Chemogenomic set

In vitro activity of closest targets:IC50(FLT1/4) = 42/74 nM, IC50(TIE2) = 59 nM, IC50(SIK) = 70 nM, IC50(FGFR1) = 78 nM

Screened at 1 µM, closest targets as % of contr.: CDK9 (10%), FLT3(D835Y) (9%), PLK1 (3%); Screened additionally against 58 kinases (radiometric kinase assay), closesttargets: IC50(FLT3) = 510 nM, IC50(MELK) = 744 nM, IC50(CK2) = 826 nM, other targets IC50 >10 µM (PMID: 21470862)

Screened at 1 µM, closest targets: IC50(PRKD2) = 139 nM, IC50(PRKD3) = 219 nM, IC50(RET) = 161 nM, IC50(FLT3) = 714 nM, IC50(LRRK2) =16 nM, IC50(PRKD1) = 35 nM, IC50(CLK1/2) = 512/ 112 nM, IC50(FGFR2) = 320 nM, IC50(PDGFRA) = 956 nM; Screened against panel of 68 receptors, ion channels and protein targets, at 10 µM, closest targets: IC50(A1A) = 914 nM, IC50(A1B) = 52 nM, IC50(A1D) = NA, IC50(A2A) = 1420 nM, IC50(AB2) = 1820 nM, IC50(imidazoline I2) = 97 nM (PMID: 22128344)

In vitro inhibition (biochemical potency): >100 fold selective for MEK, other targets >10µM

Screened at 1 µM, closest targets as % of cont.: PIK3C2B (80.4%), PIK3CA (98.6%), PIK3CD (97.9%), PIK3CG (90.6%), PIK3C3 (69.9%); IC50 follow-up of closest targets: IC50(PIK3C2B) = 292 nM, IC50(PIK3C3) = 374 nM, >1000-fold selective

Screened at 1 µM, closest targets as % of cont.: PIK3C2B (80.4%), PIK3CA (98.6%), PIK3CD (97.9%), PIK3CG (90.6%), PIK3C3 (69.9%); IC50 follow-up of closest targets: IC50(PIK3C2B) = 292 nM, IC50(PIK3C3) = 374 nM, >1000-fold selective

Screened at 1 µM, closest targets as % of cont.: PIK3C2B (80.4%), PIK3CA (98.6%), PIK3CD (97.9%), PIK3CG (90.6%), PIK3C3 (69.9%); IC50 follow-up of closest targets: IC50(PIK3C2B) = 292 nM, IC50(PIK3C3) = 374 nM, >1000-fold selective

Screened at 1 µM, closest targets as % of cont.: PIK3C2B (80.4%), PIK3CA (98.6%), PIK3CD (97.9%), PIK3CG (90.6%), PIK3C3 (69.9%); IC50 follow-up of closest targets: IC50(PIK3C2B) = 292 nM, IC50(PIK3C3) = 374 nM, >1000-fold selective

Screened at 1 µM, closest target with PoC <10%: ABL2 (5.8%)

Screened at 1 µM, closest target with PoC <10%: ABL2 (5.8%)

Screened at 10 µM, S(10) = 0.007, closest targets as % of contr.: DCAMK2 (3.6%), PLK4 (5.1%); Cellular selectivity profiling at 10 µM using KiNativ method, only ERK5 inhibited with90 % higher target occupancy;

Screened at 10 µM, > 50-fold selective

Screened at 1 µM, closest targets: Ki(LTK) = 2.7 nM, Ki(FER) = 3.3 nM, Ki(FES) = 6 nM, Ki(PTK2B) = 14 nM, Ki(TNK2) = 17 nM, Ki(PTK2) = 17 nM, Ki(NTRK1) = 24 nM, Ki(NTKR2) = 23 nM, Ki(NTRK3) = 46 nM, Ki(FRK) = 53 nM, Ki(EGFR(L858R/T790M)) = 56 nM, Ki(EGFR(T790M) = 319 nM, Ki(JAK2) = 529 nM, others >75% inhibition at 1 µM

Screened at 1 µM, closest off-targets: Ki(LTK) = 2.7 nM, Ki(FER) = 3.3 nM, Ki(FES) = 6 nM, Ki(PTK2B) = 14 nM, Ki(TNK2) = 17 nM, Ki(PTK2) = 17 nM, Ki(NTRK1) = 24 nM, Ki(NTKR2) = 23 nM, Ki(NTRK3) = 46 nM, Ki(FRK) = 53 nM, Ki(EGFR(L858R/T790M)) = 56 nM, Ki(EGFR(T790M) = 319 nM, Ki(JAK2) = 529 nM, others >75% inhibition at 1 µM

Screened at 10 µM, closest targets with < 10% of contr.: GAK, JAK1, STK16, TTK, PIK3C2G, MAP2K4; Screened against 240 kinases (Milipore panel): >10000-fold selective

Screened at 10 µM, closest targets with < 10% of contr.: GAK, JAK1, STK16, TTK, PIK3C2G, MAP2K4; Screened against 240 kinases (Milipore panel): >10000-fold selective

Screened at 10 µM, closest targets with < 10% of contr.: GAK, JAK1, STK16, TTK, PIK3C2G, MAP2K4; Screened against 240 kinases (Milipore panel): >10000-fold selective

Screened at 10 µM, closest targets with < 10% of contr.: GAK, JAK1, STK16, TTK, PIK3C2G, MAP2K4; Screened against 240 kinases (Milipore panel): >10000-fold selective

Screened at 1 µM, closest target as % of contr.: CSNK1E (39%)

Screened at 1 µM, closest targets: IC50(FLT3) = 390 nM, IC50(KIT) = 860 nM, IC50(AURKC) = 1000 nM, IC50(KDR) = 1100 nM (enzymatic assay with 100 µM ATP), other targets > 1 µM; Cellular selectivity: IC50(FLT3) > 10 µM (FLT3-ITD dependent proliferation of Ba/F3 cells); IC50(KIT) = 2.7 µM (BCR-KIT fusion dependent proliferation of Ba/F3 cells)

Screened at 10 µM, closest targets as % of contr.: GAK(4.1%), RIOK1/3 (4.1/4.2%), PDGFRB (5.3%), FLT3(D835H)(1.4%), FLT4(1.4%), KIT(D816V)(1.4%), PIP5K2B(6.6%), MAPK3K1(1.6%), PIP5K2B(6.6%), DAPK1(8.6%), FLT3(N841I) (1.7%), ROCK1 (4.7%), MST2(1.8%), SRPK1(8.9%), EPHB6(9.9%), LOK(0.0%), PLK2(0.2%), SLK(0.3%), MP2K5(0.5%), BIKE(1.0%), PIP5K1C (10%); Screened against 30 kinases, closest targets: IC50(PDGFR1b) = 160 nM, IC50(VEGFR2) = 360 nM, IC50(AURA) = 4800 nM, IC50(CDK2/cyclinA) = 7600 nM, others IC50 >10 µM (PMID: 17267659)

Screened at 10 µM, closest targets as % of contr.: GAK(4.1%), RIOK1/3 (4.1/4.2%), PDGFRB (5.3%), FLT3(D835H)(1.4%), FLT4(1.4%), KIT(D816V)(1.4%), PIP5K2B(6.6%), MAPK3K1(1.6%), PIP5K2B(6.6%), DAPK1(8.6%), FLT3(N841I) (1.7%), ROCK1 (4.7%), MST2(1.8%), SRPK1(8.9%), EPHB6(9.9%), LOK(0.0%), PLK2(0.2%), SLK(0.3%), MP2K5(0.5%), BIKE(1.0%), PIP5K1C (10%); Screened against 30 kinases, closest targets: IC50(PDGFR1b) = 160 nM, IC50(VEGFR2) = 360 nM, IC50(AURA) = 4800 nM, IC50(CDK2/cyclinA) = 7600 nM, others IC50 >10 µM (PMID: 17267659)

Screened at 10 μM, closest targets at % of contr.: DDR1 (1.1%), FLT4 (6.0%), CSF1R (6.6%), MST3 (7.0%), VEGFR1 (8.0%), YSK1 (9.4%);In-vitro potency of other targets: IC50(PDGFR, cytoplasmic domain) = 17.6 µM, IC50(c-Src) = 19.8 µM;

Screened at 10 μM, closest targets at % of contr.: DDR1 (1.1%), FLT4 (6.0%), CSF1R (6.6%), MST3 (7.0%), VEGFR1 (8.0%), YSK1 (9.4%); In-vitro potency of other targets: IC50(PDGFR, cytoplasmic domain) = 17.6 µM, IC50(c-Src) = 19.8 µM;

Screened at 10 μM, closest targets at % of contr.: DDR1 (1.1%), FLT4 (6.0%), CSF1R (6.6%), MST3 (7.0%), VEGFR1 (8.0%), YSK1 (9.4%); In-vitro potency of other targets: IC50(PDGFR, cytoplasmic domain) = 17.6 µM, IC50(c-Src) = 19.8 µM;

Screened at 1 µM, closest targets as % of contr.: ABL1-nonphosphorylated (14%), RIPK2 (30%), RPS6KA5RPS6KA5(Kin.Dom.2-C-terminal) (35%), CHEK2 (37%), PIK3CA (37%), STK38L (40%); Follow-up IC50s in cellular NanoBRET assays: all targets IC50 >25 µM (>125-fold selective);

Screened at 1 µM, closest targets as % of contr.: ABL1-nonphosphorylated (14%), RIPK2 (30%), RPS6KA5RPS6KA5(Kin.Dom.2-C-terminal) (35%), CHEK2 (37%), PIK3CA (37%), STK38L (40%); Follow-up IC50s in cellular NanoBRET assays: all targets IC50 >25 µM (>125-fold selective);

Screened at 1 µM, closest targets as % of contr.: ABL1-nonphosphorylated (14%), RIPK2 (30%), RPS6KA5RPS6KA5(Kin.Dom.2-C-terminal) (35%), CHEK2 (37%), PIK3CA (37%), STK38L (40%); Follow-up IC50s in cellular NanoBRET assays: all targets IC50 >25 µM (>125-fold selective);

Screened at 1 µM, closest targets as % of contr.: ABL1-nonphosphorylated (14%), RIPK2 (30%), RPS6KA5RPS6KA5(Kin.Dom.2-C-terminal) (35%), CHEK2 (37%), PIK3CA (37%), STK38L (40%); Follow-up IC50s in cellular NanoBRET assays: all targets IC50 >25 µM (>125-fold selective);

Screened at 100 nM, closest targets: Kd(STK10) = 5.9 nM, Ki(EPHB6) = 25 nM, Ki(ABL-1, non-phosphorylated) = 38 nM, Ki(LYN) = 40 nM; Cellular selectivity, of closest targets in HEK293T cells (NanoBRET assay): IC50(STK10) = 210 ± 97 nM (n= 2), IC50(EPHB6) = 3.9 ± 0.3 µM (n= 2), IC50(LYN) = 8.2 ± 0.86 µM (n= 2); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(OPRK1) = 124.61 nM, Ki(GABA/PBR) = 217.26 nM, Ki(TMEM97) = 983.68 nM, Ki(ADRB3) = 7099.67 nM

Screened at 100 nM, closest targets: Kd(STK10) = 5.9 nM, Ki(EPHB6) = 25 nM, Ki(ABL-1, non-phosphorylated) = 38 nM, Ki(LYN) = 40 nM; Cellular selectivity, of closest targets in HEK293T cells (NanoBRET assay): IC50(STK10) = 210 ± 97 nM (n= 2), IC50(EPHB6) = 3.9 ± 0.3 µM (n= 2), IC50(LYN) = 8.2 ± 0.86 µM (n= 2); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(OPRK1) = 124.61 nM, Ki(GABA/PBR) = 217.26 nM, Ki(TMEM97) = 983.68 nM, Ki(ADRB3) = 7099.67 nM

Screened at 100 nM, closest targets: Kd(STK10) = 5.9 nM, Ki(EPHB6) = 25 nM, Ki(ABL-1, non-phosphorylated) = 38 nM, Ki(LYN) = 40 nM; Cellular selectivity, of closest targets in HEK293T cells (NanoBRET assay): IC50(STK10) = 210 ± 97 nM (n= 2), IC50(EPHB6) = 3.9 ± 0.3 µM (n= 2), IC50(LYN) = 8.2 ± 0.86 µM (n= 2); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(OPRK1) = 124.61 nM, Ki(GABA/PBR) = 217.26 nM, Ki(TMEM97) = 983.68 nM, Ki(ADRB3) = 7099.67 nM

Screened at 100 nM, closest targets: Kd(STK10) = 5.9 nM, Ki(EPHB6) = 25 nM, Ki(ABL-1, non-phosphorylated) = 38 nM, Ki(LYN) = 40 nM; Cellular selectivity, of closest targets in HEK293T cells (NanoBRET assay): IC50(STK10) = 210 ± 97 nM (n= 2), IC50(EPHB6) = 3.9 ± 0.3 µM (n= 2), IC50(LYN) = 8.2 ± 0.86 µM (n= 2); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(OPRK1) = 124.61 nM, Ki(GABA/PBR) = 217.26 nM, Ki(TMEM97) = 983.68 nM, Ki(ADRB3) = 7099.67 nM

Screened at 1 µM, no targets within 10-fold, closest kinases IC50 >100-fold

Screened at 1 µM, >100-fold selective, closest targets: IC50(CLK4) = 225 nM, IC50(MAP3K14) = 244 nM, IC50(MAP3K5) = 428 nM, IC50(CLK2) = 605 nM, IC50(TTK) = 935 nM); PanLabs Profile showed 5 hits > 50% inhibition at 10 µM: IC50 (HTR2B) = 1.2 µM, IC50(SLC6A2) = 3.8 µM, IC50(CHRM2) = 6.4 µM, IC50(PPARG) = 6.5 µM, IC50(adenosine transporter) = 9.7 µM; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(TMEM97) = 4600.45 nM

Screened at 100 nM, closest targets as % of contr.: LATS2 (0.7%), PRKACA (28%), PRKACB (36%), LATS1 (42%), PRKG1 (49%); Screened in Upstate kinase panel against 114 kinases at 10 µM, >250 fold selective, closest targets: IC50(NTRK1) = 252 nM, IC50(FLT3) = 303 nM, IC50(PKA) = 734 nM; Screened against 45 GPCR targets (PDSP screen), closest targets: Ki(OPRK1) = 187.33 nM, Ki(SIGMAR1) = 6765.5 nM; Screened in Eurofins-Panlabs radioligand binding assay against 63 targets at 10 µM, closest targets: Ki(SLC6A3) = 0.039 µM, Ki(DAT2) = 0.027 µM;

Screened at 100 nM, closest targets as % of contr.: LATS2 (0.7%), PRKACA (28%), PRKACB (36%), LATS1 (42%), PRKG1 (49%); Screened in Upstate kinase panel against 114 kinases at 10 µM, >250 fold selective, closest targets: IC50(NTRK1) = 252 nM, IC50(FLT3) = 303 nM, IC50(PKA) = 734 nM; Screened against 45 GPCR targets (PDSP screen), closest targets: Ki(OPRK1) = 187.33 nM, Ki(SIGMAR1) = 6765.5 nM; Screened in Eurofins-Panlabs radioligand binding assay against 63 targets at 10 µM, closest targets: Ki(SLC6A3) = 0.039 µM, Ki(DAT2) = 0.027 µM;

Screened at 1 µM, >1000-fold selective, off-targets with PoC <50%

Screened at 1 µM, >1000-fold selective, off-targets with PoC <50%

Screened at 1 µM off-targets with PoC <35%

Screened at 1 µM off-targets with PoC <35%

Highly selective; screen against 45 GPCR targets, closest target: Ki(GABA/PBR) = 374.92 nM; clean in Eurofins-Panlabs radioligand binding assay, screen against 68 targets, at 10 µM

Highly selective; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(GABA/PBR) = 346.58 nM, Ki(HTR2A) = 2071.02 nM, Ki(ADRA2A) = 2355.74 nM; Screened in Eurofins-Panlabs radioligand binding assay against 68 targets at 10 µM: closest target: HTR2B (63 % of contr.)

Screened at 1 µM; 8 additional targets: AURKC, ABL1, RPS6KA6, PIKFYVE, ERBB3, RPS6KA5, LRRK2 and STK38L, initially identified, but confirmed as false positive by NanoBRET: closest targets: IC50(TYRO3) = 3.8 µM (47-fold selective), IC50(ERBB3) = 18.2 µM (227-fold selective), other targets >20 µM in NanoBRET assays.

Screened at 1 µM, closest targets as % of contr.: MAPK15 (21%), HIPK1 (31%), HIPK2 (34%), NEK7 (40%), PIP5K2B (50%), STK16 (53%), S(35) = 0.02; Follow-up IC50s in cellular NanoBRET assays: IC50(HIPK1/2) >10 µM, IC50(STK16) = 167 nM (partial inhibition), others NA;

Screened at 1 µM, closest targets as % of contr.: MAPK15 (21%), HIPK1 (31%), HIPK2 (34%), NEK7 (40%), PIP5K2B (50%), STK16 (53%), S(35) = 0.02; Follow-up IC50s in cellular NanoBRET assays: IC50(HIPK1/2) >10 µM, IC50(STK16) = 167 nM (partial inhibition), others NA;

Screened at 1 µM, closest targets as % of contr.: MAPK15 (21%), HIPK1 (31%), HIPK2 (34%), NEK7 (40%), PIP5K2B (50%), STK16 (53%), S(35) = 0.02; Follow-up IC50s in cellular NanoBRET assays: IC50(HIPK1/2) >10 µM, IC50(STK16) = 167 nM (partial inhibition), others NA;

Screened at 10 µM against 68 enzymes, transporters, GPCRs, ion chanels, closest targets: IC50(MMP2) = 44 nM, IC50(MMP10) = 13 nM, IC50(MMP8) = 15 nM, IC50(MMP13) = 67 nM, IC50(MMP14) = 250 nM, IC50(MMP3) = 360 nM, IC50(MMP9) = 460 nM, IC50(MMP7) = 600 nM, IC50(MMP16) = 940 nM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: EGFR(S752-I759del) (40%), TYK2 (41%), PNCK (46%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % inhibition: DRD2 (30%)

>2600-fold selective over other MMPs: IC50(MMP1) >10 µM, IC50(MMP2) = 18 nM, IC50(MMP3) = 600 nM, IC50(MMP7) >10 µM, IC50(MMP8) = 780 nM, IC50(MMP9) >10 µM, IC50(MMP10) = 160 nM, IC50(MMP14) >10 mM, IC50(ADAM17) >10 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: LRRK2 (43%), CDK4 (43.5%), FLT3(D835V) (44.2%), NTRK1 (46.7%); Screended against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(HRH1) = 98 nM, Ki(TMEM97) = 4553 nM; Clean profile in MSD Pharma/Taiwan screen against 118 enzymes and receptors

Highly selective (> 1000-fold) against other matrix metalloproteinases (FRET assays): IC50(MMP1) >22 µM, IC50(MMP2) = 18 µM, IC50(MMP3) >22 µM, IC50(MMP7) >22 µM, IC50(MMP8) >22 µM, IC50(MMP9) = 8.9 µM, IC50(MMP10) = 16 µM, IC50(MMP12) >22 µM, IC50(MMP14) = 8.3 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 100 nM, closest targets as % of contr.: CDK19 (0%), CDKL3 (0%), CDK3 (43%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: DRD5 (60% inhibition, Ki >10 µM); Screened against 56 targets (Invitrogen Panel) at 10 µM, closest targets: IC50(AURKA) = 1.3 µM, IC50(MAPKAPK2) = 1.1 µM, IC50(PRKAA1) = 1.9 µM, IC50(PRKACA) = 1.6 µM;

Selective in SGC bromodomain panel; Screened in Invitrogen kinase panel against 40 kinases at 10 µM, clean profile; No activity in a panel of 25 PDEs, ion channels and GPCRS, IC50 >50 µM; IC50(YEATS2/4) > 40 µM (HTRF assay);

Selective in SGC bromodomain panel; Screened in Invitrogen kinase panel against 40 kinases at 10 µM, clean profile; No activity in a panel of 25 PDEs, ion channels and GPCRS, IC50 >50 µM; IC50(YEATS2/4) > 40 µM (HTRF assay);

> 30-fold selective over 21 serine proteases (for all IC50 > 30 µM); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: RPS6KA4 (33.5%), PRKG1 (45.5%), PRKACB (48.7%); Screened against 45 GPCR targets (PSDP screen) at 10 µM, closest target: Ki(HRH3) = 3.07 µM, Ki(DTR1A) = 4.98 µM, Ki(ADRB1) = 5.45 µM; Radioligand binding assays (Lead Profiling Screen, Eurofins) against 64 receptors and transporters at 10 µM: > 30-fold selective