Chemogenomic set

Screened at 1 µM, closest targets as % of control: BTK (1%), CASK (41%); Kd follow-up of closest targets: Kd(BTK) = 1.5 nM, Kd(MINK1) = 40 µM; Screened at 1 µM (Kinase panel, UBI KinaseProfiler, 205 kinases Millipore screen, using Km = ATP), closest target as % of control: BTK (99%), FES(53%); Kinase panel (Enzymatic assay, 47 kinases, using Km = ATP) in-vitro potency of closest targets: IC50(BLK) = 7573 nM, IC50(BMX) = 1870 nM, IC50(ITK) = 4270 nM, IC50(PDGFRB) = 2440 nM, IC50(SRC) = 3650 nM, IC50(NTRK1) = 1885 nM, IC50(FLT4) = 3287 nM, others IC50 >10 µM; Screened at 1 µM against 82 non-kinase targets (CEREP screen), closest targets as % of inhibition: Adenosine A1 (31%), Adenosine A2A (34%), https://pubmed.ncbi.nlm.nih.gov/21113169/;

In-vitro potency of closest target: IC50(CDK11) = 1.7 nM

Screened at 10 µM, S(10) = 0.003, closest target: IC50(PAK2) = 72 nM; Screened against 27 receptors (Novartis panel) at 10 µM, closest target: IC50(H1) = 13 µM, IC50(M1) = 13 µM; Screened against panel of 48 bromodomains at 10 µM in DSF-assay, all targets dTm < 1 K clean profile

Screened at 1 µM, S(10) = 0.02, follow-up Kds for closest targets: Kd(PIP5K1C) = 260 nM, Kd(RIOK1) = 72 nM, Kd(RIOK3) = 290 nM; Kd(RIOK3) = 290 nM, Kd(CDKL2) = 880 nM, Kd(MYLK2) = 960 nM;

Screened at 1 µM, S(10) = 0.02, follow-up Kds for closest targets: Kd(PIP5K1C) = 260 nM, Kd(RIOK1) = 72 nM, Kd(RIOK3) = 290 nM; Kd(RIOK3) = 290 nM, Kd(CDKL2) = 880 nM, Kd(MYLK2) = 960 nM;

Screened at 1 µM, off-target PoC of 12%

Screened at 1 µM, closest targets as % of contrl.: ALK(C1156Y) (0.3%), CAMKK1/2 (3.7/ 3.4%), PIP5K2C (4.2%), PLK2/3 (1.2/ 0.5%), RPS6KA4(KD2-C) (0.3%); follow-up IC50s of closest targets: IC50(PLK2) = 3.5 nM, IC50(PLK3) = 9 nM; Screened in Dundee panel against 30 kinases at 10 µM, clean profile; Screened in SafetyScreen44™ (Eurofins) against 64 targets (GPCRS, ion channels, transporters) at 10 µM, closest target as % of contr. ALPHA1A/R (13%);

Screened at 1 µM, closest targets as % of contrl.: ALK(C1156Y) (0.3%), CAMKK1/2 (3.7/ 3.4%), PIP5K2C (4.2%), PLK2/3 (1.2/ 0.5%), RPS6KA4(KD2-C) (0.3%), follow-up IC50s of closest targets: IC50(PLK2) = 3.5 nM, IC50(PLK3) = 9 nM; Screened in Dundee panel against 30 kinases at 10 µM, clean profile; Screened in SafetyScreen44™ (Eurofins) against 64 targets (GPCRS, ion channels, transporters) at 10 µM, closest target as % of contr. ALPHA1A/R (13%);

Screened at 1 µM, S(10) = 0.05, closest targets as % of contr.: ABL (M351T phosph.) (5.5%), ABL (Q252H phosph.) (6.8%), ABL (T315I phosph.) (1.6%), AURKA (0.05%), AURKB (3.4%), DCAMKL1 (8.3%), MAP3K2 (2.6%), MAP3K3 (2%), JAK1 (JH2 pseudokinase domain) (4.3%), STK24 (0.9%), STK26 (0.6%), PIK3CG (0.15%), PIP5K1C (10%), PIP5K2C (8%), RIPK4 (9.5%), TAOK1 (2.6%), TAOK2 (3.4%), TAOK3 (4.5%), TYK2 (JH1 catalytic domain, 2.7%), ULK2 (4.8%); Follow-up Kds and IC50s: Kd/IC50(ABL, M351T phosph.) = 42/ 17.6 nM, KD/IC50(ABL (Q252H phosph.) = 24/ 11.6 nM, Kd/IC50(ABL, T315I phosph.) = 14/ 16.6 nM, Kd/IC50 (AURKA) = 13/ 19.4 nM, Kd/IC50(AURKB) = 40/ 19.4 nM, Kd/IC50(DCAMKL1) = 25/ 27.9 nM, Kd/IC50(MAP3K2) = 19/ 79.6 nM, Kd/IC50(MAP3K3) = 27/ 38.4 nM, Kd/IC50(JAK1, JH2 pseudokinase domain) = 22/ 38.7 nM, Kd/IC50(STK24) = 18/ 44.8 nM, Kd/IC50(STK26) = 16/ 27.3 nM, Kd/IC50(PIK3CG) = 4.8/ 38.4 nM, Kd/IC50(PIP5K1C) = 77/ 436 nM, Kd(PIP5K2C) = 34 nM, Kd(RIPK4) = 140 nM, Kd/IC50(TAOK1) = 180/ 165 nM, Kd/IC50(TAOK2) = 100/ 134 nM, Kd/IC50(TAOK3) = 610/ 254 nM, Kd/IC50(TYK2, JH1 catalytic domain) = 610/ 254 nM, Kd/IC50(ULK2) = 35/ 41.5 nM;

Screened at 1 µM, S(10) = 0.05, closest targets as % of contr.: ABL (M351T phosph.) (5.5%), ABL (Q252H phosph.) (6.8%), ABL (T315I phosph.) (1.6%), AURKA (0.05%), AURKB (3.4%), DCAMKL1 (8.3%), MAP3K2 (2.6%), MAP3K3 (2%), JAK1 (JH2 pseudokinase domain) (4.3%), STK24 (0.9%), STK26 (0.6%), PIK3CG (0.15%), PIP5K1C (10%), PIP5K2C (8%), RIPK4 (9.5%), TAOK1 (2.6%), TAOK2 (3.4%), TAOK3 (4.5%), TYK2 (JH1 catalytic domain, 2.7%), ULK2 (4.8%); Follow-up Kds and IC50s: Kd/IC50(ABL, M351T phosph.) = 42/ 17.6 nM, KD/IC50(ABL (Q252H phosph.) = 24/ 11.6 nM, Kd/IC50(ABL, T315I phosph.) = 14/ 16.6 nM, Kd/IC50 (AURKA) = 13/ 19.4 nM, Kd/IC50(AURKB) = 40/ 19.4 nM, Kd/IC50(DCAMKL1) = 25/ 27.9 nM, Kd/IC50(MAP3K2) = 19/ 79.6 nM, Kd/IC50(MAP3K3) = 27/ 38.4 nM, Kd/IC50(JAK1, JH2 pseudokinase domain) = 22/ 38.7 nM, Kd/IC50(STK24) = 18/ 44.8 nM, Kd/IC50(STK26) = 16/ 27.3 nM, Kd/IC50(PIK3CG) = 4.8/ 38.4 nM, Kd/IC50(PIP5K1C) = 77/ 436 nM, Kd(PIP5K2C) = 34 nM, Kd(RIPK4) = 140 nM, Kd/IC50(TAOK1) = 180/ 165 nM, Kd/IC50(TAOK2) = 100/ 134 nM, Kd/IC50(TAOK3) = 610/ 254 nM, Kd/IC50(TYK2, JH1 catalytic domain) = 610/ 254 nM, Kd/IC50(ULK2) = 35/ 41.5 nM;

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

inactive at 3 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

inactive at 3 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

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

fold-activation(PPARD) = 19.05, inactive at 1 µM for UL48, RXRA, HNF4A, NR2C1, NR2E1, NR4A1, NR5A1

fold-activation(PPARD) = 19.05, 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

Screened at 10 µM against receptors, ion channels and enzymes, closest targets as % of contr.: A1 receptor (48%), Cl- channel (GABA-gated) (23%), PDE2A1 (49%), PDE5 (2%), >10000-fold selective; In vitro activity of closest targets (TR-FRET assays): IC50(BRPF1) = 3.15 µM, IC50(BRPF3) = 5.55 µM, IC50(TAF1L, BD2) = 0.106 µM, IC50(BRD4, BD1) >15 µM, IC50(BRD4,BD2) >20 µM; Screened in DSF assay against 48 bromodomains at 10 µM, closest targets: dTm(CECR2) = 3.5 K, dTm(EP300) = 3.5 K, dTm(BRD7) = 2.3 K; In vitro follow up ITC: Kd(CECR2) = 1.39 µM; Screened against31 bromodomains in (BROMOscan DiscoverX) at 100 nM, closest targets as % contr.: CREBBP (30%), TAF1L(BD2) (0.7%); Screened against 300 kinases at 10 µM, >10000-fold selective (PMID: 28402630)

Screened at 10 µM against receptors, ion channels and enzymes, closest targets as % of contr.: A1 receptor (48%), Cl- channel (GABA-gated) (23%), PDE2A1 (49%), PDE5 (2%), >10000-fold selective; In vitro activity of closest targets (TR-FRET assays): IC50(BRPF1) = 3.15 µM, IC50(BRPF3) = 5.55 µM, IC50(TAF1L, BD2) = 0.106 µM, IC50(BRD4, BD1) >15 µM, IC50(BRD4,BD2) >20 µM; Screened in DSF assay against 48 bromodomains at 10 µM, closest targets: dTm(CECR2) = 3.5 K, dTm(EP300) = 3.5 K, dTm(BRD7) = 2.3 K; In vitro follow up ITC: Kd(CECR2) = 1.39 µM; Screened against31 bromodomains in (BROMOscan DiscoverX) at 100 nM, closest targets as % contr.: CREBBP (30%), TAF1L(BD2) (0.7%); Screened against 300 kinases at 10 µM, >10000-fold selective (PMID: 28402630)

Screeed at 10 µM in thermal shift assays, dTm(BAZ2A) = 5.2 K, dTm(BAZ2B) = 3.8 K, dTm(CECR2) = 2.0 K; Follow-up in vitro potency (ITC): Kd(CECR2) = 1.55 µM (15-fold selective); Greater 100-fold selective for BRD4 (IC50 >50 µM, BRD4 AlphaScreen assay); Clean profile in CEREP screen against 55 targets (receptors, ion chanels) at 10 µM;

Screeed at 10 µM in thermal shift assays, dTm(BAZ2A) = 5.2 K, dTm(BAZ2B) = 3.8 K, dTm(CECR2) = 2.0 K; Follow-up in vitro potency (ITC): Kd(CECR2) = 1.55 µM (15-fold selective); Greater 100-fold selective for BRD4 (IC50 >50 µM, BRD4 AlphaScreen assay); Clean profile in CEREP screen against 55 targets (receptors, ion chanels) at 10 µM;

Highly selective in methyltransferase screen at 1 µM and 10 µM; Inactive against protein arginine methyltransferases and DNA methyltransferases; Selective against a panel of 9 epigenetic readers and chromatin binders; Screened against 125 nonepigenetic targets (CEREP panel: GPCRS, ion channels, transporters) at 10 µM, closest targets >50% inhibition: Adenosin A1 receptor (98%, Ki = 21 nM), Adenosin A2A receptor (98%, Ki = 28 nM), Cl-chanel (GABA-gated, 90%, Ki = 1.8 µM), Adenosin A2 receptor (74%, 2 µM), kappa (KOP, 62%, Ki >10 µM), NK2 (61%, Ki = 4.1 µM), delta2 (DOP, 51%, Ki = 9 µM)

Highly selective in methyltransferase screen at 1 µM and 10 µM; Inactive against protein arginine methyltransferases and DNA methyltransferases; Selective against a panel of 9 epigenetic readers and chromatin binders; Screened against 125 nonepigenetic targets (CEREP panel: GPCRS, ion channels, transporters) at 10 µM, closest targets >50% inhibition: Adenosin A1 receptor (98%, Ki = 21 nM), Adenosin A2A receptor (98%, Ki = 28 nM), Cl-chanel (GABA-gated, 90%, Ki = 1.8 µM), Adenosin A2 receptor (74%, 2 µM), kappa (KOP, 62%, Ki >10 µM), NK2 (61%, Ki = 4.1 µM), delta2 (DOP, 51%, Ki = 9 µM)

>100-fold selective against other HMTs, selective against 44 GPCRS, ion channels and transporters at 1 µM, closest targets as % of contr.: H3 (93%, Ki = 45 nM), µ opioid (74%, Ki >10 µM), a2C (67%, Ki > 10 µM), a1D (59%, Ki = 4.5 µM); Selective against 50 kinases at 10 µM

>100-fold selective against other HMTs, selective against 44 GPCRS, ion channels and transporters at 1 µM, closest targets as % of contr.: H3 (93%, Ki = 45 nM), µ opioid (74%, Ki >10 µM), a2C (67%, Ki > 10 µM), a1D (59%, Ki = 4.5 µM); Selective against 50 kinases at 10 µM

Screened at 2 µM via biolayer interferometry (BLI), weak inhibition of BET bromodomains at 2.0 µM (BRD2, BRD3, BRD4, BRDT on both BD1 and BD2), clean at screen with 200 nM; DSF assay screen against 43 bromodomain family members, closest targets: dTM(BRD2, BD1) = 1.35 K, dTM(BRD3, BD1) = 1.55 K, dTM(BRD4, BD1) = 2.09 K, dTm(BRD4, BD2) = 0.58 K; In vitro follow-up (ITC): Kd(BRD4, BD1) = 5.6 µM, Kd(BRD4, BD2) = 20 µM; Screened in CEREP selectivity panel against 104 targets (nuclear receptors, ion channels, and 32 enzyme assays (including 10 kinases, 9 proteases, and 5 phosphodiesterases) at 10 µM, closest targets as % of inhibition: a1A (89%), M2 (67%);

Screened at 2 µM via biolayer interferometry (BLI), weak inhibition of BET bromodomains at 2.0 µM (BRD2, BRD3, BRD4, BRDT on both BD1 and BD2), clean at screen with 200 nM; DSF assay screen against 43 bromodomain family members, closest targets: dTM(BRD2, BD1) = 1.35 K, dTM(BRD3, BD1) = 1.55 K, dTM(BRD4, BD1) = 2.09 K, dTm(BRD4, BD2) = 0.58 K; In vitro follow-up (ITC): Kd(BRD4, BD1) = 5.6 µM, Kd(BRD4, BD2) = 20 µM; Screened in CEREP selectivity panel against 104 targets (nuclear receptors, ion channels, and 32 enzyme assays (including 10 kinases, 9 proteases, and 5 phosphodiesterases) at 10 µM, closest targets as % of inhibition: a1A (89%), M2 (67%);

Screened at 50 µM, >1000-fold selective over other methyltransferases (G9a, EZH2, EHMT1, SUV39H2, EZH1, SUV420H1, SUV420H2, SETD8, SETD2, PRMT1, PRMT3, PRMT5, PRMT8, SETDB1, MLL1, DOT1L, WHSC1, SMYD2, DNMT1); Screened against 137 ion channels, GPCRs, and enzymes at 10 µM, closest targets: Ki(A2B) = 1.3 µM, Ki(BB3) = 8.1 µM, Ki(motilin) = 7.2 µM, Ki(KOP) = 3.8 µM

Screened at 1 µM, 5 µM and 20 µM, closest target at 20 µM: METTL21a (60% of contr.); Selective against panel of 250 enzymes (kinases, GPCRs, ion channels, transporters) at 10 µM, closest targets as % of contr.: 5HT2B (69%, Ki >15 µM), BRSK1 (56%), DLK1 (60%), MSK2 (55%), PKG2 (58%), PRKX (61%);

Screened at 1 and 10 µM, closest target NSD3 (75% at 10 µM), GSK591 is highly selective

Screened in DSF assay at 10 µM, dTm(PBRM1,BD5) = 9.1 K, dTm(SMARCA2) = 8.2 K, dTM(SMARCA4) = 7.0 K, other targets dTm <1 K; Screened in CEREP panel against 102 cellular receptors and 30 enzymes, closest targets: IC50(A1A) = 0.44 µM, IC50(A1B) = 3.6 µM, IC50(kKOP) = 13 µM, IC50(5-HAT2B) = 4.0 µM;

Screened in DSF assay at 10 µM, dTm(PBRM1,BD5) = 9.1 K, dTm(SMARCA2) = 8.2 K, dTM(SMARCA4) = 7.0 K, other targets dTm <1 K; Screened in CEREP panel against 102 cellular receptors and 30 enzymes, closest targets: IC50(A1A) = 0.44 µM, IC50(A1B) = 3.6 µM, IC50(kKOP) = 13 µM, IC50(5-HAT2B) = 4.0 µM;

Screened in DSF assay at 10 µM, dTm(PBRM1,BD5) = 9.1 K, dTm(SMARCA2) = 8.2 K, dTM(SMARCA4) = 7.0 K, other targets dTm <1 K; Screened in CEREP panel against 102 cellular receptors and 30 enzymes, closest targets: IC50(A1A) = 0.44 µM, IC50(A1B) = 3.6 µM, IC50(kKOP) = 13 µM, IC50(5-HAT2B) = 4.0 µM;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

Screened at 10 µM in DSF-assay, closest target: dTm(CREBBP) = 1.18 K;

DSF-assay, dTm(CECR2) = 12.52 K, closest targets, dTM 1-2 K: BRD4(BD2), BRD7, FALZ, SMARCA4, TAF1 (BD2);

No targets confirmed outside BRD family; Screened in BROMOscan panel against 46 targets, closest target: IC50(TRIM24) = 1600 nM, 500-fold selective; Clean profile in CEREP screen against 55 ligand receptors, ion channels and transporters, at 10 µM

No targets confirmed outside BRD family; Screened in BROMOscan panel against 46 targets, closest target: IC50(TRIM24) = 1600 nM, 500-fold selective; Clean profile in CEREP screen against 55 ligand receptors, ion channels and transporters, at 10 µM

No targets confirmed outside BRD family; Screened in BROMOscan panel against 46 targets, closest target: IC50(TRIM24) = 1600 nM, 500-fold selective; Clean profile in CEREP screen against 55 ligand receptors, ion channels and transporters, at 10 µM

Screened at 1 µM, in vitro follow-up of closest targets: Kd(RIPK2) = 110 nM, Kd(ADCK3) = 190 nM, Kd(NLK) = 520 nM, Kd(ACVR1) = 980 nM; Cellular selectivity: IC50(RIPK2) = 360 nM (NanoBRET assay), others not tested;

Screened at 10 µM in DSF assay, dTM (CREBBPA/EP300A) = 9.6/ 10.4 K, closest targets: dTM(BRD4) = 2.0 K, dTM(BRD3) = 1.9 K, dTm(BRD2) = 1.4 K, dTm (BRD3) = 1.2 K; Follow-up Kds via ITC: Kd(BRD4 at BD1/ BD2) = 0.854/ 4.88 µM, Ki(BRD3 at BD1/ BD2) = 1.09/ 2.19 µM, Kd(BRD2 at BD1/BD2) = 1.01/ 1.76 µM, Kd(BRD3 at BD1/BD2) = 1.09/ 2.19µM; Screened in CEREP panel against 104 receptors, ion channels and other enzymes, closest targets: Ki(alpha2A) = 570 nM, Ki(alpha2C) = 110 nM, Ki(PAF) = 540 nM;

Screened at 10 µM in DSF assay, dTM (CREBBPA/EP300A) = 9.6/ 10.4 K, closest targets: dTM(BRD4) = 2.0 K, dTM(BRD3) = 1.9 K, dTm(BRD2) = 1.4 K, dTm (BRD3) = 1.2 K; Follow-up Kds via ITC: Kd(BRD4 at BD1/ BD2) = 0.854/ 4.88 µM, Ki(BRD3 at BD1/ BD2) = 1.09/ 2.19 µM, Kd(BRD2 at BD1/BD2) = 1.01/ 1.76 µM, Kd(BRD3 at BD1/BD2) = 1.09/ 2.19µM; Screened in CEREP panel against 104 receptors, ion channels and other enzymes, closest targets: Ki(alpha2A) = 570 nM, Ki(alpha2C) = 110 nM, Ki(PAF) = 540 nM;

Selective over 15 other lysine, arginine, and DNA methyltransferases; Less than 20% inhibition on 50 kinase targets at 10 µM; Tested in PDSP screen: less than 50% inhibition on 40 7TM targets at 10 µM, greater than 50% inhibition of 4 7TM targets at 10 µM, closest targets: Ki(H3) = 300 nM, Ki(NET) = 1500nM, Ki(Sigma 1) =4700 nM, Ki(Sigma 2) = 65 nM (Radioligand binding assay);

Selective over 15 other lysine, arginine, and DNA methyltransferases; Less than 20% inhibition on 50 kinase targets at 10 µM; Tested in PDSP screen: less than 50% inhibition on 40 7TM targets at 10 µM, greater than 50% inhibition of 4 7TM targets at 10 µM, closest targets: Ki(H3) = 300 nM, Ki(NET) = 1500nM, Ki(Sigma 1) =4700 nM, Ki(Sigma 2) = 65 nM (Radioligand binding assay);

Screened at 50 µM, >1000-fold selective (IC50 >50 µM); Screened against 8 Methyl-Lysine/Arginine binder (DSF assay), dTm(WDR5) = 18.1 K, closest target: dTm(TDRD3) = 1.8 K; Screened in CEREP panel against 215 targets (kinases, GPCRs, ion channels, transporters) at 1 µM: no kinase with >50% inhibition (IC50 >1 µM)

Selectivity on methyltransferases measured by scintillation proximity assay (SPA), selective <50 µM

Selectivity on methyltransferases measured by scintillation proximity assay (SPA), selective <50 µM

Screened against 55 human recombinant receptors at 10 µM, closest targets as % inhibition: 5-HT1A (49%), 5-HAT (74%), dopamine transporter (39%); Screened against 45 other enzymes (GPCRs, kinases, transporters, ion channels, nuclear receptors), >1000-fold selective over other closely related FAD utilizing enzymes (i.e. LSD2, MAO-A, MAO-B)

Selective; <50% inhibition at 250 mM against 10 HMT targets; >50-fold selective against other members of human MBT family: IC50(L3MBTL1) = 2 µM, IC50(L3MBTL4) = 11 µM, IC50(SFMBT) >30 µM, IC50(MBTD1) = 6 µM (Alphascreen assay); Histone demethylases: < 50% inhibition at 10 mM versus 35 targets; Bromodomains: Tm shift < 0.5 °C at 10 mM versus 12 targets (DSF assay); Kinases: < 15% inhibition at 10 mM versus 49 kinases, closest target as % of contr.: FLT3 (64%); NIMH Psychoactive Drug Screening Program Selectivity Panel (Radioligand binding assay): Ki(M1) = 97 nM, Ki(M2) = 72 nM, Ki(M3) = 890 nM, Ki(M4) = 400 nM, Ki(M5) = 4.3 µM, Ki(Alpha 2C) = 860 nM

Selective over other methyltransferases (radioisotope-based filter binding assays), closest target: IC50(EZH1) = 240 nM, others <60 µM; Screened against 44 receptors and enzymes (internal screening at GSK), closest target: IC50(A1b) = 10 µM, others >10 µM, >100-fold selective

Screened against G9a, EHMT1, PRMT3/5, SETDB1, SETD7/8, MLL, EZH2, SUV39H2, SUV420H2; Clean profile in Ricerca selectivity panel at 1 µM against 29 diverse receptors

Screened in DSF assay at 10 µM: highly selective, other targets <2 K; Screened against 130 enzymes (GPCR panel and Eurofins panel) at 10 µM, closest targets: opioid receptors, Ki(MU) = 100 nM, Ki(OPRL1) = 840 nM, Ki(kappa) = 1100 nM, and 5-HAT transporter, Ki = 220 nM

Screened in DSF assay at 10 µM: highly selective, other targets <2 K; Screened against 130 enzymes (GPCR panel and Eurofins panel) at 10 µM, closest targets: opioid receptors, Ki(MU) = 100 nM, Ki(OPRL1) = 840 nM, Ki(kappa) = 1100 nM, and 5-HAT transporter, Ki = 220 nM

Screened at 1 µM and 50 µM against epigenetic methyltransferases including lysine, arginine, and DNA methyltransferases: clean selectivity profile with all targets >80% remaining activity, https://www.thesgc.org/chemical-probes/A-395; Screened in DSF assays, at 0.5 mM and 1 mM, against 8 epigenetic ‘reader domain' proteins: dTm(53BP1) <2 K, dTm(BRPF3) <2 K, dTm(DNMT3A) <2 K, dT,(RBBP1) <2 K, dTm(RBPF1) <2 K, dTm(UHRF1) <2 K, dTm(WDR5) <2 K, dTm(WHSC1L1) <2 K, https://pubmed.ncbi.nlm.nih.gov/28135237/;

Screened at 1 µM, closest target as % of contr.: RIOK3 (46%); Screened at 10 µM against Eurofins-Panlabs screen against 68 targets, closest targets as % of inhibition: SLC6A2 (NET, 78%), SLC6A3 (DAT, 71 %); Screened at 1 µM, 10 µM, and 50 µM, against 32 methyltransferases (enzyme activity assays, including closely related family members SMYD3, SUV420H1, and SUV420H2) >100-fold selective; IC50(SMYD3) = 3 μM, IC50(PAR1) = 1.7 µM

Screened at 1 µM and 10 µM, highly selective for SMYD3 with no activity on other methyltransferases; Screened against 31 kinases in enzyme assays, closest target T-Fyn (HTRF assay) = 5.71 µM, others >20 µM;

Screened at 10 µM in DSF assays, dTm(BAZ2A) = 4.1 K, dTm(BAZ2B) = 2.7 K, closest targets: dTm(TAF1L) = 3.4 K, dTm(BRD9) = 2.3 K; In vitro follow up (biolayer interferometry experiment) at 0.2 µM: Kd(BAZ2B) = 0.1 µM, Kd(BRD9) = 1.2 µM, Kd(TAF1L) = 3.19 µM, (ITC experiment): Kd(TAF1L) = 3.2 µM, Kd(BRD9) = 1.1 µM; Screened in CEREP panel against 55 receptors and ion channels at 10 µM, closest target as % of contr.: Melatonin (MT-1) receptor(4%)

Screened at 10 µM in DSF assays, dTm(BAZ2A) = 4.1 K, dTm(BAZ2B) = 2.7 K, closest targets: dTm(TAF1L) = 3.4 K, dTm(BRD9) = 2.3 K; In vitro follow up (biolayer interferometry experiment) at 0.2 µM: Kd(BAZ2B) = 0.1 µM, Kd(BRD9) = 1.2 µM, Kd(TAF1L) = 3.19 µM, (ITC experiment): Kd(TAF1L) = 3.2 µM, Kd(BRD9) = 1.1 µM; Screened in CEREP panel against 55 receptors and ion channels at 10 µM, closest target as % of contr.: Melatonin (MT-1) receptor(4%)

>1600-fold selective, closest targets: pKd(BRPF2/BRD2) = 6.29, pKd(BRPF3) = 6.00, pKd(BRD9) = 6.00, pKd(BRD7) = 6.09; Screened against 48 ion channels, GPCRs and other enzymes, all pXC50 <5, compound is highly selective;

Screened in DSF assay at 10 µM, closest target: dTm(CECR2) = 2 K, follow up in Alpha Screen: IC50(CECR2) = 2.32 µM

Screened in DSF assay at 10 µM, closest target: dTm(CECR2) = 2 K, follow up in Alpha Screen: IC50(CECR2) = 2.32 µM

In-vitro potencies of related targets: IC50(IDH1,WT) = 466.5 nM, IDH2 IC50(IDH2,WT) = 1360 nM, IC50(IDH2,R140Q) = 1916 nM, IC50(IDH2,R172S) = 997 nM; Selective in vitro for IDH1 over other classes of proteins (7TMs, ion channels, kinases)

In-vitro potencies of related targets: IC50(IDH1,WT) = 466.5 nM, IDH2 IC50(IDH2,WT) = 1360 nM, IC50(IDH2,R140Q) = 1916 nM, IC50(IDH2,R172S) = 997 nM; Selective in vitro for IDH1 over other classes of proteins (7TMs, ion channels, kinases)

In-vitro potencies of related targets: IC50(IDH1,WT) = 466.5 nM, IDH2 IC50(IDH2,WT) = 1360 nM, IC50(IDH2,R140Q) = 1916 nM, IC50(IDH2,R172S) = 997 nM; Selective in vitro for IDH1 over other classes of proteins (7TMs, ion channels, kinases)

Screened at 1 µM and 10 µM, selective over other targets including closely related arginine methyltransferases PRMT4, PRMT6, PRMT7;

Screened at 10 µM, closest target as % of control: NK3 (66%); Screened at 10 µM against 48 BRDs in DSF assays: clean selectivity profile with dTm <1 K for all BRDs, including other members of sub-family IV, except BRD7/9;
In-vitro potency (TR-FRET binding assay): IC50(BRD9) = 325 nM;
Disruption of BRD9 interactions with chromatin at a concentration of 0.8 µM (FRAP assay);
In-cellular potency (NanoBRET assay, HEK293T cells): IC50(BRD7) = 3.3 µM, IC50(BRD9) = 6.2 µM;

Screened at 10 µM, closest target as % of control: NK3 (66%); Screened at 10 µM against 48 BRDs in DSF assays: clean selectivity profile with dTm <1 K for all BRDs, including other members of sub-family IV, except BRD7/9;
In-vitro potency (TR-FRET binding assay): IC50(BRD9) = 325 nM;
Disruption of BRD9 interactions with chromatin at a concentration of 0.8 µM (FRAP assay);
In-cellular potency (NanoBRET assay, HEK293T cells): IC50(BRD7) = 3.3 µM, IC50(BRD9) = 6.2 µM;

Screened at 10 µM, closest targets as % control binding: Cl-channel, GABA gated (70%), Adenosine A1 (82%); Selectivity against histone demethylases, in-vitro potency (AlphaScreen assay): IC50(KDM4A) = 100 µM, IC50(KDM4C) = 100 µM, IC50(KDM4E) >100 µM, IC50(KDM6B) >100 µM, IC50(KDM3A) >100 µM, IC50(KDM2A) = 52 µM, IC50(KDM5C) = 159 µM

In-vitro potencies of closest targets: IC50(PRMT6) = 1.3 µM, IC50(PRMT8) = 8.1 µM, IC50(PRMT4) <0.01 µM, IC50(PRMT1) >10 µM, IC50(PRMT3) >10 µM, IC50(PRMT5) >10 µM, IC50(PRMT7) >10 µM, IC50(PRMT9) >10 µM, IC50(G9a) >10 µM, IC50(GLP) >10 µM, IC50(SETDB1) >10 µM, IC50(SUV39H1) >10 µM, IC50(SUV39H2) >10 µM, IC50(PRDM9) >10 µM, IC50(SETD) >10 µM, IC50(SETD7) >10 µM, IC50(MLL1) >10 µM, IC50(MLL3) >10 µM, IC50(SETD2) >10 µM, IC50(PRC2) >10 µM, IC50(SUV420H1) >10 µM, IC50(SUV420H2) >10 µM, IC50(SMYD2) >10 µM, IC50(SMYD3) >10 µM, IC50(DNMT1) >10 µM, IC50(DNMT3A/3L) >10 µM, IC50(DNMT3B/3L) >10 µM, IC50(NSD1) >10 µM, IC50(NSD2) >10 µM, IC50(NSD3) >10 µM, IC50(ASH1L) >10 µM, IC50(DOT1L) >10 µM

Screened against 71 receptors and ion channels and against 25 other enzymes at 10 µM, closest targets as % of contr.: Adenosine A1 receptor (14%), Benzodiazepine receptor (47%), PDE2A1 (h) (25%), PDE3A (h) (48%) and PDE4D2 (h) (28%); Selectivity >30 fold over all other bromodomain family members except BRD7: Kd(BRD7) = 340 nM (ITC)

Screened at 1 µM, no significant activities below 50%

Screened at 1 µM, no significant activities below 50%

Screened in DSF assay at 10µM, dTm(PRDMT9) = 5.2K, closest target dTm(PRDMT7) = 5.0K; Screened against 108 enzymes and receptors (Eurofins Panlabs Discovery Services), closest targets>50% at 10µM: MOR, A2AB, M2, H3; In vitro follow up with GloSensor assay (PDSP screen): EC50>10µM for all targets

Screened at 0.1 µM and 1 µM, all values determined for active compound SGC8158, closest targets: IC50(PRMT4) = 3.6 nM, IC50(PRMT5-MEP50) = 44 nM, IC50(PRMT9) = 138 nM; Clean in radiometric assays against 35 methyltransferases including PRMTs: IC50(PRMT5) = 0.15 µM, IC50(PRMT4) = 0.14 µM, IC50 (PRMT9) = 0.16 µM, IC50(BCDIN3D) = 1.1 µM, IC50(PRMT3) = 1.6 µM, IC50(DOT1L) = 1.7 µM, IC50(PRMT8) = 15 µM, IC50(PRMT6) = 15 µM, IC50(PRMT1) = 12 µM

Screened at 1 µM, closest targets as % inhibition: ABL1(49%) and ABL2 (49%);

Screened at 10 µM, no cross reactivity among proteases owing to allosteric mechanism; Screened against 468 kinases(KinomeScan DiscoverX) at 1 µM, closest targets as % contr.: FLT3(N841I) (23%), CDK8 (39%), HIPK1 (40%), NEK3 (40%), MAST1 (43%), PIK3CA(E545K) (48%); Clean profile in screen against 45 GPCRs (PDSP screen) at 10 µM; Screened in kinase panel against 50 targets at 10 µM, closest targets: IC50(FLT3) = 3.5 µM; Screened against panel of 110 receptors, ion channels, enzymes and transporters, at 10 µM, closest target as % of inhibition: adenosine transporter AdT (52%);

Screened at 10 µM, closest targets in the GPCR scan: Ki(SIGMAR1) = 1431.53 nM, Ki(TMEM97) = 2051.16 nM, Ki(OPRK1) = 7634.84 nM; hTSH antagonism: IC50 = 24 µM; hFSH antagonism: IC50 > 16 µM; Screened against 468 kinases (KinomeScan (DiscoverX)) at 1 µM, closest target as % of contr.: CDKL2 (33%)

>700-fold selective over the BET family of bromodomains; In-vitro potencies (enzymatic assay) of related targets: IC50(BRD9) = 0.73 μM, IC50(BRD4, BD1) = 11 µM, IC50(BRD4, BD2) = 13 µM, IC50(CECR2) = 7.7 µM, IC50(TAF1, BD2) = 17 µM, IC50(BAZ2B, BRG1, BRPF1, TAF1(BD1)) >20 µM;

>700-fold selective over the BET family of bromodomains; In-vitro potencies (enzymatic assay) of related targets: IC50(BRD9) = 0.73 μM, IC50(BRD4, BD1) = 11 µM, IC50(BRD4, BD2) = 13 µM, IC50(CECR2) = 7.7 µM, IC50(TAF1, BD2) = 17 µM, IC50(BAZ2B, BRG1, BRPF1, TAF1(BD1)) >20 µM;

290-fold selectivity relative to the BD1 domains of BRD2, BRD3 and BRD4; Clean in screen against 75 kinases

290-fold selectivity relative to the BD1 domains of BRD2, BRD3 and BRD4; Clean in screen against 75 kinases

290-fold selectivity relative to the BD1 domains of BRD2, BRD3 and BRD4; Clean in screen against 75 kinases

290-fold selectivity relative to the BD1 domains of BRD2, BRD3 and BRD4; Clean in screen against 75 kinases

>70-fold selective over other bromodomains tested, closest targets: Kd(CECR2) = 140 nM, Kd(BRD7) = 390 nM, Kd(CREBBP) = 740 nM, Kd(EP300) = 770 nM, others >1.5 µM; Inactive against a panel of 49 human receptors, ion channels, kinases and other enzymes