10µMeachoftheco-agonists potentiationofchannelactivity byincubationwithapositive The graphshowstheconcentration-dependent Example ofNMDAreceptor subtype2D This isalowthroughputplatformmostsuitableforconfirminghitsorleadoptimization. be expressedviamRNAinjectionintooocytes;thus,thereisnoneedforgenerationofstablecelllines. orligand-gatedionchannels.Ionchannelsofinterest,andmutantformsthereof,voltage-gated can Two-electrode voltageclampsusesXenopuslaevisoocytestotesttheactivityofinhibitorsagainst Two-Electrode Voltage Clamp Assay principle Current [uA] membrane. passing throughthechannelsexpressedoncell electrodes tocontrolandmeasurethecurrent Oocyte impaledwithvoltageandcurrent üüüüüü ü ü üüü ü ü üüü ü ü üü ü ü ü ü ü ü ü=üüü üü üüüü ü ü üü ü ü ü ü ü ü ü ü ü ü ü electrode electrode tomeasure ü ü ground internal potential üü üü xenopus egg to injectcurrent electrode channels ion

45 Target-Specific Assays Ion Channel Assays 46 Target-Specific Assays GPCR Assays G-P discovery researchintheareaofGPCRbiologyandpharmacology. increasingly importanttoolsindrugdiscovery. Reaction Biologyoffersservicestoprogressdrug advances inGPCRpharmacology, includingbiasedsignalingandallostericmodulators,havebecome GPCRs representthelargestindividualfamilyoftargetsforcurrentlyapprovedmedications.Recent 6. 5. 4. 3. 2. 1. specific needs: willenabledrugscreening withassaystailoredtoyour Our dedicatedteamofGPCRexperts

• •

updates onthestudyprogress. During everystepoftheprocess,youwillbeinclose contactwithyourprojectmanagerfor We guaranteefastturn-around timesfordatageneration. for yourproject. assayswillapplytonewly developed The samehighstandardsweuseforouroff-the-shelf We willacquireorgenerateacelllineappropriatetotheprojectneeds. oriented workrightfromthestart. Make usfamiliarwiththegoalsforyourresearchprojectanddefinetimelinestoensuregoal- Define theneedsandscopeforprojecttogetherwithourassaydevelopmentteam. 1-monophosphate (IP1)generation translocation,cAMPgenerationandinositol including calciummobilization,ß-arrestin Transmembrane signalingassayf We offerassaydevelopment,high-throughputscreening, SARsupportservices. rotein -C oupled R ormats canbereadilyestablishedforyourreceptorofinterest ecptor

( GPC R) A ssa ys

assays regular signal. wherein cellularcAMPcompeteswithalabeledprobe tobindananti-cAMP-cryptate generatingaFRET cAMP canbeaccuratelymeasuredbyavarietyofstandarddetectionmethodsincludingcompetitiveimmunoassay GPCRsofinterest arestimulatedwithanagonisttoaffectcellularactivation. Cells engineeredtoexpressGs-coupled Agonist-induced cAMPgeneration binding to an anti-IP1-cryptate generatingaFRETsignal. binding toananti-IP1-cryptate generated IP1ismeasuredusingancompetitiveimmunoassaywhereincellularcompeteswithalabeledfor GPCRsofinterestarestimulatedwithanagonistforIP1accumulation.The Cells engineeredtoexpressGq-coupled Agonist-induced IP1generation

FRET ratio 12000 FRET ratio 3000 6000 9000 10000 15000 20000 25000 5000 0 -13 0 -12 (n=4) (n=4) -12 -10 compound [logM] -11 compound [logM] -10 -8 -9 -6 -8 IP-1 standardcurve agonist cAMP standardcurve agonist -7 -4 -6 -2 anti-IP1-cryptate with labeledIP1 anti-cAMP-cryptate with labeledcAMP FRET FRET PIP2 ATP IP2 cAMP adenylate IP3 cyclase

PLC

activation inhibition cAMP anti-cAMP-cryptate with cellularcAMP anti-IP1-cryptate with cellularIP1 ligand ligand no FRET no FRET cAMP

47 Target-Specific Assays GPCR Assays 48 Target-Specific Assays GPCR Assays proteins that further mediate cell signaling pathways independent of G-proteins. proteins thatfurthermediatecellsignalingpathwaysindependentofG-proteins. bindtoactivatedGPCRsmediatedesensitizationandinternalizationofGPCRs.Theyarescaffolding ß-Arrestins Agonist-induced ß-arrestintranslocation concentration-response ofagoniststimulationforcalciummobilizationisshownontheright. Test compoundsareaddedandfluorescencechangesinthecellsmonitoredovertime(leftfigure).The AM. dyeFluo-8 CHO cellsengineeredtoexpressthereceptorofinterestareloadedwithcalcium-sensitive FDSS-based calciummobilization (undisclosed) in responsetotreatmentwithagonist Example ofß-arrestintranslocation

relative light units 2.5 5.0 7.5 × × × 10 10 10 0 5 5 5

-11 normalized fluorescence ratio 0.6 0.0 0.2 0.4 -12 -10 compound [logM] -10 compound [logM] -9 -8 -8 β -6 -arrestin (n=4) -7 -4 peptide agonist ATP receptors withthefollowingadvantages: Reaction Biologyoffersasuiteofassaystosupportthediscoverynewdrugsmodulatenuclear such asgrowth,proliferation,metabolism,orhomeostasisonatranscriptionallevel. DNA andmodulatetheexpressionoftheirtargetgenestoregulateavarietycellularmechanisms hormones orvitamins.Uponligandbinding, nuclearreceptorstranslocateintothenucleustobind Nuclear receptorsaretranscriptionfactorsthatregulatedbysmallhydrophobicligandssuchas N • • •

uclear luminescence measurements weretaken. various concentrationsfor24 hoursbefore line wasstimulatedagonist6a-FIT-R in The androgenreceptorre AR AgonistAssay: Custom-assay development translocation, activity, structuralchanges,dimerization, etc. Any classofinhibitorcanbetestedsuchasmodulators and complexenvironmentofintactcells Cell-based assaysallowfordrugtestinginthephysiological R ecptor porter cell A ssa ys AR AntagonistAssay: before luminescencereadout. of antagonistEnzalutamidefor 24hours agonist 6a-FIT-R andvariousconcentrations was stimulatedwithafixedconcentration of The androgenreceptorre Pregnane Xreceptor(PXR) Farnesoid Xreceptor(FXR) Androgen receptor(AR) Nuclear Receptors porter cellline

49 Target-Specific Assays Additional Assays 50 Target-Specific Assays Additional Assays drugs pharmacokineticsandresponses. compound profilingagainstthe6mostimportantCYPisoforms thataffect interactions.Reactionessential forpredictingdrug-drug Biologyprovides Understanding acompound’sinhibitoryactivityagainstkeyCYPproteinsis CYPs arehemeproteinsthatplaykey rolesinthemetabolismofdrugs. Cytochrome P450-CYP isoforms. Reaction BiologyprovidescompoundscreeningforbothHSP90aandHSP90b regulatory moleculesincludingmanytyrosinekinasesandtranscriptionfactors. HSP90 isanATP-dependent molecularchaperonethatstabilizesseveral Heat shockprotein90–HSP90 A dditional A ssa ys

CYP 3A4 CYP 2D6 CYP 2C9 CYP 2C19 CYP 19A CYP 1A2 CYP HSP90b HSP90a HSP90 of yourinterest. We havedevelopedover1,000assays. Useourvastexperiencetodevelopanassayfor thetarget C Assay formats availablefor customizedassaydevelopment Assay formats Electrophysiology MSD Flow cytometry Isothermal titrationcalorimetry Microscale thermophoresis Surface plasmonresonance Thermal shiftassay ELISA AlphaScreen AlphaLisa Transcription factors RNAse H2 GABA aminotransferase Ectonucleotide pyrophosphatase DNA cytidinedeaminase Dihydroorotate dehydrogenase reductase Aldo-keto Examples ofassaysdevelopedforcustomers include enzymes,protein-proteininteraction,GPCRs,nuclearreceptors,ionchannels,andmore. Reaction Biologyprovidesproteinproductionandassaydevelopmentbased oncustomerrequests.Targets ustomized A ssa

y D Radiometric assaysusing IncuCyte FRET Fluorescence Polarization NanoBRET Fluorescent peptidescreening ADP-Glo HTFR Autoradiogram afterSDS-PAGE USP proteases Sentrin-specific RASGRP PP2A/CIP2A RNA epigeneticenzymes RNA polymerase evlopmnt 3 H, H, 32 P or 33 P

51 Target-Specific Assays Custom Assay Development 52 Target-Specific Assays L ET‘S Reaction Biology|www.reactionbiology.com |[email protected] BIOPHYSICAL ASSAYS RECOMBINANT PROTEINS Microscale Thermophoresis TitrationIsothermal Calorimetry ShiftAssay Thermal PlasmonResonance Surface production Custom-tailored protein Inhibitors Substrates Epigenetic proteins Kinase proteins

D ISCOVER # ofkinases

T OGETHER IN VIVOPHARMACOLOGY TARGET-SPECIFIC ASSAYS Flow cytometry models Proprietary Metastasis models models Orthotopic Syngeneic models Xenograft models In VivoHollowFiberModel Receptor Biology Protein: ProteinInteractionassays Enzymatic activitytesting Biochemical andcell-basedassays for kinase,epigeneticandproteases in industry Largest assayportfolio . CELLULAR ONCOLOGY SAFETY &ADME-Tox Angiogenesis assay Invasion andmigrationassays 3D Tumor Spheroidassays Drug combinationscreening 2D and3Dproliferationassays Maximum-tolerated dose PK/PD studies CYP inhibition Cardiac SafetyPanel # ofkinases