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  • Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe ... · We set up an IC50 kinase profiling...
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Introduction Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe, Christoph Schächtele, and Michael H.G. Kubbutat Reaction Biology Europe GmbH, Freiburg, Germany. Introduction Protein kinases belong to one of the largest families of evolutionarily related proteins. The human genome encodes more than 500 kinases. Due to the structural similarity, especially within the ATP-binding site, many kinase inhibitors showlimited selectivity. Still, sufficient selectivity within the human kinases is critical e.g. to reduce the risk of adverse side effects during treatment. Therefore, measuring and improving the selectivity of a compound within the kinome is of pivotal importance during drug discovery and optimization phase in the development of therapeutically relevant kinase inhibitors. Broad profiling of kinase inhibitors in biochemical activity assays of several hundred kinases is nowadays well established. Usually, kinase profiling is done using one or two concentrations of a test compound and measurement of the relative inhibition of the kinase activity compared to a high and low control. However, due to the limited dynamic range of this approach, and the challenge of selecting the most appropriate compound concentration, this profiling approach oftengives limited information with respect to the potency of compounds against on- and off-target kinases. We set up an IC50 kinase profiling approach that consists of measuring the impact of a compound on the activity of 320 human protein kinases at six different concentrations with the 33 PanQinase TM assay format. Here, we present data showing the effect of compound concentration on the selectivity score in traditional profiling settings. IC50 kinase profiles of different approved and clinical stage kinase inhibitors demonstrate that an IC50 based profiling allows a more accurate determination of selectivity of a compound based on the comparison of the IC50 values against the on-target kinases in relation to the IC50 values of the off-target kinases providing significantly improved guidance in the further optimization of the test compound. 33 PanQinase TM : Principle of the assay The assay is based on radiolabelled 33 P-γ- ATP. Kinase and substrate are incubated in presence of ATP containing 33 P-γ-ATP as tracer in 96 well FlashPlates (Perkin Elmer). After stopping the kinase reaction, the proteins are immobilized to the plate surface and bound radioactivity is quantified by scintillation counting. All assay steps are performed using a robotic pipetting system 96 well FlashPlate per well: • 10 μl ATP/ 33 P-γ-ATP mix • 25 μl 2.5x assay buffer • 5 μl compound in 10% DMSO • 10 μl kinase-substrate mix • - mix on a plate shaker • - incubate for 60 min at 30°C • - stop reaction with 50 μl 2% (v/v) H 3 PO 4 • mix on a plate shaker • wash 2 times with 200 μl 0.9% (w/v) NaCl • count dry plate with a scintillation counter Final assay conditions were modified for those kinases requiring specific cofactors for kinase activity. • 70 mM HEPES pH 7.5 • 3 mM MgCl 2 • 3 mM MnCl 2 • 3 μM Na-orthovanadate • 1.2 mM DTT • 1 % (v/v) DMSO • ATP*, substrate and kinase at variable concentration *: [ATP]: apparent K M [ATP] of the respective kinase Kinase and substrate combinations are selected for optimal results under the assay conditions described above. Kinase concentration is typically 1-40 nM (assuming 100% pure and active protein preparations), substrate concentration is 10-30 times higher than the kinase concentration. Results ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AKT1AKT3AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT CSK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY RK4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 JAK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b eta p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 PRKX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SRC SRPK1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 T TB K 1 T TK T YK 2 VEGF-R1 VEGF-R3VRK2W NK1W NK3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AKT1AKT3AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT CSK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY RK4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 JAK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b eta p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 PRKX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SRC SRPK1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 T TB K 1 T TK T YK 2 VEGF-R1 VEGF-R3VRK2W NK1W NK3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AKT1AKT3AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT CSK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY RK4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 JAK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b eta p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 PRKX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SRC SRPK1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 T TB K 1 T TK T YK 2 VEGF-R1 VEGF-R3VRK2W NK1W NK3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AKT1AKT3AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT CSK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY RK4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 JAK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b eta p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 PRKX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SRC SRPK1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 T TB K 1 T TK T YK 2 VEGF-R1 VEGF-R3VRK2W NK1W NK3 ZAK 0.1 nM 1 nM 10 nM 100 nM 1000 nM 10000 nM ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK ABL1 ACK1ACV-R1B ACV-R2B AK T1 AK T3 AM PK - alp h a1 ASK1 Aurora-B AXL BMPR1A B-RAF BRSK1 BTK CAMK1D CAMK2B CAMK2G CAMKK1 CDC42BPA CDC7/DBF4 CDK1/CycB1 CDK16/Cyc Y CDK2/CycA2 CDK3/Cy cC CDK4/Cy cD1 CDK5/p25NCK CDK6/Cy cD1 CDK7/Cy cH/M AT1 CDK9/Cy cK CHK1 CK 1- al ph a 1 CK 1- e psi lon CK 1- ga mma 2 CK 2- al ph a 1 CLK1 CLK3 COT C SK DAPK2 DCAM KL 2 DM PK DY RK1 A DY RK2 DY R K4 E GF- R E IF2 AK 3 E PH A2 E PH A4 E PH A6 E PH A8 E PH B2 E PH B4 ERBB4 E RK 2 E RK 7 FE R FG F- R1 FG F- R3 FG R FRK GRK2 GRK4 GRK6 GS G2 GS K3 - b et a HI PK 1 HI PK 3 HRI IK K -a lp h a IK K -e p silo n IN SR- R IRAK4 J AK1 J AK3 J NK 2 K IT LI MK 1 LRRK2 LY N M AP3 K 10 M AP3 K 7/M AP3 K 7 IP 1 M AP4 K 2 M AP4 K 5 M APK AP K3 M ARK 1 M ARK 3 M ATK M EK 2 M EK K 2 M ELK M ET M KK 4 M KK 7 M KN K2 M ST 1 M ST 3 mT OR M YLK M YLK 3 NE K 11 NE K 3 NE K 6 NE K 9 NL K p 38 - b et a p 38 - ga mma P AK2 P AK4 P AK7 PBK P DGF R-b e ta P HK G1 P IM 1 P IM 3 P KC- al ph a P KC- b et a 2 P KC- e psi lon P KC- ga mma PKC-mu P KC- t he t a P KM YT 1 PLK3 PRK2 P RKG 1 P R KX RAF1 Y340D/Y341D RIPK2 ROCK1 RON RPS6KA1 RPS6KA3 RPS6KA5 S6 K SAK SG K2 SI K1 SI K3 SN ARK SR C SR P K 1 ST K 17 A ST K 25 ST K 39 TAOK2 T BK 1 T GFB - R 1 T IE2 T LK 2 TRK-A TRK-C T SK 2 TTBK1 T TK T YK 2 VEGF-R1 VE GF- R3 VRK2W NK 1W NK 3 ZAK 0.1 nM 1 nM 10 nM 100 nM 1000 nM 10000 nM 320 kinases were tested with Crizotinib (left) and Dinaciblib (right) in 6 concentrations. For each compound concentration, the percent inhibition was determined for the respective kinase relative to the non-inhibited positive control. The circles represent from the inside 25, 50, 75, and 100 % inhibition. ACK1 ALK CAMK2G CAMKK2 CLK2 FAK FER FES IGF1-R INS-R INSR-R LRRK2 LTK MKNK2 ROS TNK1 TSSK1 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 pIC50 Kinase Ceritinib CDK19/CycC CDK8/CycC DDR2 ERK7 FGF-R2 FLT3 HIPK4 MUSK PDGFR-alpha PDGFR-beta RAF1 RET TAOK2 TRK-A TRK-B VEGF-R1 VEGF-R2 ZAK 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 pIC50 Kinase pIC50 Threshold Value Sorafenib ALK AXL FAK LCK LTK MAP4K5 MERTK MET MUSK RON ROS SLK TRK-A TRK-B TRK-C 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 pIC50 Kinase Crizotinib IC 50 values were determined for compound Crizotinib, Ceritinib, and Sorafenib by determining the residual activities in the presence of 6 increasing, logarithmically diluted concentrations of the respective compounds. From these six values IC 50 values were calculated using Graphpad Prism 5.04. The mathematical model used was "Sigmoidal response (variable slope)" with parameters "top" fixed at 100 % and "bottom" at 0 %. pIC 50 (negative decimal logarithm of IC 50 values) were depicted in the graphs for better visualization of the range of the compound potency. An arbitrary threshold value 20 fold below the pIC 50 of the most sensitive kinase was set to select kinases showing significant inhibition by the respective compound. Red circles indicate the primary target of the respective compound. Summary & Conclusion 1. Apparent kinase inhibitor selectivity depends on the compound concentration used to measure percental inhibition of kinases 2. Accuracy and reproducibility of profiling are limited by the dynamic range and assay variability of single compound concentration measurement. 3. IC 50 selectivity profiling based on six compound concentrations represents a significant improvement to assess differences in the potency of a given compound and allows a more precise judgment of the selectivity of protein kinase inhibitors 0,0 0,1 0,2 0,3 0,4 0,5 0,1 1 10 100 1000 10000 Selectivity Score S 50 Compound concentration [nM] Sorafenib Crizotinb Selectivity scores were determined for Sorafenib, Crizotinib and Ponatinib for a panel of 320 protein kinases at different compound concentrations based on residual activities. Selectivity scores were calculated using the formula: (count of data points < 50 %) / (total number of data points). 0,036 0,048 0,077 0,081 0,130 0,188 0,188 0,217 0,270 0,0 0,1 0,2 0,3 10x 20x 50x IC50 Selectivity Score Threshold x IC50 (target) Sorafenib (VEGFR2) Crizotinib (ALK) Ponatinib (ABL1) Selectivity scores were determined for Sorafenib, Crizotinib and Ponatinib using a panel of 320 protein kinases based on IC 50 values calculated from six different compound concentrations in the assay. Selectivity scores were compared with different threshold values of 10/20/50 fold of the target IC 50 value based on the calculation: count of IC 50 values < threshold) / total number of IC 50 values determined R² = 0,9614 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00 The pIC50 profile of compound Ponatinib was determined twice in independent experiments and the resulting pIC50 values for each kinase were compared pIC50 values of experiment 2 pIC50 values or experiment 1

Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe ... · We set up an IC50 kinase profiling approach that consists of measuring the impact of a compound on the activity of

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Page 1: Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe ... · We set up an IC50 kinase profiling approach that consists of measuring the impact of a compound on the activity of

Intro

du

ction

Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe, Christoph Schächtele, and Michael H.G. Kubbutat

Reaction Biology Europe GmbH, Freiburg, Germany.

Introduction

Protein kinases belong to one of the largest families of evolutionarily related proteins. The human genome encodes more than 500 kinases. Due to the

structural similarity, especially within the ATP-binding site, many kinase inhibitors show limited selectivity. Still, sufficient selectivity within the human kinases

is critical e.g. to reduce the risk of adverse side effects during treatment.

Therefore, measuring and improving the selectivity of a compound within the kinome is of pivotal importance during drug discovery and optimization

phase in the development of therapeutically relevant kinase inhibitors.

Broad profiling of kinase inhibitors in biochemical activity assays of several hundred kinases is nowadays well established. Usually, kinase profiling is done

using one or two concentrations of a test compound and measurement of the relative inhibition of the kinase activity compared to a high and low control.

However, due to the limited dynamic range of this approach, and the challenge of selecting the most appropriate compound concentration, this profiling

approach oftengives limited information with respect to the potency of compounds against on- and off-target kinases.

We set up an IC50 kinase profiling approach that consists of measuring the impact of a compound on the activity of 320 human protein kinases at six

different concentrations with the33

PanQinaseTM

assay format. Here, we present data showing the effect of compound concentration on the selectivity

score in traditional profiling settings. IC50 kinase profiles of different approved and clinical stage kinase inhibitors demonstrate that an IC50 based

profiling allows a more accurate determination of selectivity of a compound based on the comparison of the IC50 values against the on-target kinases in

relation to the IC50 values of the off-target kinases providing significantly improved guidance in the further optimization of the test compound.

33PanQinase

TM: Principle of the assay

The assay is based on radiolabelled33

P-γ-

ATP. Kinase and substrate are incubated in

presence of ATP containing33

P-γ-ATP as

tracer in 96 well FlashPlates (Perkin Elmer).

After stopping the kinase reaction, the

proteins are immobilized to the plate surface

and bound radioactivity is quantified by

scintillation counting.

All assay steps are performed using a robotic

pipetting system 96 well FlashPlate per well:

• 10 µl ATP/33

P-γ-ATP mix

• 25 µl 2.5x assay buffer

• 5 µl compound in 10% DMSO

• 10 µl kinase-substrate mix

• - mix on a plate shaker

• - incubate for 60 min at 30°C

• - stop reaction with 50 µl 2% (v/v) H3PO

4

• mix on a plate shaker

• wash 2 times with 200 µl 0.9% (w/v) NaCl

• count dry plate with a scintillation counter

Final assay conditions were modified for those kinases

requiring specific cofactors for kinase activity.

• 70 mM HEPES pH 7.5

• 3 mM MgCl2

• 3 mM MnCl2

• 3 µM Na-orthovanadate

• 1.2 mM DTT

• 1 % (v/v) DMSO

• ATP*, substrate and kinase at variable concentration

*: [ATP]: apparent KM[ATP] of the respective kinase

Kinase and substrate combinations are selected for optimal results under the assay conditions described above. Kinase concentration is typically 1-40 nM (assuming

100% pure and active protein preparations), substrate concentration is 10-30 times higher than the kinase concentration.

Results

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

EPH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST 1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

STK17A

ST K25

ST K39

T AOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-AT RK-C

T SK2T TBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

EPH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST 1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

STK17A

ST K25

ST K39

T AOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-AT RK-C

T SK2T TBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

EPH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST 1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

STK17A

ST K25

ST K39

T AOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-AT RK-C

T SK2T TBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK

ABL1

ACK1 ACV-R1BACV-R2BAKT1 AKT3AM PK-alpha1

ASK1Aurora -B

AXLBMPR1A

B-RAFBRSK1

BTKCAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

EPH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST 1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

STK17A

ST K25

ST K39

T AOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-AT RK-C

T SK2T TBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK

0.1 nM 1 nM 10 nM

100 nM 1000 nM 10000 nM

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

E PH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3

AM PK-alpha1ASK1

Aurora -BAXL

BMPR1AB-RAF

BRSK1BTK

CAMK1D

CAMK2B

CAMK2G

CAMKK1

CDC42BPA

CDC7/DBF4

CDK1/Cy cB1

CDK16/Cyc Y

CDK2/Cy cA2

CDK3/Cy cC

CDK4/Cy cD1

CDK5/p25NCK

CDK6/Cy cD1

CDK7/Cy cH/M AT1

CDK9/Cy cK

CHK1

CK1-al pha 1

CK1-epsi lon

CK1-ga mma 2

CK2-al pha 1

CLK1

CLK3

COT

CSK

DAPK2

DCAM KL 2

DM PK

DY RK1A

DY RK2

DY RK4

EGF-R

E IF2AK3

EPH A2

E PH A4

EPH A6

EPH A8

EPH B2

EPH B4

ERBB4

ERK2

ERK7

FER

FG F-R1

FG F-R3

FG R

FRK

GRK2

GRK4

GRK6

GS G2

GS K3-beta

HI PK1

HI PK3HRI

IKK-a lphaIKK-epsilon

IN SR-RIRAK4

JAK1JAK3

JNK2KITLI MK1LRRK2LY NM AP3K10

M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3

M ATKM EK2

M EKK2M ELK

M ETM KK4

M KK7M KN K2

M ST1

M ST 3

mT OR

M YLK

M YLK3

NEK11

NEK3

NEK6

NEK9

NL K

p38-beta

p38-ga mma

PAK2

PAK4

PAK7

PBK

PDGF R-beta

PHKG1

PIM 1

PIM 3

PKC-al pha

PKC-beta 2

PKC-epsi lon

PKC-ga mma

PKC-mu

PKC-theta

PKM YT 1

PLK3

PRK2

PRKG 1

PRKX

RAF1 Y 340D/Y341D

RIPK2

ROCK1

RON

RPS6KA1

RPS6KA3

RPS6KA5

S6K

SAK

SG K2

SI K1

SI K3

SN ARK

SRC

SRPK1

ST K17A

ST K25

ST K39

TAOK2

T BK1

T GFB-R1

T IE2T LK2

T RK-ATRK-C

T SK2TTBK1

T TKT YK2

VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK

0.1 nM 1 nM 10 nM

100 nM 1000 nM 10000 nM

320 kinases were tested with Crizotinib (left) and Dinaciblib (right) in 6 concentrations. For each compound concentration, the percent inhibition was

determined for the respective kinase relative to the non-inhibited positive control. The circles represent from the inside 25, 50, 75, and 100 % inhibition.

ACK1

ALK

CAMK2G

CAMKK2

CLK2

FAKFER

FES IGF1-RINS-R

INSR-R

LRRK2

LTK

MKNK2

ROS TNK1

TSSK1

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

pIC

50

Kinase

Ceritinib

CDK19/CycC

CDK8/CycCDDR2

ERK7

FGF-R2

FLT3

HIPK4

MUSKPDGFR-alpha PDGFR-beta

RAF1

RET

TAOK2

TRK-ATRK-BVEGF-R1

VEGF-R2

ZAK

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

pIC

50

Kinase

pIC50 Threshold Value

Sorafenib

ALK

AXLFAK

LCK

LTK

MAP4K5MERTK

MET

MUSK RON

ROS

SLK

TRK-A

TRK-BTRK-C

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

pIC

50

Kinase

Crizotinib

IC50

values were determined for compound Crizotinib, Ceritinib, and

Sorafenib by determining the residual activities in the presence of 6 increasing,

logarithmically diluted concentrations of the respective compounds. From these six

values IC50

values were calculated using Graphpad Prism 5.04. The mathematical

model used was "Sigmoidal response (variable slope)" with parameters "top" fixed at

100 % and "bottom" at 0 %. pIC50

(negative decimal logarithm of IC50

values) were

depicted in the graphs for better visualization of the range of the compound

potency. An arbitrary threshold value 20 fold below the pIC50

of the most sensitive

kinase was set to select kinases showing significant inhibition by the respective

compound. Red circles indicate the primary target of the respective compound.

Summary & Conclusion

1. Apparent kinase inhibitor selectivity depends on the compound concentration used to measure

percental inhibition of kinases

2. Accuracy and reproducibility of profiling are limited by the dynamic range and assay variability

of single compound concentration measurement.

3. IC50

selectivity profiling based on six compound concentrations represents a significant

improvement to assess differences in the potency of a given compound and allows a more

precise judgment of the selectivity of protein kinase inhibitors

0,0

0,1

0,2

0,3

0,4

0,5

0,1 1 10 100 1000 10000

Sele

ctiv

ity

Sco

re S

50

Compound concentration [nM]

Sorafenib Crizotinb

Selectivity scores were

determined for Sorafenib, Crizotinib

and Ponatinib for a panel of 320

protein kinases at different compound

concentrations based on residual

activities. Selectivity scores were

calculated using the formula:

(count of data points < 50 %) / (total

number of data points).

0,0360,048

0,0770,081

0,130

0,1880,188

0,217

0,270

0,0

0,1

0,2

0,3

10x 20x 50x

IC5

0 S

elec

tivi

ty S

core

Threshold x IC50 (target)

Sorafenib (VEGFR2) Crizotinib (ALK) Ponatinib (ABL1)

Selectivity scores were determined

for Sorafenib, Crizotinib and Ponatinib using a

panel of 320 protein kinases based on IC50

values calculated from six different compound

concentrations in the assay. Selectivity scores

were compared with different threshold values

of 10/20/50 fold of the target IC50

value

based on the calculation:

count of IC50

values < threshold) / total

number of IC50

values determined

R² = 0,9614

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00

The pIC50

profile of compound

Ponatinib was determined

twice in independent

experiments and the

resulting pIC50 values for

each kinase were

compared

pIC50 values of experiment 2

pIC

50

val

ues

or

exp

erim

ent

1

SKRIPSI - core.ac.uk Rumus Kimia Flavonoid ... IC50 ekstrak akar jarak merah remaserasi kinetik sebesar 98,63

SKRIPSI - core.ac.uk Rumus Kimia Flavonoid ... IC50 ekstrak akar jarak merah remaserasi kinetik sebesar 98,63

Documents
PATHE Handbook

PATHE Handbook

Documents
SILLY LOVE SONGS / COOK OF THE HOUSE EMI-PATHE : SP 474

SILLY LOVE SONGS / COOK OF THE HOUSE EMI-PATHE : SP 474

Documents
Y.HMJ.media.bufvc.ac.uk/newsonscreen2/Pathe/109290/NoS_109290_other… · R.M.Bradbury, News Sei RECEIVED! 14JUL1960//-/^ Lee ''PEACE PURSUING" DISTRICT CONVENTION OF JEHOVAH'S WITNESSES

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