Chemogenomic set

No closely related mammalian protein known; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: ABL1(Q252H)-nonphosphorylated (0%), GRK7 (0%), CHUK (0%), PK5 (0%), JAK1 (33%), PRKCE (44%); Screened against 546 targets (Invitrogen kinase panel) at 3 µM: all kinases showed < 30% inhibition; Screened against 68 targets (Eurofins-Panlabs panel) at 10 µM: clean profile; Screened against 137 targets in CEREP screen at 20 µM, closest targets as % inhibition: FLT1 (64%), ABL1 (67%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % of inhibition: SLC6A3 (61%);

No near family aspartyl proteases detected, closest target: IC50(SLC18A2) = 213 nM, 6 additional targets between 2-10 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: CIT (0%), EGFR (0%), IKBKE (0%), LATS2 (0%), AKT1 (5%), PIK3CA(C420R) (36%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(SLC6A3) = 3137 nM;

Targets tested in PDE activity assays, closest targets: Ki(PDE6) = 44 nM (44-fold selective), Ki(PDE5A) = 116 nM (116-fold selective), Ki(PDE298) = 298 nM (300-fold selective), others >1000-fold selective

Biochemical bovine OxPhos activity assay: >30 µM for Complex 2, 3, 4 and 5; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets:Ki(HTR2B) = 558.19 nM, Ki(TMEM97) = 1118.32 nM, Ki(SIGMAR1) = 1179.84 nM, Ki(ADRA2C) = 3499.92 nM; Screened in Eurofins-CEREP, GPCR panel at 10 µM, closest target as % inhibition: HTR2B (73.5%); Selectivity > 500 fold against numerous kinases tested;

No near family aspartyl proteases, closest targets: IC50(SLC6A4) = 13 nM, IC50(SLC6A3) = 536 nM, IC50(Na channel site 2) = 1000 nM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: ACVR2A (0%), EGFR (0%), MAP4K4 (0%), MAP4K5 (5.8%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(SLC6A4) = 24 nM, Ki(SLC6A3) = 191 nM, Ki(SLC6A2) = 808 nM;

rel. activation(RXRA)=19.35% to bexarotene, inactive at 1 µM for UL48, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

rel. activation(RXRA)=19.35% to bexarotene, inactive at 1 µM for UL48, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

rel. activation(RXRA)=19.35% to bexarotene, inactive at 1 µM for UL48, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

Screened at 10 µM, closest targets as % of contr.: RPS6KA5 (76%); Screened against 50 related targets (GPCRs, ion channels, transporters): in-vitro potency of closest targets IC50(hPDE4D) = 4.7 µM, IC50(5HT2B) = 7.7 µM; No activity when tested against CYP isoforms (CYP3A3, CYP2D9, CYP2D6,CYP2C9)

Screened at 1 µM, closest targets as % of control: PIP5K1C (0%), MEK5 (0.95%), RIPK4 (4.5%), MLK3 (99%); In-vitro potency of closest targets (enzymatic assay, Reaction Biology): IC50(PIP5K1C) = 11 µM, IC50(MEK5) = 0.025 µM, IC50(RIPK4) = 5.69 µM, IC50(MLK3) = 2.75 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01803#; Screened at 10 µM in Eurofins Cerep-Panlabs Safety Screen 44 (GPCRS, Transporters, Ion Channels, Nuclear Receptors, other enzymes), closest targets as % of inhibition or stimulation: dopamine transporter (100%), hERG channel (89%), norepinephrine transporter (87%), M1 (84%), M2(70%), M3(74%), acetylcholinesterase (84%), https://www.thesgc.org/chemical-probes/cs-640;

Screened at 1 µM, closest targets as % of control: PIP5K1C (0%), MEK5 (0.95%), RIPK4 (4.5%), MLK3 (99%); In-vitro potency of closest targets (enzymatic assay, Reaction Biology): IC50(PIP5K1C) = 11 µM, IC50(MEK5) = 0.025 µM, IC50(RIPK4) = 5.69 µM, IC50(MLK3) = 2.75 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01803#; Screened at 10 µM in Eurofins Cerep-Panlabs Safety Screen 44 (GPCRS, Transporters, Ion Channels, Nuclear Receptors, other enzymes), closest targets as % of inhibition or stimulation: dopamine transporter (100%), hERG channel (89%), norepinephrine transporter (87%), M1 (84%), M2(70%), M3(74%), acetylcholinesterase (84%), https://www.thesgc.org/chemical-probes/cs-640;

Screened at 1 µM, closest targets as % of control: PIP5K1C (0%), MEK5 (0.95%), RIPK4 (4.5%), MLK3 (99%); In-vitro potency of closest targets (enzymatic assay, Reaction Biology): IC50(PIP5K1C) = 11 µM, IC50(MEK5) = 0.025 µM, IC50(RIPK4) = 5.69 µM, IC50(MLK3) = 2.75 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01803#; Screened at 10 µM in Eurofins Cerep-Panlabs Safety Screen 44 (GPCRS, Transporters, Ion Channels, Nuclear Receptors, other enzymes), closest targets as % of inhibition or stimulation: dopamine transporter (100%), hERG channel (89%), norepinephrine transporter (87%), M1 (84%), M2(70%), M3(74%), acetylcholinesterase (84%), https://www.thesgc.org/chemical-probes/cs-640;

Screened at 1 µM, closest targets as % of control: PIP5K1C (0%), MEK5 (0.95%), RIPK4 (4.5%), MLK3 (99%); In-vitro potency of closest targets (enzymatic assay, Reaction Biology): IC50(PIP5K1C) = 11 µM, IC50(MEK5) = 0.025 µM, IC50(RIPK4) = 5.69 µM, IC50(MLK3) = 2.75 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01803#; Screened at 10 µM in Eurofins Cerep-Panlabs Safety Screen 44 (GPCRS, Transporters, Ion Channels, Nuclear Receptors, other enzymes), closest targets as % of inhibition or stimulation: dopamine transporter (100%), hERG channel (89%), norepinephrine transporter (87%), M1 (84%), M2(70%), M3(74%), acetylcholinesterase (84%), https://www.thesgc.org/chemical-probes/cs-640;

Screened at 1 µM, closest targets as % of control: STK2 (3.83%), SRPK1 (8.76%), SRPK2 (13.94%), PIM1 (54.68%), PKCe(55.07%), PKN1(55.56%); In-vitro potency of closest targets (Radioactive assay, Reaction Biology): IC50(CLK1) >10 µM, IC50(CLK2) >10 µM, IC50(CLK3) >10 µM, IC50(CLK4) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.2c01705#;

Screened at 1 µM, closest targets as % of control: STK2 (3.83%), SRPK1 (8.76%), SRPK2 (13.94%), PIM1 (54.68%), PKCe(55.07%), PKN1(55.56%); In-vitro potency of closest targets (Radioactive assay, Reaction Biology): IC50(CLK1) >10 µM, IC50(CLK2) >10 µM, IC50(CLK3) >10 µM, IC50(CLK4) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.2c01705#;

Screened at 1 µM, closest targets as % of control: STK2 (3.83%), SRPK1 (8.76%), SRPK2 (13.94%), PIM1 (54.68%), PKCe(55.07%), PKN1(55.56%); In-vitro potency of closest targets (Radioactive assay, Reaction Biology): IC50(CLK1) >10 µM, IC50(CLK2) >10 µM, IC50(CLK3) >10 µM, IC50(CLK4) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.2c01705#;

Screened at 1 µM, no kinases with <45% of control; In-vitro potency (Eurofins Enzymatic assay): IC50(CLK3) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, IC50(HIPK1) >10 µM, IC50(HIPK2) >10 µM, IC50(HIPK3) >10µM, IC50(HIPK4) >10 µM, IC50(PIM2) >10 µM, https://www.thesgc.org/chemical-probes/SGC-CLK-1;

Screened at 1 µM, no kinases with <45% of control; In-vitro potency (Eurofins Enzymatic assay): IC50(CLK3) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, IC50(HIPK1) >10 µM, IC50(HIPK2) >10 µM, IC50(HIPK3) >10µM, IC50(HIPK4) >10 µM, IC50(PIM2) >10 µM, https://www.thesgc.org/chemical-probes/SGC-CLK-1;

Screened at 1 µM, no kinases with <45% of control; In-vitro potency (Eurofins Enzymatic assay): IC50(CLK3) >10 µM, IC50(DYRK1A) >10 µM, IC50(DYRK1B) >10 µM, IC50(DYRK2) >10 µM, IC50(DYRK3) >10 µM, IC50(HIPK1) >10 µM, IC50(HIPK2) >10 µM, IC50(HIPK3) >10µM, IC50(HIPK4) >10 µM, IC50(PIM2) >10 µM, https://www.thesgc.org/chemical-probes/SGC-CLK-1;

Screened at 100 nM, closest targets as % of control: STK38L (24%), SRPK2 (34%), PIK3CA (44%), TNIK(45%), LRRK2(51%), NEK1(51%), RIOK1(54%), EIF2AK2 (55%), FYN (56%), STK32A (57%), RIPK2 (59%), FRK (60%), NIM1K (60%), PRPF4B (60%)

Screened at 100 nM, closest targets as % of control: STK38L (24%), SRPK2 (34%), PIK3CA (44%), TNIK(45%), LRRK2(51%), NEK1(51%), RIOK1(54%), EIF2AK2 (55%), FYN (56%), STK32A (57%), RIPK2 (59%), FRK (60%), NIM1K (60%), PRPF4B (60%)

Screened at 100 nM, closest targets as % of control: STK38L (24%), SRPK2 (34%), PIK3CA (44%), TNIK(45%), LRRK2(51%), NEK1(51%), RIOK1(54%), EIF2AK2 (55%), FYN (56%), STK32A (57%), RIPK2 (59%), FRK (60%), NIM1K (60%), PRPF4B (60%)

Screened at 100 nM, closest targets as % of control: STK38L (24%), SRPK2 (34%), PIK3CA (44%), TNIK(45%), LRRK2(51%), NEK1(51%), RIOK1(54%), EIF2AK2 (55%), FYN (56%), STK32A (57%), RIPK2 (59%), FRK (60%), NIM1K (60%), PRPF4B (60%)

Screened at 1 µM, closest targets as % of contr.: PIM2 (0%), VRK2 (29%), KIT(V55D,V654A) (41%), NLK (41%); Screened at 10 µM against 45 GPCRs (PDPS screen), closest targets as % of inhibition: HTR1E (31.76%), HTR2C (31.08%), ADRA1B (30.19%);

Screened at 1 µM, closest targets as % of control: NEK1(45%), STK38L (47%), BMX(50%)

Screened at 1 µM, closest targets as % of control: NEK1(45%), STK38L (47%), BMX(50%)

Various assays types used, i.e. TR-FRET, Luminescence assay, FLINT, Electrophysiological assay, LEADseeker SPA; No activation across HDACs 1–11 at 100 μM; Selective for PADI4 over PADI1-3 (tested with recombinant enzymes and cell lysates).

Screened at 1 µM, closest target as % of inhibition: CHRM2 (40.7%)

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

Screened at 1 μM inhibition, closest targets as % of residual activity: MAP4K5 (8%), PAK3 (9%), PAK2 (10%), NLK (13%), PKN3 (35%), PAK1 (36%), MAP2K4 (37%), TIE2 (39%), MST4 (45%), MELK (48%); In-vitro follow-up of closest targets (33PanQinase assay, Reaction Biology): IC50(MAP4K5) = 210 nM, IC50(PAK3) = 140 nM, IC50(PAK2) = 41 nM, IC50(NLK) = 13 nM, IC50(PKN3) = 1400 nM, IC50(PAK1) = 580 nM, IC50(MAP2K4) = 830 nM, IC50(TIE2) = 3100 nM, IC50(MST4) = 1600 nM, IC50(MELK) = 2200 nM (https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144); In-cellulo follow-up of closest targets (NanoBRET assay in HEK293T cells): IC50(MAP4K5) = 13 µM, IC50(NLK) = 0.25 µM, IC50(PKN3) = 6.7 µM, IC50(TIE2) = 6 µM, IC50(MST4) = 34 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144;

Screened at 1 μM inhibition, closest targets as % of residual activity: MAP4K5 (8%), PAK3 (9%), PAK2 (10%), NLK (13%), PKN3 (35%), PAK1 (36%), MAP2K4 (37%), TIE2 (39%), MST4 (45%), MELK (48%); In-vitro follow-up of closest targets (33PanQinase assay, Reaction Biology): IC50(MAP4K5) = 210 nM, IC50(PAK3) = 140 nM, IC50(PAK2) = 41 nM, IC50(NLK) = 13 nM, IC50(PKN3) = 1400 nM, IC50(PAK1) = 580 nM, IC50(MAP2K4) = 830 nM, IC50(TIE2) = 3100 nM, IC50(MST4) = 1600 nM, IC50(MELK) = 2200 nM (https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144); In-cellulo follow-up of closest targets (NanoBRET assay in HEK293T cells): IC50(MAP4K5) = 13 µM, IC50(NLK) = 0.25 µM, IC50(PKN3) = 6.7 µM, IC50(TIE2) = 6 µM, IC50(MST4) = 34 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144;

Screened at 1 μM inhibition, closest targets as % of residual activity: MAP4K5 (8%), PAK3 (9%), PAK2 (10%), NLK (13%), PKN3 (35%), PAK1 (36%), MAP2K4 (37%), TIE2 (39%), MST4 (45%), MELK (48%); In-vitro follow-up of closest targets (33PanQinase assay, Reaction Biology): IC50(MAP4K5) = 210 nM, IC50(PAK3) = 140 nM, IC50(PAK2) = 41 nM, IC50(NLK) = 13 nM, IC50(PKN3) = 1400 nM, IC50(PAK1) = 580 nM, IC50(MAP2K4) = 830 nM, IC50(TIE2) = 3100 nM, IC50(MST4) = 1600 nM, IC50(MELK) = 2200 nM (https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144); In-cellulo follow-up of closest targets (NanoBRET assay in HEK293T cells): IC50(MAP4K5) = 13 µM, IC50(NLK) = 0.25 µM, IC50(PKN3) = 6.7 µM, IC50(TIE2) = 6 µM, IC50(MST4) = 34 µM, https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02144;

Clean selectivity profile with no target activity >50%; Screened in enzymatic assays against other transaminases: IC50(GOT1/2) >50 µM; Screened at 6 µM against 30 proteases, all targets IC50 >10 µM; Screened at 2 µM against 45 kinases (enzymatic assays), clean selectivity profile with all kinases IC50 >7 µM

Clean selectivity profile with no target activity >50%; Screened in enzymatic assays against other transaminases: IC50(GOT1/2) >50 µM; Screened at 6 µM against 30 proteases, all targets IC50 >10 µM; Screened at 2 µM against 45 kinases (enzymatic assays), clean selectivity profile with all kinases IC50 >7 µM

Screened at 10 µM, in vitro-potencies of closest targets: Ki(SLC6A3) = 1377.15 nM, Ki(HTR2A) = 2576.32 nM; Selectivity against family members in biochemical assays, all >30 fold: IC50(hTRPV1) >25 µM, IC50(hTRPV4) >25 µM, IC50(hTRPC3) >25 µM, IC50(hTRPC5) = 5.6 µM, IC50(hTRPC6) >25 µM, IC50(hKCNK9, TASK-3) >30 µM, IC50(hCACNA1H, Cav3.2) >25 µM; Selectivity screen in Eurofins Lead Profiling Screen, GPCR Profiling Screen and Bayer Kinase Panel was performed, in-vitro potencies of closest targets: Ki (SLC6A3, human) = 0.9 μM, Ki(PGR, human) = 4 μM, EC50(ESR1) = 2.1 μM

Screened at 10 µM, closest target as % of inhibition: HTR3A (38%); Screened against other human prostanoid receptor (binding assay, Eurofins/Panlabs): IC50(PTGFR) = 22 nM, IC50(EP1) >10 µM, IC50(EP2) >10 µM, IC50(EP3) >10 µM, IC50(EP4) >10 µM, IC50(IP) >10 µM, IC50(DP) >10 µM, IC50(CRTH2) >10 µM, >420-fold selective; Screened in cell-based assay (Eurofins/Panlabs): IC50(TBXA2R) = 2.2 µM, 200-fold selective; Screened at 10 µM against 77 other targets (Leadprofiling Screen, Panlabs), closest target as % of inhibition: TBXAS1 (33%); Screened at 10 µM against 8 ion channels (Ion channel profiler, Eurofins): IC20(Nav1.5) >10 μM, IC20(Kv4.3/KChIP2) >10 μM, IC20(Cav1.2) >10 μM, IC20(Kv1.5) >10 μM, IC20(KCNQ/minK) >10 μM, IC20(herG) >10 μM, IC20(HCN4) >10 μM, IC20(Kir2.1) >10 μM

Screened at 10 µM, in-vitro potency of closest target: IC50(CTSG, cathepsin G) = 110 nM, >100-fold selective; Screened at 10 µM against 44 targets outside target family (Eurofins Safety Screen44TM), clean selectivity profile; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(HTR1D) = 1130.61 nM, Ki(OPRK1) = 4948.48 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 6.4, pKd(TAF1L, BD2) = 5.7, pKd(BRD9) = 5.7, pKd(CECR2) = 5.5, pKd(BAZ2A) = 5.5, pKd(BAZ2B) = 5.1; Selective for the BD2 domain of BET proteins; Clean in selectivity screen against 48 targets (GPCRs, ion-channels, other targets); Chemoproteomics data (3 vectors) showed excellent non BCPs selectivity; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 257.47 nM, Ki(DRD4) = 982.65 nM, Ki(DRD2) = 964.05 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 6.4, pKd(TAF1L, BD2) = 5.7, pKd(BRD9) = 5.7, pKd(CECR2) = 5.5, pKd(BAZ2A) = 5.5, pKd(BAZ2B) = 5.1; Selective for the BD2 domain of BET proteins; Clean in selectivity screen against 48 targets (GPCRs, ion-channels, other targets); Chemoproteomics data (3 vectors) showed excellent non BCPs selectivity; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 257.47 nM, Ki(DRD4) = 982.65 nM, Ki(DRD2) = 964.05 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 6.4, pKd(TAF1L, BD2) = 5.7, pKd(BRD9) = 5.7, pKd(CECR2) = 5.5, pKd(BAZ2A) = 5.5, pKd(BAZ2B) = 5.1; Selective for the BD2 domain of BET proteins; Clean in selectivity screen against 48 targets (GPCRs, ion-channels, other targets); Chemoproteomics data (3 vectors) showed excellent non BCPs selectivity; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 257.47 nM, Ki(DRD4) = 982.65 nM, Ki(DRD2) = 964.05 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 6.4, pKd(TAF1L, BD2) = 5.7, pKd(BRD9) = 5.7, pKd(CECR2) = 5.5, pKd(BAZ2A) = 5.5, pKd(BAZ2B) = 5.1; Selective for the BD2 domain of BET proteins; Clean in selectivity screen against 48 targets (GPCRs, ion-channels, other targets); Chemoproteomics data (3 vectors) showed excellent non BCPs selectivity; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 257.47 nM, Ki(DRD4) = 982.65 nM, Ki(DRD2) = 964.05 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 1621 nM, Kd(BRD2, BD2) = 35 nM (64-fold selective), Kd(BRD3, BD1) = 2082 nM, Kd(BRD3, BD2) = 32 nM (63-fold selective), Kd(BRD4, BD1) = 769 nM, Kd(BRD4, BD2) = 9 nM (85-fold selective), Kd(BRDT, BD1) = 2454 nM, Kd(BRDT, BD2) = 15 nM (164-fold selective); In-vitro potencies (SPR measurements): Kd(BRD4, BD1) >10 µM, Kd(BRD4, BD2) = 30 nM, excellent selectivity against other BCPs, Kd >30 µM for TAF1(BD2), BRD9, TAF1L(BD2), BAZ2A, CECR2, BAZ2B; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(HTR1D) = 3438.75 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 1621 nM, Kd(BRD2, BD2) = 35 nM (64-fold selective), Kd(BRD3, BD1) = 2082 nM, Kd(BRD3, BD2) = 32 nM (63-fold selective), Kd(BRD4, BD1) = 769 nM, Kd(BRD4, BD2) = 9 nM (85-fold selective), Kd(BRDT, BD1) = 2454 nM, Kd(BRDT, BD2) = 15 nM (164-fold selective); In-vitro potencies (SPR measurements): Kd(BRD4, BD1) >10 µM, Kd(BRD4, BD2) = 30 nM, excellent selectivity against other BCPs, Kd >30 µM for TAF1(BD2), BRD9, TAF1L(BD2), BAZ2A, CECR2, BAZ2B; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(HTR1D) = 3438.75 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 1621 nM, Kd(BRD2, BD2) = 35 nM (64-fold selective), Kd(BRD3, BD1) = 2082 nM, Kd(BRD3, BD2) = 32 nM (63-fold selective), Kd(BRD4, BD1) = 769 nM, Kd(BRD4, BD2) = 9 nM (85-fold selective), Kd(BRDT, BD1) = 2454 nM, Kd(BRDT, BD2) = 15 nM (164-fold selective); In-vitro potencies (SPR measurements): Kd(BRD4, BD1) >10 µM, Kd(BRD4, BD2) = 30 nM, excellent selectivity against other BCPs, Kd >30 µM for TAF1(BD2), BRD9, TAF1L(BD2), BAZ2A, CECR2, BAZ2B; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(HTR1D) = 3438.75 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 1621 nM, Kd(BRD2, BD2) = 35 nM (64-fold selective), Kd(BRD3, BD1) = 2082 nM, Kd(BRD3, BD2) = 32 nM (63-fold selective), Kd(BRD4, BD1) = 769 nM, Kd(BRD4, BD2) = 9 nM (85-fold selective), Kd(BRDT, BD1) = 2454 nM, Kd(BRDT, BD2) = 15 nM (164-fold selective); In-vitro potencies (SPR measurements): Kd(BRD4, BD1) >10 µM, Kd(BRD4, BD2) = 30 nM, excellent selectivity against other BCPs, Kd >30 µM for TAF1(BD2), BRD9, TAF1L(BD2), BAZ2A, CECR2, BAZ2B; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(HTR1D) = 3438.75 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 13 nM, Kd(BRD3, BD1) = 5 nM, Kd(BRD4, BD1) = 5.9 nM, Kd(BRDT, BD1) = 18 nM; Selectivity screen against 50 targets (GPCRs, ion-channels, other targets), in-vitro potencies of closest targets: pIC50(CHRNA1) = 6, pIC50(CYP3A4) = 6; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 485.92 nM, Ki(DRD4) = 856.99 nM, Ki(GABAA) = 1595.89 nM, Ki(GABAA/BZP) = 1970.29 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 13 nM, Kd(BRD3, BD1) = 5 nM, Kd(BRD4, BD1) = 5.9 nM, Kd(BRDT, BD1) = 18 nM; Selectivity screen against 50 targets (GPCRs, ion-channels, other targets), in-vitro potencies of closest targets: pIC50(CHRNA1) = 6, pIC50(CYP3A4) = 6; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 485.92 nM, Ki(DRD4) = 856.99 nM, Ki(GABAA) = 1595.89 nM, Ki(GABAA/BZP) = 1970.29 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 13 nM, Kd(BRD3, BD1) = 5 nM, Kd(BRD4, BD1) = 5.9 nM, Kd(BRDT, BD1) = 18 nM; Selectivity screen against 50 targets (GPCRs, ion-channels, other targets), in-vitro potencies of closest targets: pIC50(CHRNA1) = 6, pIC50(CYP3A4) = 6; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 485.92 nM, Ki(DRD4) = 856.99 nM, Ki(GABAA) = 1595.89 nM, Ki(GABAA/BZP) = 1970.29 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): Kd(BRD2, BD1) = 13 nM, Kd(BRD3, BD1) = 5 nM, Kd(BRD4, BD1) = 5.9 nM, Kd(BRDT, BD1) = 18 nM; Selectivity screen against 50 targets (GPCRs, ion-channels, other targets), in-vitro potencies of closest targets: pIC50(CHRNA1) = 6, pIC50(CYP3A4) = 6; Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(DRD3) = 485.92 nM, Ki(DRD4) = 856.99 nM, Ki(GABAA) = 1595.89 nM, Ki(GABAA/BZP) = 1970.29 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 7.3, pKd(TAF1L, BD2) = 6.4, selective for the BD1 domain of BET proteins; Follow-up of closest targets (FRET assay): pIC50(TAF1, BD1) = 5.0 ± 0.1, n = 3 (BRD4, BD1 selectivity = 500-fold); Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(DRD4) = 1013.12 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 7.3, pKd(TAF1L, BD2) = 6.4, selective for the BD1 domain of BET proteins; Follow-up of closest targets (FRET assay): pIC50(TAF1, BD1) = 5.0 ± 0.1, n = 3 (BRD4, BD1 selectivity = 500-fold); Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(DRD4) = 1013.12 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 7.3, pKd(TAF1L, BD2) = 6.4, selective for the BD1 domain of BET proteins; Follow-up of closest targets (FRET assay): pIC50(TAF1, BD1) = 5.0 ± 0.1, n = 3 (BRD4, BD1 selectivity = 500-fold); Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(DRD4) = 1013.12 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 7.3, pKd(TAF1L, BD2) = 6.4, selective for the BD1 domain of BET proteins; Follow-up of closest targets (FRET assay): pIC50(TAF1, BD1) = 5.0 ± 0.1, n = 3 (BRD4, BD1 selectivity = 500-fold); Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest target: Ki(DRD4) = 1013.12 nM

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 5.4, pKd(EP300) = 5.1, pKd(CREBBP) = 5.0, pKd(EP300) = 5.1; Cross screening in liability panel against 48 targets (GPCRs, transporter, ion-channels, and other targets), closest target: pEC50(CB2 receptor) <5; Selective for BD2 domain of BET proteins; Screened at 10 µM against 45 GPCRs (PDSP screen), closest target as % inhibition: OPRK1(40%);

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 5.4, pKd(EP300) = 5.1, pKd(CREBBP) = 5.0, pKd(EP300) = 5.1; Cross screening in liability panel against 48 targets (GPCRs, transporter, ion-channels, and other targets), closest target: pEC50(CB2 receptor) <5; Selective for BD2 domain of BET proteins; Screened at 10 µM against 45 GPCRs (PDSP screen), closest target as % inhibition: OPRK1(40%);

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 5.4, pKd(EP300) = 5.1, pKd(CREBBP) = 5.0, pKd(EP300) = 5.1; Cross screening in liability panel against 48 targets (GPCRs, transporter, ion-channels, and other targets), closest target: pEC50(CB2 receptor) <5; Selective for BD2 domain of BET proteins; Screened at 10 µM against 45 GPCRs (PDSP screen), closest target as % inhibition: OPRK1(40%);

In-vitro potencies of closest targets in BROMOScan (DiscoverX): pKd(TAF1, BD2) = 5.4, pKd(EP300) = 5.1, pKd(CREBBP) = 5.0, pKd(EP300) = 5.1; Cross screening in liability panel against 48 targets (GPCRs, transporter, ion-channels, and other targets), closest target: pEC50(CB2 receptor) <5; Selective for BD2 domain of BET proteins; Screened at 10 µM against 45 GPCRs (PDSP screen), closest target as % inhibition: OPRK1(40%);

Screened at 20 µM, dTm(SMARCA2) = 7.7 K, dTm(SMARCA4) = 7.4 K, dTm(PBRM1,BD5) = 11.3 K, other targets <2.7 K; Screened at 20 µM against 85 protein kinases (DSF assay), clean selectivity profile with all targets dTm <0.5 K

Screened at 20 µM, dTm(SMARCA2) = 7.7 K, dTm(SMARCA4) = 7.4 K, dTm(PBRM1,BD5) = 11.3 K, other targets <2.7 K; Screened at 20 µM against 85 protein kinases (DSF assay), clean selectivity profile with all targets dTm <0.5 K

Screened at 20 µM, dTm(SMARCA2) = 7.7 K, dTm(SMARCA4) = 7.4 K, dTm(PBRM1,BD5) = 11.3 K, other targets <2.7 K; Screened at 20 µM against 85 protein kinases (DSF assay), clean selectivity profile with all targets dTm <0.5 K

Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potency of closest targets: Ki(HTR7) >10 µM, Ki(GABA/PBR) >10 µM, Ki(HTR1D) = 6453.57 nM; Selectivity within target family (GLS2 enzyme activity assay): IC50 >30 µM (> 1000 fold); Selectivity outside target family (probe selectivity observed in Proteome Integral Stability Assay): closest targets in CEREP binding assay at 10 μM (percent inhibition of control specific binding): ADORA3 (agonist radioligand) = 76.6%, CHRM1 (antagonist radioligand) = 92.6%, CHRM2 (antagonist radioligand) = 71.5%

Screened at 10 µM, in-vitro potencies of closest targets: Ki(TMEM97) = 1476.05 nM, Ki(SIGMAR1) = 1828.52 nM, Ki(GABA/PBR) = 1805.52 nM, Ki(OPRK1) = 3005.38 nM; Screened in enzymatic assays against other CCR family members: inactive on hCCR3, IC50(hCCR2) >30 µM, IC50(hCCR4) >30 µM, IC50(hCCR%) >30 µM, IC50(hCCR6) >30 µM, IC50(hCCR7) >30 µM, IC50(hCCR8) >30 µM, IC50(hCCR9) >30 µM, IC50(hCCR10) >30 µM, IC50(hCXCR1) >30 µM, IC50(hCXCR2) >30 µM, IC50(hCXCR3) >30 µM, IC50(hCXCR4) >30 µM, IC50(hCXCR5) >30 µM

Screened at 1 µM in quantitative proteomic studies, clean selectivty profile

Screened at 1 µM, 10 µM, and 50 µM (methyltransferase activity of G9a, GLP, SUV39H1, SUV39H2, SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 pentameric complex, MLL3 pentameric complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT4, PRMT5-MEP50 complex, PRMT6, PRMT7, PRMT8, PRMT9, PRDM9, SETD2, SMYD2, SMYD3, BCDIN3D, METTL3-14, and DNMT1), closest targets SUV420H1 (60% activity at 1 µM), SUV420H2 (62% activity at 1 µM); In-cellular follow-up by western blot assay (effect on H4K20me2/3 cellular mark modulated by SUVH420H1/H2 using MCF7 cells): EC50(SUVH420H1) >50 µM, EC50(SUV420H2) >50 µM; Screened at 1 µM against 40 kinases (enzymatic assay), closest target as % of inhibition: MAP4K4 (10.42%), clean selectivity profile; Screened against 15 diverse protein targets, all targets >10 µM, clean selectivity profile

Screened at 10 µM, in-vitro potencies of closest targets: Ki(HTR3A) = 584.83 nM, Ki(TMEM97) = 1099.22 nM, Ki(ADRA2A) = 8468.01 nM

Screened at 10 µM, in-vitro potency of closest target: Ki(HRH2) = 9781.38 nM; Selectivity against family members in biochemical assays, all >30 fold: IC50(hTRPV1) >25 µM, IC50(hTRPV4) >25 µM, IC50(hTRPC3) n.d., IC50(hTRPC5) = 10.0 µM, IC50(hTRPC6) >30 µM, IC50(hKCNK9 (TASK-3)) n.d., IC50(hCACNA1H (Cav3.2)) n.d.

Screened at 10 µM, in-vitro potency of closest target: KI(GABA/BZP) = 2539.22 nM

Screened at 10 µM, in-vitro potency of closest target: KI(GABA/BZP) = 2539.22 nM

Screened at 10 µM, in-vitro potency of closest target: KI(GABA/BZP) = 2539.22 nM

Screened at 10 µM, in-vitro potency of closest target: KI(GABA/BZP) = 2539.22 nM

Screened at 10 µM, in-vitro potency of closest target: Ki(GABA/BZP) = 473.91 nM

Screened at 10 µM, in-vitro potency of closest target: Ki(GABA/BZP) = 473.91 nM

Screened at 10 µM, in-vitro potency of closest target: Ki(GABA/BZP) = 473.91 nM

Screened at 10 µM, in-vitro potency of closest target: Ki(GABA/BZP) = 473.91 nM

rem. activity(RORA) = 0.56, rem. activity(RORB) = 0.53, rem. activity(RORC) = 0.46, rel. activity(RXRA)=30.83% to bexarotene 1 µM, rem. activity(NR2E1) = 0.42, rem. activity(NR5A1) = 0.42, inactive at 1 µM for UL48, HNF4A, NR2C1, NR4A1

rem. activity(RORA) = 0.56, rem. activity(RORB) = 0.53, rem. activity(RORC) = 0.46, rel. activity(RXRA)=30.83% to bexarotene 1 µM, rem. activity(NR2E1) = 0.42, rem. activity(NR5A1) = 0.42, inactive at 1 µM for UL48, HNF4A, NR2C1, NR4A1

rem. activity(RORA) = 0.56, rem. activity(RORB) = 0.53, rem. activity(RORC) = 0.46, rel. activity(RXRA)=30.83% to bexarotene 1 µM, rem. activity(NR2E1) = 0.42, rem. activity(NR5A1) = 0.42, inactive at 1 µM for UL48, HNF4A, NR2C1, NR4A1

fold-activation(NR5A1)=2.0, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1

fold-activation(NR5A1)=2.0, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1

fold-activation(PPARD) = 6.61, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARD) = 6.61, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARG) = 2.82, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARG) = 2.82, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARD) = 19.95, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARD) = 19.95, 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