Chemogenomic set

Screened at 3 µM against 75 ion channels, receptors, GPCRs and enzymes (ligand binding assay), closest target as % inhibition: PRCP (97%), >100 000-fold selective for FNTB over the closely related enzyme PGGT1B (GGTase I); Screened against 468 kinases (KinomeScan DiscoverX), at 1 µM, closest targets as % of contr.: EPHA1 (38%), NEK7 (46%), BRSK2 (48%), GRK3 (48%), follow-up Kd values: IC50(EPHA1/NEK7/BRSK2/GRK3) >10 µM; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(HRH1) >10 µM;

Screened at 10 µM, closest target as % of inhibition: CNR1 (88 %); CNR1 functional test (GTPyS binding): IC50 = 10.6 µM; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(TMEM97) = 1203.22 nM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: CDK11A (0%), DCLK2(0%), MAP3K12(0%), CDK18(0%), CDK4(18%), TYK2(53%);

Screened at 1 µM, closest target: IC50(RPS6KA3) = 0.9 µM, >1000 fold selective against all other kinases; Eurofins-Panlabs radioligand binding assay (screened against 68 targets, at 10 µM): clean profile; Screened against 45 GPCR targets (PDSP screen) at 10 µM: clean profile

Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: MAP2K5 (16%), NTRK3 (32%), AURKA (43%), LATS1 (48%); Screened against 30 GPCR, ion channels, receptors and other enzymes (MDS Pharma Service panel, radioligand binding assays) at 10 µM, closest target as % inhibition: Imidazoline I2, central (19%); Screened against 45 GPCRs (PDSP screen) at 10 µM, closest targets: SLC6A3 (77%)

Solute carrier family 9 isoform selectivity: > 30-fold selective versus SLC9A2 (IC50 = 231 nM) and >1000-fold versus SLC9A3 (IC50 >16 µM); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(DAT) = 1523 nM; Selective in KinomeScan (DiscoverX) against 468 targets at 1 µM; Screened in Eurofins-Panlabs panel (68 targets), closest target M2 (67 % inhibition at 10 µM)

Screened at 10 µM, closest targets: Ki(GABA/PBR) = 82 nM, Ki(TMEM97) = 605 nM, Ki(SIGMAR1) = 1158 nM; Radioligand binding assay: IC50(CNR2) = 4.1 µM (PMID: 31859507); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % contr.: DYRK1B (35%), STK11 (43%), EIF2AK4 (48%), PIK3C2B (48%), PRKCE (48%); Screened against 168 enzymes and receptors (MSD Pharma/Taiwan screen) at 10 µM, closest targets: IC50(ALOX5) = 1.34 µM, IC50(EGFR) = 3.26 µM, IC50(MAPK14) = 3.68 µM, IC50(MAPK3) = 8.61 µM; Radioligand binding assays (MDS Pharma Service): Ki(SLC18A2) = 0.648 µM, Ki(ADORA3) = 0.773 µM, Ki(SLC6A3) = 0.799 µM, Ki(ADRA2C) = 1.13 µM;

Screened at 10 µM, closest targets: Ki(SLC6A3) = 283 nM, Ki(GABA/PBR) = 1115 nM, Ki(DRD2) = 3746 nM; Prostanoid receptor binding assay: Ki(TBXA2R)= 3804 nM, >100-fold selective against related receptors; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contrl.: ROCK1 (34%), DYRK1B (44%)

Screened at 0.1 µM, closest targets: Kd(LATS2) = 55 nM, Kd(MAPK15) = 30 nM, Kd(TGFBR2) = 54 nM, Kd(ROCK2) = 82 nM, Kd(ULK1) = 140 nM

Screened at 0.1 µM, closest targets: Kd(LATS2) = 55 nM, Kd(MAPK15) = 30 nM, Kd(TGFBR2) = 54 nM, Kd(ROCK2) = 82 nM, Kd(ULK1) = 140 nM

Screened at 10 µM, closest targets as % inhibition: ADRA1A (84%), ADRA2B (80%); In vitro follow up: Ki(ADRA1A) >2.7 µM (> 1700 fold), Ki(ADRA1B) >1.2 µM (>750 fold); Screened against 468 kinases (KinomeScan, DiscoverX) at 1 µM, closest targets as % contr.: MAPK6 (0%), TYRO3 (33%);

Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: CDK8 (36%), EIF2AK4 (47%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(GABAA/BZP) = 934 nM; Selectivity against recently discovered D-Dopachrome Tautomerase (DDT) is unknown.

Selective across the Nudix family when tested in Tm shift assay; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(GABAA/BZP) = 1527 nM; Screened against 30 kinases at 40 µM (Bayer in-house kinase panel), all targets IC50 >8 µM (PMID: 28679043)

Highly selective in panel of GPCRs, ion channels, transporters, enzymes and proteases, at 10 µM: METAP2/1 >1000-fold; Selectivity against other proteases: < 1000 -fold; Clean in KinomeScan (DiscoverX) against 468 targets at 1 µM, closest targets as % of contr.: LATS2 (26%), EPHB6 (36%), WNK2 (41%), GRK2 (42%), RPS6KA3 (48%), SGK1 (49%); Clean in screen against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(SLC6A3) = 1679 nM, Ki(GABAA/BZP) = 4538 nM

Screened at 10 µM, panel of ion channels, receptors, enzymes, > 100-fold selective (including closely related TRPV1, TRPA1 channels); Clean in CardiacProfiler ion channel panel (Millipore) at 10 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: MAPK14 (0%), GRK3 (40%), TAOK3 (41%), PIK3CA(Q546K) (42%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki (GABA/PBR) = 1022.12 nM

Screened at 100 nM, closest targets as % of contr.: MAP2K5 (8.5%), MAP3K19 (10%), SYK (31%), FLT3(D835V) (32%), STK17A (40%), TAOK3 (41%), PIK3CA(Q546K) (45%); In vitro follow-up of closest targets: IC50(FLT3) = 123 nM, IC50(MAP2K5) = 847 nM (Bayer in-house data), IC50(FLT3) = 123 nM, IC50(RPS6KA6) = 276 nM, IC50(DRAK1) = 311 nM, IC50(ULK1) = 7930 nM (PMID: 31859507); Kd(STK17A) = 74 nM, Kd(MAP3K19) = 9.6 nM (DiscoverX), IC50(STK17A) = 310 nM (Eurofins kinase panel); Cellular selectivity (NanoBRET assay, HEK293T cells): IC50(MAP3K19) >20 µM, IC50(STK17A) >20 µM, IC50(MAP2K5) >20 µM, IC50(FLT3) >20 µM; Screened against 45 GPCR targets (PDSP screen), closest targets: Ki(TMEM97) = 1155.57 nM , Ki(HTR2B) = 1305.2 nM;

Screened at 100 nM, closest targets as % of contr.: MAP2K5 (8.5%), MAP3K19 (10%), SYK (31%), FLT3(D835V) (32%), STK17A (40%), TAOK3 (41%), PIK3CA(Q546K) (45%); In vitro follow-up of closest targets: IC50(FLT3) = 123 nM, IC50(MAP2K5) = 847 nM (Bayer in-house data), IC50(FLT3) = 123 nM, IC50(RPS6KA6) = 276 nM, IC50(DRAK1) = 311 nM, IC50(ULK1) = 7930 nM (PMID: 31859507); Kd(STK17A) = 74 nM, Kd(MAP3K19) = 9.6 nM (DiscoverX), IC50(STK17A) = 310 nM (Eurofins kinase panel); Cellular selectivity (NanoBRET assay, HEK293T cells): IC50(MAP3K19) >20 µM, IC50(STK17A) >20 µM, IC50(MAP2K5) >20 µM, IC50(FLT3) >20 µM; Screened against 45 GPCR targets (PDSP screen), closest targets: Ki(TMEM97) = 1155.57 nM , Ki(HTR2B) = 1305.2 nM;

Functional proteomic screen, testet at 10 µM and 100 µM, PF-04457845 showed exquisite selectivity for FAAH relative to other mammalian serine hydrolases (no targets up to 100 µM); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: KIT(L576P) (0%), TAOK3 (42%), PIK3CA (45%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(HTR2A) = 248.71 nM, Ki(TMEM97) = 436.72 nM, Ki(SIGMAR1) = 472.06 nM, Ki(ADRA2C) = 595.66 nM, Ki(GABA/ PBR) = 1140.08 nM; Screened in CEREP ligand profiling, panel of 68 ion channels, GPCRS and transporters at 10 µM, closest target: Ki(HTR2A) =1.6 µM;

Selectivity against other glucose transporters (CellTiter-Glo® Luminescent Cell Viability Assay, Promega): IC50(SLC2A4) = 270 nM, IC50(SLC2A3) = 1.6 µM, IC50(SLC2A2) = 9.4 µM, >100-fold selective; Screened against 468 targets in KinomeScan (DiscoverX) at 1 µM, closest targets as % of contr.: WNK4 (0%), DDR1 (1.5%); Screened against 45 GPCRs (PDSP screen) at 10 µM, closest target: Ki(HRH2) = 8078 nM; Screened in Eurofins-Panlabs radioligand binding assay against 68 enzymes, closest targets: IC50(ADORA3) =1.14 µM, IC50(PTGER4) = 1.12 µM;

Screened at 10 µM (radioligand binding assay) against 75 enzymes, GPCRS, transporters, and ion channels, closest targets as % inhibition: to SLC6A3 (43%), SLC6A2 (32%), MTNR1A (31%), >1000-fold selective over other TRP channels (TRPV1, TRPV3, TRPV4, or TRPM8); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest target as % of contr.: ULK2 (46%) ; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(SIGMAR1) =505.48 nM;

Screened against 67 GPCRs, ion channels, kinases and transporters at 10 µM, closest targets: IC50(SLC6A3) = 2.17 µM, IC50(P2RX1) >50 µM, IC50(P2RX2) >30 µM, IC50(P2RX3) = 8.3 µM, IC50(P2RX7) = 10.6 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: WNK2 (0%), WNK4 (0%), BUB1 (1.6%), EGFR(L747-T751del,Sins) (47%); Screened against 45 GPCR targets at 10 µM (PDPS screen), closest targets: Ki(TMEM97) = 4.34 µM, Ki(ADRA2B) = 4.58 µM, Ki(HTR2C) = 6.52 µM;

Screened against 47 receptors, ion channel and other enzymes at 10 µM, closest target as % inhibition: CACNA1C (72%), >1000 fold selective against closely related ITGB2 and ITGB1; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: STK39 (24%), EIF2AK2 (34%), STK38L (49%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: TMEM97 (26%);

Screened at 1 µM, in-vitro potencies of closest targets: Kd(RPS6KA3) = 52 nM, Kd(RPS6KA2) = 24 nM, Kd(RPS6KA6) = 36 nM, Kd(FLT3) = 124 nM, Kd(RPS6KA5) = 43 nM, Kd(MST3) = 94 nM

Screened at 1 µM, in-vitro potencies of closest targets: Kd(RPS6KA3) = 52 nM, Kd(RPS6KA2) = 24 nM, Kd(RPS6KA6) = 36 nM, Kd(FLT3) = 124 nM, Kd(RPS6KA5) = 43 nM, Kd(MST3) = 94 nM

Screened at 1 µM, closest targets as % contr.: PIK3CA (33.9%), DDR1 (36.7%), JAK1 (38.6%), PNCK (39.5%), MAPK9 (39.6%), all >20 µM in NanoBRET cellular assays (HEK293T cells)

Screened at 1 µM, closest targets as % contr.: PIK3CA (33.9%), DDR1 (36.7%), JAK1 (38.6%), PNCK (39.5%), MAPK9 (39.6%), all >20 µM in NanoBRET cellular assays (HEK293T cells)

Screened at 1 µM, closest targets as % of control: DYRK1B (44%), DYRK1A (48%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(ADRA2C) = 625.6 nM, Ki(ADRA2B) = 1439.1 nM, Ki(HTR2B) = 1537.5 nM, Ki(ADRB1) = 2370.3 nM; Screened in Eurofins-Panlabs radioligand binding assays against 43 targets at 10 µM, closest targets: IC50(ADRA2B) = 4.7 nM, IC50(HTR2B) = 5.33 nM, IC50(ADRB1) =9.62 nM;

> 100 fold selective against related nuclear receptors (PGR, NR3C2, ESR1, AR); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % of contr: DRD5 (34%); Screened against 49 targets (Eurofins-Panlabs panel at Boehringer Ingelheim) at 10 µM: clean profile; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: LATS2(40%)

Screened at 10 µM; >100 fold selective against related nuclear receptors (PGR, NR3C2, ESR1, AR); Screened at 10 µM against 45 GPCRs, closest targets >30% of inhibition: DRD5 (34%), HTR3A (32%); Screened at 1 µM against 468 kinases (KinomeScan, DiscoverX), closest targets <45% of control: LATS2 (40%), NDR2 (45%), NEK1 (45%)

High selectivity over other acyltransferases, >100-fold over mouse MGAT1 (IC50 = 2.6 µM), >1000-fold over human DGAT1 (IC50 > 30 µM), human DGAT2 (IC50 > 30 µM), human ACAT1 (IC50 = 19 µM); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: MARK3 (12%), FLT3(ITD,F691L) (32%), SRPK2 (34%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target: Ki(HTR1D) = 4.65 µM

Screened at 1 µM, closest targets as % of contrl.: NEK1 (40%), STK38L (41%), EIF2AK2 (46%); Screen against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % inhibition: CHRM4 (49%); Clean in Takeda in-house kinase panel against 344 targets, at 1 µM; compound is protein-protein inhibitor targetting non-conserved sites.

Screened at 10 µM, closest target as % of inhibition: HTR7 (21%); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest target as % of contr.: STK38L (49%); Clean in Diversity panel screen (by Ricerca) against>100 targets, at 10 µM

Screened at 1 µM, closest targets as % of contr.: ASK (13%), NEK1 (44%); Screened at 10 µM against 45 GPCRs (PDSP screen), in-vitro potencies of closest targets: Ki(GABA/PBR) = 2.56 µM, Ki(SIGMAR1) =3.55 µM; Screened at 10 µM against 44 other enzymes (CEREP selectivity screen), closest targets as % inhibition: CACNA1C (80%), SCN1B (66%), SLC6A2 (61%), HTR2A (55%)

Screened at 1 µM, closest targets as % of contr.: MYO3B (38%), MATK(41%), DYRK1B (46%); Screened at 10 µM against 45 GPCRs (PDSP screen), closest targets as % of inhibition: ADRA1A(25.68%), HRH1(23.9%), TMEM97(18.5%)

Screened at 1 µM, closest targets as % of contr.: STK33 (45%), MYO3B (47%), FLT3(ITD,D835V) (48%), BMX (49%); Screened against 358 kinases (Eurofins panel) at 1 µM, closest target as % of contr.: CK2A2 (67%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest targets: Ki(HTR2A) = 133.44 nM, Ki(ADRA2C) = 130.87 nM, Ki(HRH2) = 139.82 nM, Ki(HTR1D) = 181.12 nM, Ki(TMEM97) = 179.81 nM, Ki(CHRM1) = 237.75 nM, Ki(ADRA1D) = 337.65 nM, further GPCRs and transporters inhibited but not considered causative for on-target and downstream cellular effects or antiproliferative activity; Lead profiling screen (Eurofins) against 77 GPCRs, transporters, nuclear receptors and enzymes at 10 µM, closest targets as % inhibition: HTR2A (110%), ADRA1A (106%), OPRK1 (105%),HTR2C (104%), HTR2B (100%); Screened against 358 targets (Eurofins Kinase panel) at 1 µM: clean profile (all tested kinases retain activity > 67%), GEFs: IC50 > 20 µM on SOS2 (KRAS(G12C)–SOS2(cat) activation assay) and MCF2L (CDC42 activation by DBS assay)

Screened at 100 nM, closest targets as % of contr.: DYRK1A (0.3%), DYRK1B (4.4%), MAP2K5 (4.9%), KIT (14%), MAP3K5 (19%), PDGFRB (24%), PDGFRA (29%); IC50 follow up in cellular NanoBRET assays: IC50(DYRK1A) = 32 nM, IC50(DYRK1B) = 67 nM, IC50(MAP2K5) >10 µM, IC50(KIT) = 2.44 µM, IC50(MAP3K5) >10 µM, IC50(PDGFRB) = 3 µM, IC50(PDGFRA) = 2.52 µM

Screened at 100 nM, closest targets as % of contr.: DYRK1A (0.3%), DYRK1B (4.4%), MAP2K5 (4.9%), KIT (14%), MAP3K5 (19%), PDGFRB (24%), PDGFRA (29%); IC50 follow up in cellular NanoBRET assays: IC50(DYRK1A) = 32 nM, IC50(DYRK1B) = 67 nM, IC50(MAP2K5) >10 µM, IC50(KIT) = 2.44 µM, IC50(MAP3K5) >10 µM, IC50(PDGFRB) = 3 µM, IC50(PDGFRA) = 2.52 µM

Screened at 100 nM, closest targets as % of contr.: DYRK1A (0.3%), DYRK1B (4.4%), MAP2K5 (4.9%), KIT (14%), MAP3K5 (19%), PDGFRB (24%), PDGFRA (29%); IC50 follow up in cellular NanoBRET assays: IC50(DYRK1A) = 32 nM, IC50(DYRK1B) = 67 nM, IC50(MAP2K5) >10 µM, IC50(KIT) = 2.44 µM, IC50(MAP3K5) >10 µM, IC50(PDGFRB) = 3 µM, IC50(PDGFRA) = 2.52 µM

Screened at 100 nM, closest targets as % of contr.: DYRK1A (0.3%), DYRK1B (4.4%), MAP2K5 (4.9%), KIT (14%), MAP3K5 (19%), PDGFRB (24%), PDGFRA (29%); IC50 follow up in cellular NanoBRET assays: IC50(DYRK1A) = 32 nM, IC50(DYRK1B) = 67 nM, IC50(MAP2K5) >10 µM, IC50(KIT) = 2.44 µM, IC50(MAP3K5) >10 µM, IC50(PDGFRB) = 3 µM, IC50(PDGFRA) = 2.52 µM

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF assays, dTm values of closest targets: dTm(EP300) = 2.71 K, dTm(CREBBP) = 2.66 K, dTm of main targets: dTm(BRD2, BD1) = 4.61 K, dTm(BRD2, BD2) = 5.32 K, dTm(BRD3, BD1) = 5.24 K, dTm(BRD3, BD2) = 5.45 K, dTm(BRD4, BD1) = 6.84 K, dTm(BRD4, BD2) = 3.79 K, dTm(BRDT, BD1) = 2.08 K; Screened at 1 µM against 38 protein kinases (Invitrogen kinase panel), closest targets as % of inhibition: NTRK1 (28.5%), CHEK1 (16%); Screened at 10 µM against 14 membrane receptors, closest targets as % of inhibition: PDE3B (46.3%), H1 (6.5%)

Screened in DSF-panel, closest target: dTm(BRD4, BD1) = 2.1 K; In vitro follow-up with AlphaScreen: IC50(BRD4, BD1) >10 µM, ITC: Kd(BRD4, BD1) = 3.9 µM; Screened in CEREB panel against 56 GPCRs, ion channels and transporters at 10 µM, closest targtes as % inhibition: A1 (82%), 5-HT6 (54%), A3 (53%);

Screened in DSF-panel, closest target: dTm(BRD4, BD1) = 2.1 K; In vitro follow-up with AlphaScreen: IC50(BRD4, BD1) >10 µM, ITC: Kd(BRD4, BD1) = 3.9 µM; Screened in CEREB panel against 56 GPCRs, ion channels and transporters at 10 µM, closest targtes as % inhibition: A1 (82%), 5-HT6 (54%), A3 (53%);

Screened in DSF-panel, closest target: dTm(BRD4, BD1) = 2.1 K; In vitro follow-up with AlphaScreen: IC50(BRD4, BD1) >10 µM, ITC: Kd(BRD4, BD1) = 3.9 µM; Screened in CEREB panel against 56 GPCRs, ion channels and transporters at 10 µM, closest targtes as % inhibition: A1 (82%), 5-HT6 (54%), A3 (53%);

Selective against other available HATs (MYST3, MYST4, PCAF, HAT1, TIP60 and GCN5L2); Screened in KinomeScan (DiscoverX) against 468 targets at 10 µM, clostest targets as % of contr.: PRKCE (22%), LATS2 (47%), ABL1(H396P)-nonphosphorylated (49%); Screened against 45 GPCR targets, closest targets: Ki(SLC6A4) = 223.27 nM, Ki(GABA/PBR) = 1107.62 nM, Ki(HTR2B) = 1292.04 nM, Ki(SLC6A3) = 1817.04;Cerep ligand profiling against 83 targets, at 10 µM, closest targets as % inhibition: SLC6A4 (99%), SLC6A3 (94%);

Selective against other available HATs (MYST3, MYST4, PCAF, HAT1, TIP60 and GCN5L2); Screened in KinomeScan (DiscoverX) against 468 targets at 10 µM, clostest targets as % of contr.: PRKCE (22%), LATS2 (47%), ABL1(H396P)-nonphosphorylated (49%); Screened against 45 GPCR targets, closest targets: Ki(SLC6A4) = 223.27 nM, Ki(GABA/PBR) = 1107.62 nM, Ki(HTR2B) = 1292.04 nM, Ki(SLC6A3) = 1817.04;Cerep ligand profiling against 83 targets, at 10 µM, closest targets as % inhibition: SLC6A4 (99%), SLC6A3 (94%);

> 200-fold selective on all other HATs tested; No significant activity on 166 targets at 10 µM (kinases, GPCRs, HDACs, phosphatases etc.), no target was affected by more than 50%, closest hits in the GPCR scan: Ki(DRD2) = 397.52 nM, GABAA/BZP (75.65 % inhibition)

> 200-fold selective on all other HATs tested; No significant activity on 166 targets at 10 ?M (Kinases, GPCRs, HDACs, phosphatases etc.), no target was affected by more than 50%, closest hits in the GPCR scan: Ki(DRD2) = 397.52 nM, GABAA/BZP (75.65 % inhibition)

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM in DSF-assay, closest target: dTM(CREBBP) = 1.04 K, inactive in CEREP assay; Screened at 1 µM in CEREP panel against 55 ligand receptors, ion channels and transporters, clean profile;

Screened at 10 µM, closest target: Ki(TMEM97) = 1107.9 nM; Screened against 122 enzymes, receptors, transporters and ion channels (Leadprofiling screen, Ricerca) at 10 µM, closest targets: IC50(MAPK3) = 4.87 µM, IC50(MAPK14) = 10.7 µM, IC50(CNR1) = 3.44 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % contr.: CDK5 (0%), CLK4 (0%), MAP3K13(0%), PRKAA1(39%)

Screened at 10 µM against 75 receptors, ion channels and other enzymes, closest targets as % inhibition: EDNRB (56%), OPRD1 (62 %); in vitro follow up: Ki(EDNRB) = 13000 nM; Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % inhibition: HRH1(54%); In vitro follow up: Ki(HRH1) >10 µM; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets: CDK5 (0%), CLK4 (0%), MAP3K13 (0%);

Screened at 1 µM, closest target: Ki(OPRD1) = 47.5 nM; Potency against NPFFR2 (GPR74) and NPFFR1 (GPR147) >30 fold;

Screened at 10 µM (binding assay), closest targets as % inhibition: EDNRA (97.1%), BZD peripheral (72.1%), CCK (63.1%), Cl- channel (77.4%); In vitro follow up of closest target: IC50(EDNRA) = 4.28 µM (>600-fold selective, EDNRB- dependent phosphatidylinositol hydrolysis assay in MMQ cells); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets: FLT3(ITD,F691L) (13%), NEK5(29%); Screened against 45 GPCR targets (PDSP screen) at 10 µM, closest target as % inhibition: HRH1 (36%); Screened in Eurofins-Cerep panel against 7 targets at 10 µM (enzymes and uptake assays): clean profile;

Screened at 10 µM, no target >50% of inhibition; F-04418948 displayed >2000-fold functional selectivity for the human EP2 receptor over antagonist activity against the human EP1, EP3, EP4, DP1 and CRTH2 receptors; Selectivity over human IP, FP, TP and LTB4 receptors; Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: PRKAA2 (28%), TAOK3 (35%), PIK3CA (42%); Screened in CEREP ligand profiling (at 10 µM, against 125 targets), closest targets: IC50(NQO2) = 0.53 µM, IC50(PDE3) = 3.5 µM, IC50(LTB4R) = 9.7 µM

Screened at 10 µM, closest target: Ki(ADRB1) = 1.88 µM; Screened in CEREP panel of CCRs at 10 µM: >1000-fold selective over other CCRs (CCR10, CCR9, CCR6, CCR4, all >90% of control); Screened against 69 receptors (Eurofins-Panlabs screen) at 10 µM, closest target as % inhibition: DATRANS (71%); Screened against 468 kinases (KinomeScan DiscoverX) at 1 µM, closest targets as % of contr.: NRD2 (34%), LRRK2 (43%)

Closest targets: Ki(DRD2) = 369.37 nM, Ki(TMEM97) = 489.44 nM, Ki(HTR6) = 748.23 nM; Screened against 45 GPCR targets (PDSD screen) at 10 µM, closest targets: Ki(D2) = 369.37 nM, Ki(5-HT6)= 748.23 nM, Ki(Sigma2) = 489.44 nM, Ki(DAT) = 1109 nM; Screened against 468 kinases (KINOMEscan DiscoverX) at 10 µM, closest targets: Kd(BTK) = 27 µM, Kd(MAPK8) = 19 µM, Kd(MAPK10) >30 µM, PMID: 28288109;

Highly selective in GPCR screen at 10 µM, clostest target: Ki(5-HT1A) = 849.9 nM