Chemogenomic set

Screened at 1 µM (activity assay in HTLA cells), closest targets: Ki(DRD4 )= 4.9 nM, Ki(HTR1A) = 223 nM, Ki(HTR2B) = 236.67 nM, Ki(NMDA) = 1004 nM, Ki(HRH2) = 1909.67, Ki(SLC6A3) = 2116.67 nM; Screened against D2-like family: > 3300-fold selective (DRD2 Ki > 10 µM, DRD3 Ki >10 µM)

Screened against human CYP epoxygenases (Boehringer panel) CYP2J2, CYP2C9, CYP2C19 and IL2: IC50 >1 µM for related targets (> 100-fold selective); Screened against 67 targets (Eurofins-Panlabs) at 10 µM, closest targets: TBXAS1 (96% inhibition, IC50 = 132 nM), 5-Lipoxygenase (66% inhibition, IC50 = 5.92 µM); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(SLC6A3) = 4.48 µM, Ki(SIGMAR1) = 6.28 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: CHEK2 (0%), TYRO3 (0%), JAK1 (JH2domain-pseudokinase) (37%)

Screened at 10 µM (binding assay), closest targets as % inhibition: BZD peripheral (95.5%), ADORA3 (63.9%) and ADRA2B (63.5%); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest target: HIPK4 (51%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(GABA/PBR) = 19.59 nM, Ki(SIGMAR1) = 314.63 nM, Ki(TMEM97) = 1192.34;Screened in Eurofins-Cerep panel against 7 targets at 10 µM (enzymes and uptake assays): clean profile;

inactive at 1 µM for VDR, NR1I2, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(RARB)=13.49 , inactive at 1 µM for RARG, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(NR2C1) = 1.5, fold-activation(NR2E1) = 2.03, inactive at 1 µM for RARA, UL48, RXRA, HNF4A, NR4A1, NR5A1

fold-activation(NR2C1) = 1.5, fold-activation(NR2E1) = 2.03, inactive at 1 µM for RARA, UL48, RXRA, HNF4A, NR4A1, NR5A1

fold-activation(PPARA) = 14.79, fold-activation(PPARG) = 10.47, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(NR2C1) = 1.98, fold-activation(NR2E1) = 1.82, inactive at 3 µM for UL48, RXRA, HNF4A, NR4A1, NR5A1

fold-activation(NR2C1) = 1.98, fold-activation(NR2E1) = 1.82, inactive at 3 µM for UL48, RXRA, HNF4A, NR4A1, NR5A1

inactive at 1 µM for NR1H3, NR1H2, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 10 µM for VDR, NR1I3, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

1.2 fold activation for VDR, active at 1 µM for VP16, inacitve at 1 µM for NR1I3 and RXRA

inactive at 1 µM for VDR, NR1I3, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARG) = 3.39, inactive at 1 µM for PPARD, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for NR1I2, NR1I3, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for NR1I2, NR1I3, UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

In vitro follow up of closest targets: Kd(IKKB) = 170 nM, Kd(YSK4) = 200 nM, Kd(CDK11A) = 399 nM, Kd(CDK11B) = 420 nM, Kd(CDK13) = 800 nM, Kd(CDK7) = 680 nM, Kd(MYLK4) = 700 nM; S(300nM) = 0.0052, S(3µM) = 0.0440;

Closest targets: Kd(GAK) = 79 nM, Kd(BLK) = 220 nM, Kd(ABL,F317L) = 230 nM, Kd(IRAK1) = 240 nM, KD(EPHA6) = 340 nM, Kd(HIPK4) = 360 nM, Kd(ABL,Q252H) = 380 nM, Kd(ABL,E255K) = 420 nM, Kd(PHKG2) = 470 nM,Kd(ABL,H396P) = 500 nM, Kd(ABL,phos.) = 570 nM, Kd(LCK) = 570 nM, Kd(ABL,F317L, nonphos.) = 580 nM, Kd(ABL,F317I, phos.) = 750 nM, Kd(ABL,Q252H, nonphos.) = 790 nM, Kd(ABL,Y253F, phos.) = 830 nM, Kd(ABL,T315I, phos.) = 870 nM, Kd(DYRK1A) = 970 nM, others >1 µM; Screened also against 52 kinases, closest targets: IC50(LYN) = 736 nM, IC50(ABL,T315I)= 1180 nM, others >2 µM (PMID: 18408761)

Closest targets: Kd(GAK) = 79 nM, Kd(BLK) = 220 nM, Kd(ABL,F317L) = 230 nM, Kd(IRAK1) = 240 nM, KD(EPHA6) = 340 nM, Kd(HIPK4) = 360 nM, Kd(ABL,Q252H) = 380 nM, Kd(ABL,E255K) = 420 nM, Kd(PHKG2) = 470 nM,Kd(ABL,H396P) = 500 nM, Kd(ABL,phos.) = 570 nM, Kd(LCK) = 570 nM, Kd(ABL,F317L, nonphos.) = 580 nM, Kd(ABL,F317I, phos.) = 750 nM, Kd(ABL,Q252H, nonphos.) = 790 nM, Kd(ABL,Y253F, phos.) = 830 nM, Kd(ABL,T315I, phos.) = 870 nM, Kd(DYRK1A) = 970 nM, others >1 µM; Screened also against 52 kinases, closest targets: IC50(LYN) = 736 nM, IC50(ABL,T315I)= 1180 nM, others >2 µM (PMID: 18408761)

Closest targets: Kd(GAK) = 79 nM, Kd(BLK) = 220 nM, Kd(ABL,F317L) = 230 nM, Kd(IRAK1) = 240 nM, KD(EPHA6) = 340 nM, Kd(HIPK4) = 360 nM, Kd(ABL,Q252H) = 380 nM, Kd(ABL,E255K) = 420 nM, Kd(PHKG2) = 470 nM,Kd(ABL,H396P) = 500 nM, Kd(ABL,phos.) = 570 nM, Kd(LCK) = 570 nM, Kd(ABL,F317L, nonphos.) = 580 nM, Kd(ABL,F317I, phos.) = 750 nM, Kd(ABL,Q252H, nonphos.) = 790 nM, Kd(ABL,Y253F, phos.) = 830 nM, Kd(ABL,T315I, phos.) = 870 nM, Kd(DYRK1A) = 970 nM, others >1 µM; Screened also against 52 kinases, closest targets: IC50(LYN) = 736 nM, IC50(ABL,T315I)= 1180 nM, others >2 µM (PMID: 18408761)

Closest targets with Kd <100 nM: NTRK2, targets with 100 nM < Kd < 1 µM: NTRK1, NTRK3, others >1 µM

S(Kd < 300 nM) =0.0026, S(Kd < 3µM) = 0.0078

S(Kd < 300 nM) =0.0026, S(Kd < 3µM) = 0.0078

Screened at 10 µM, closest targets as % inhibition: PDGFRA(V561D) (96%), RPS6KA6 (92%), RPS6KA3(92%), PKN2(92%), RPS6KA1(91%), PIM1 (90%); Screened against 48 kinases (GlaxoSmithKline panel), closest target: IC50(NEK2) = 158 nM;

Screened at 10 µM, closest targets as % of contr.: PLK1(4.3%); In vitro follow up of closest targets: IC50(GAK) = 140 nM, IC50(PLK1/3/4) = 61/ 1600/ 3100 nM, IC50(STK33) = 5000 nM

Screened at 10 µM, closest targets as % of contr.: PLK1(4.3%); In vitro follow up of closest targets: IC50(GAK) = 140 nM, IC50(PLK1/3/4) = 61/ 1600/ 3100 nM, IC50(STK33) = 5000 nM

Screened at 10 µM, closest targets as % of contr.: ALK(8.9%), CLK1(7.3%), ERK2(6%), GAK(5.4%), IGF1R(0.9%), INSR(0.15%), INSRR(2%), JNK1(5.2%), LTK(1.2%), MEK4(6.2%), PRK2(0.1%), PTK2B(0.5%), RIOK3(0.5%); In vitro follow up of closest targets: IC50(ALK) = 21 nM, IC50(CLK1) = 1900 nM, IC50(ERK2) = 2900 nM, IC50(FAK) = 440 nM, IC50(GAK) = 1100 nM, IC50(IGF1R) = 750 nM, IC50(INSR) = 470 nM, IC50(INSRR) = 1200 nM, IC50(LTK) = 29 nM, IC50(PYK2) = 280 nM, IC50(TNK1) = 2600 nM, IC50(TNK2) = 3100 nM;

Screened at 0.5 µM, closest targets: IC50(DAPK3) = 17 nM, IC50(FLT3) = 35 nM, IC50(TBK1) = 35 nM, IC50(CLK3) = 41 nM, IC50(HIPK3) = 45 nM, IC50(PIM1) = 46 nM, IC50(CDK1/cyclinB) = 56 nM (radiometric filter-binding assay, conc. ATP = Km); Follow-up in cellular assays: IC50(FLT3/ PIM1/ CDK1) >10 µM;

Screened at 1 µM, closest targets as % inhibition: MAP4K2 (99%), MET(86%); Screened also with ProfilerPro® Kinase Selectivity Assay Kit (Caliper Life Sciences) at 1 µM, 88 kinases <50% inhibition, clean in IC50 follow up assay: IC50 >10 µM (PMID: 21425998)

Screened at 1 µM, closest targets as % inhibition: MAP4K2 (99%), MET(86%); Screened also with ProfilerPro® Kinase Selectivity Assay Kit (Caliper Life Sciences) at 1 µM, 88 kinases <50% inhibition, clean in IC50 follow up assay: IC50 >10 µM (PMID: 21425998)

Screened at 1 µM, closest targets as % inhibition: MAP4K2 (99%), MET(86%); Screened also with ProfilerPro® Kinase Selectivity Assay Kit (Caliper Life Sciences) at 1 µM, 88 kinases <50% inhibition, clean in IC50 follow up assay: IC50 >10 µM (PMID: 21425998)

Screened at 1 µM and 10 µM, no target >50% inhibition; Screened in Dundee panel (51 kinases at 10 µM), no target >50% inhibition; Screened in Pfizer panel (48 kinases), no target >50% inhibition; Screened in Millipore panel (105 kinases at 1 µM an 10 µM), closest targets: ALK, EpHB2, INSR; IC50 follow-up showed all IC50 >10 µM; PF-04217903 is >1000-fold selective for c-Met;

Screened at 20 µM, closest targets with dTm >5K: dTm(STK6) = 8.23K, dTm(STK10) = 7.64 K, dTm(PLK4) = 5.33 K, further targets dTm 3-5K: dTm(BMX) = 4.14 K, dTm(ULK3) = 4.13 K, dTm(SLK) = 3.82 K, dTm(AURKB) = 3.81 K, dTm(ABL1) = 3.52 K; HotSpot assay (Reaction Biology), 30 targets, in-vitro potencies of closest targets: IC50(ABL1) = 705.9 nM, IC50(BTK) = 871.7 nM, IC50(LCK) = 432.3 nM, IC50(LYN) = 346.7 nM, https://pubmed.ncbi.nlm.nih.gov/22566699/;

Screened at 20 µM, closest targets with dTm >1K: dTm(MARK3) = 1.76 K, dTm(MST4) = 1.33 K, dTm(GSK3B) = 1.33 K; Screened against 88 targets in functional assays (AstraZeneca): >200-fold with exception of PIK3 isoforms, PIK3CA (86-fold) and PIK3CG (108-fold), as well as DNA-PK (42-fold), IC50(PIK3CA) = 0.87 µM (AlphaScreen), IC50(PIK3CG) = 1.09 µM (AlphaScreen), IC50(DNA-PK) = 0.42 µM (ELISA), https://pubmed.ncbi.nlm.nih.gov/22906130/;

Screened at 20 µM, closest targets with dTm >1K: dTm(MARK3) = 1.76 K, dTm(MST4) = 1.33 K, dTm(GSK3B) = 1.33 K; Screened against 88 targets in functional assays (AstraZeneca): >200-fold with exception of PIK3 isoforms, PIK3CA (86-fold) and PIK3CG (108-fold), as well as DNA-PK (42-fold), IC50(PIK3CA) = 0.87 µM (AlphaScreen), IC50(PIK3CG) = 1.09 µM (AlphaScreen), IC50(DNA-PK) = 0.42 µM (ELISA), https://pubmed.ncbi.nlm.nih.gov/22906130/;

Screened at 10 µM, Ambit score <10 at 10 µM, off-targets with PoC <50%

Screened at 1 µM, in-vitro potency of closest targets: IC50(ASK2)= 510 nM, IC50(MEKK1) >10 µM, IC50(TAK1) >10 µM, IC50(IKKB) >10 µM, IC50(ERK1) >10 µM, IC50(JNK1) >10µM, IC50(MAPK14) >10 µM, IC50(GSK3B) >10 µM, IC50(PRKCT) >10 µM; Screened against 12 kinases (Invitrogen, enzymatic assays), in-vitro potency of closest targets: IC50(CDK2) = 0.158 µM, IC50(EGFR) <20 µM, IC50(EPHA3) <20 µM, IC50(ERBB4) <20 µM, IC50(GSK3B) = 0.832 µM, IC50(ITK) = 5.5 µM, IC50(MER) = 6.5 µM, IC50(MAPK14) <20 µM, IC50(PRKAA1) = 13.8 µM, IC50(RET) = 11.5 µM, IC50(ROCK1) = 4.9 µM, IC50(RON) <20 µM, https://pubmed.ncbi.nlm.nih.gov/28291695/; Screened at 20 µM against 100 kinases, DSF panel, closest targets with dTm >5 K: dTm(MAP3K5) = 12.41 K, dTm(CLK1) = 5.46 K, closest targets with dTm 3-5 K: dTm(CAMKK2) = 4.55 K, dTm(RPS6KA5) = 3.13 K, in-house data SGC Frankfurt;

Screened at 10 µM (Ambit phage-based competition binding assay), closest targets as % of contr.: FRK (0.55%), FLT4 (0.85%), FLT3 (0.05%), EPHA3 (0.1%), CSF1R (0.8%), BLK (0.35%), STK10 (0%), AURKB (0%), KIT (0%), LCK (0.2%), MUSK (0%), PDGFRA/B (0.2/0.7%), TEK (0.15%), NTRK1/2/3 (0.55/ 0.1/ 0.1%), KDR (0.2%), MKNK2 (0.75%); In vitro follow up of closest targets: IC50(MERTK) = 14 nM, IC50(FLT3) = 16 nM, IC50(AURKB) = 78 nM, IC50(LCK) = 120 nM, IC50(KDR) = 180 nM, IC50(NTRK1/2) = 290/ 190 nM, IC50(PKA) = 550, IC50 >2 µM for: BTK, CDK2E, EGFR, GSK3B, IGFR1R, IKK1/2, INSR, JAK2, MAPKAPK2, MAPK14, PIM1, PLK1/3

Screened at 10 µM (Ambit phage-based competition binding assay), closest targets as % of contr.: FRK (0.55%), FLT4 (0.85%), FLT3 (0.05%), EPHA3 (0.1%), CSF1R (0.8%), BLK (0.35%), STK10 (0%), AURKB (0%), KIT (0%), LCK (0.2%), MUSK (0%), PDGFRA/B (0.2/0.7%), TEK (0.15%), NTRK1/2/3 (0.55/ 0.1/ 0.1%), KDR (0.2%), MKNK2 (0.75%); In vitro follow up of closest targets: IC50(MERTK) = 14 nM, IC50(FLT3) = 16 nM, IC50(AURKB) = 78 nM, IC50(LCK) = 120 nM, IC50(KDR) = 180 nM, IC50(NTRK1/2) = 290/ 190 nM, IC50(PKA) = 550, IC50 >2 µM for: BTK, CDK2E, EGFR, GSK3B, IGFR1R, IKK1/2, INSR, JAK2, MAPKAPK2, MAPK14, PIM1, PLK1/3

Screened at 10 µM (Ambit phage-based competition binding assay), closest targets as % of contr.: FRK (0.55%), FLT4 (0.85%), FLT3 (0.05%), EPHA3 (0.1%), CSF1R (0.8%), BLK (0.35%), STK10 (0%), AURKB (0%), KIT (0%), LCK (0.2%), MUSK (0%), PDGFRA/B (0.2/0.7%), TEK (0.15%), NTRK1/2/3 (0.55/ 0.1/ 0.1%), KDR (0.2%), MKNK2 (0.75%); In vitro follow up of closest targets: IC50(MERTK) = 14 nM, IC50(FLT3) = 16 nM, IC50(AURKB) = 78 nM, IC50(LCK) = 120 nM, IC50(KDR) = 180 nM, IC50(NTRK1/2) = 290/ 190 nM, IC50(PKA) = 550, IC50 >2 µM for: BTK, CDK2E, EGFR, GSK3B, IGFR1R, IKK1/2, INSR, JAK2, MAPKAPK2, MAPK14, PIM1, PLK1/3

Screened at 10 µM (Ambit phage-based competition binding assay), closest targets as % of contr.: FRK (0.55%), FLT4 (0.85%), FLT3 (0.05%), EPHA3 (0.1%), CSF1R (0.8%), BLK (0.35%), STK10 (0%), AURKB (0%), KIT (0%), LCK (0.2%), MUSK (0%), PDGFRA/B (0.2/0.7%), TEK (0.15%), NTRK1/2/3 (0.55/ 0.1/ 0.1%), KDR (0.2%), MKNK2 (0.75%); In vitro follow up of closest targets: IC50(MERTK) = 14 nM, IC50(FLT3) = 16 nM, IC50(AURKB) = 78 nM, IC50(LCK) = 120 nM, IC50(KDR) = 180 nM, IC50(NTRK1/2) = 290/ 190 nM, IC50(PKA) = 550, IC50 >2 µM for: BTK, CDK2E, EGFR, GSK3B, IGFR1R, IKK1/2, INSR, JAK2, MAPKAPK2, MAPK14, PIM1, PLK1/3

Screened at 1 µM, closest targets: IC50( FLT3) = 338 nM, IC50(FLT4) = 274 nM, IC50(CSF1R) = 488.9 nM, IC50(TYK2) = 116 nM

Screened at 1 µM, closest targets: IC50( FLT3) = 338 nM, IC50(FLT4) = 274 nM, IC50(CSF1R) = 488.9 nM, IC50(TYK2) = 116 nM

Screened at1 µM, closest targets as % of kinase activity.: STK33 (33%), ULK2 (37%)

Screened at1 µM, closest targets as % of kinase activity.: STK33 (33%), ULK2 (37%)

Screened at 0.1 µM, closest targets with PoC <50%, TAK1-TAB1

Screened at 1 µM, >1000-fold selective against other targets; Screened in Dundee panel against 140 kinases and 19 lipid kinases at 1 µM, >1000-fold selective; Screened in AstraZeneca panel against 8 lipid kinases, closest target: IC50(PIP5K1C) = 382 µM, IC50(PI4K3B) = 1.069 µM, IC50(PI3Kd) = 1.896 µM, others >2 µM; Screened in ProQuinase panel against 13 lipid kinases at 1 µM, closest targets: IC50(PIP5K1A) = 4.93 µM, IC50(PIP5K1C) = 3.37 µM;

Screened at 1 µM, closest targets as % of contr.: ABL (12%), BRK (13%), FYN (18%), KDR (17%)