lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@H]1Oc2cc(cnc2N)-c2c(nn(C)c2C#N)CN(C)C(=O)C2C=CC(F)=CC21
InChIKey: VXYZCOCRVAQWBG-VCANKDNSSA-N
Target: Leukocyte tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29376
Assay: Inhibition of GST-tagged recombinant LTK (450 to 864 residues) (unknown origin) expressed in Insect cell
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 408.44, ALogP 2.33, HBA 7, HBD 1, PSA 110.06, QED 0.72
Compound (SMILES): CNC(=O)C(Cc1ccc(C(=O)c2ccccc2)cc1)NC(=O)c1ccc(OC(C)C)c(Cl)c1
InChIKey: KEJPESDCHUPRQN-UHFFFAOYSA-N
Target: Centromere-associated protein E (SINGLE PROTEIN), Homo sapiens
UniProt: Q02224
Assay: Binding affinity to human CENP-E I182L/T183A/K184T mutant assessed as inhibition constant
Measurement: Ki = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 478.98, ALogP 4.45, HBA 4, HBD 2, PSA 84.5, QED 0.45
Compound (SMILES): COC(=O)[C@H](CO)NC(=O)[C@H](CCCCN(C)C1CC2CCC1C2)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)c1ccc(C(C)(C)C)cc1
InChIKey: UKWNWTBRKZUHGX-GKUWNYOMSA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Selectivity interaction (SPR) EUB0001090a CBX8
Measurement: Kd = 458 nM (pChEMBL 6.340000152587891)
Descriptors: MW 847.11, ALogP 3.79, HBA 9, HBD 6, PSA 195.27, QED 0.08
Compound (SMILES): CN(C)Cc1ccc(S(=O)(=O)n2ccc(/C=C/C(=O)NO)c2)cc1.Cl
InChIKey: BVXPKDRKHXARHY-HAAWTFQLSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Affinity Biochemical interaction: (detection of AMC fluorescence generated by tryptic cleavage of deacetylated peptide Ac-NH-GGK(Ac)-AMC, enzymatic assay) EUB0002304aCl HDAC1
Measurement: IC50 = 42.5 nM (pChEMBL 7.369999885559082)
Descriptors: MW 349.41, ALogP 1.31, HBA 6, HBD 2, PSA 91.64, QED 0.47
Compound (SMILES): CC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@H](C=O)CC(=O)O.O=C(O)C(F)(F)F
InChIKey: VQTZMPRKMMMAQG-YPFLJBJZSA-N
Target: Caspase-14 (SINGLE PROTEIN), Homo sapiens
UniProt: P31944
Assay: Selectivity interaction (Enzymatic assay) EUB0002198aAD CASP14
Measurement: IC50 = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 538.56, ALogP -1.5, HBA 8, HBD 7, PSA 236.75, QED 0.11
Compound (SMILES): C#CCNC(=O)[C@H](CO)NC(=O)[C@H](CCCCN(CC)CC)NC(=O)[C@H](NC(=O)[C@@H](NC(=O)c1ccc(Oc2ccccc2)cc1)C1CCCC1)[C@@H](C)CC
InChIKey: HEDLPSLAOJYDMT-GUKRFWIHSA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Affinity Biochemical interaction: (Fluorescence polarisation (fluorescein conjugated)) EUB0002326a CBX8
Measurement: Kd = 800 nM (pChEMBL 6.099999904632568)
Descriptors: MW 760.98, ALogP 3.52, HBA 8, HBD 6, PSA 178.2, QED 0.07
Compound (SMILES): CCCOc1cc(OCCCCN(C)C)cc(Oc2cc3c(cc2NS(=O)(=O)c2ccc(OC)c(OC)c2)n(C)c(=O)n3C)c1.O=C(O)C(F)(F)F
InChIKey: GAOCLDPTFLDJIP-UHFFFAOYSA-N
Target: Transcription intermediary factor 1-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: O15164
Assay: Affinity Biochemical interaction: (AlphaScreen) EUB0002253aAD TRIM24
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 642.78, ALogP 5, HBA 11, HBD 1, PSA 122.49, QED 0.17
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)CCl)[C@@H](C)O
InChIKey: UCWNTWGHVCSMJP-WPUDHWPRSA-N
Target: Caspase-5 (SINGLE PROTEIN), Homo sapiens
UniProt: P51878
Assay: Affinity Biochemical interaction: (enzymatic assay (fluorogenic substrate cleavage)) EUB0002176a CASP5
Measurement: IC50 = 329 nM (pChEMBL 6.480000019073486)
Descriptors: MW 569.06, ALogP -0.1, HBA 7, HBD 6, PSA 191, QED 0.15
Compound (SMILES): CC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C=O)CC(=O)O)C(C)C
InChIKey: MXGJLIBJKIIQSF-FPXQBCRKSA-N
Target: Caspase-5 (SINGLE PROTEIN), Homo sapiens
UniProt: P51878
Assay: Selectivity interaction (Eurofins (enzymatic assay)) EUB0002201a CASP5
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 500.55, ALogP -0.81, HBA 7, HBD 6, PSA 208.07, QED 0.15
Compound (SMILES): Cl.N=C(N)NC(=O)c1cnn(-c2cccc3ncccc23)c1C1CC1
InChIKey: JTEXNYIOROVPIR-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Selectivity interaction (Panel of neurotransmitter receptors, receptors, ion channels/regulatory sites) EUB0002781aCl OPRM1
Measurement: Ki = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 320.36, ALogP 1.92, HBA 5, HBD 3, PSA 109.68, QED 0.51
Compound (SMILES): COC(=O)CC[C@H](NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)OCc1ccccc1)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(=O)OC)C(=O)CF
InChIKey: NLZNSSWGRVBWIX-KRCBVYEFSA-N
Target: Caspase-5 (SINGLE PROTEIN), Homo sapiens
UniProt: P51878
Assay: Affinity Biochemical interaction: (enzymatic assay (fluorogenic substrate cleavage)) EUB0002202a CASP5
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 763.78, ALogP 1.48, HBA 11, HBD 6, PSA 239.77, QED 0.06
Compound (SMILES): Cc1csc(CCCNCc2cc(=O)c3cc(F)ccc3[nH]2)n1
InChIKey: HWQOQIALQACUAC-UHFFFAOYSA-N
Target: D(4) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21917
Assay: Agonist activity at DRD4 expressed in human HEK293T cells assessed as cAMP accumulation incubated for 15 mins by split-luciferase-based biosensor analysis
Measurement: EC50 = 33.4 nM (pChEMBL 7.480000019073486)
Descriptors: MW 331.42, ALogP 3.15, HBA 4, HBD 2, PSA 57.78, QED 0.68
Compound (SMILES): COc1ccccc1-c1cc(Nc2cccc(NS(C)(=O)=O)c2)ncn1
InChIKey: GZMVECPBSUHMMJ-UHFFFAOYSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51948
Assay: Inhibition of CDK7/Cyclin H/MAT1 (unknown origin) using ULight MBP peptide as substrate in presence of ATP incubated for 1 hr by FRET based LANCE Ultra KinaSelect screen assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 370.43, ALogP 3.27, HBA 6, HBD 2, PSA 93.21, QED 0.69
Compound (SMILES): Cn1cc(-c2cnc3nnn(Cc4cc5cccnc5cc4F)c3n2)cn1
InChIKey: JLNMOXLBPUEIPE-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition of His6-tagged human c-Met Y1230H mutant expressed in Sf9 cells pre-incubated with enzyme for 30 mins prior to addition of ATP using (Glu4Tyr) or Met2 peptide substrate by coupled enzymatic assay
Measurement: Ki = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 360.36, ALogP 2.36, HBA 8, HBD 0, PSA 87.2, QED 0.49
Compound (SMILES): C[C@@H](Oc1cc(-c2cnn(C3CCNCC3)c2)cnc1N)c1c(Cl)c(F)cc2c1OCCO2
InChIKey: FMEVJCDQZZZMHA-CYBMUJFWSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition of His6-tagged human c-Met Y1230H mutant expressed in Sf9 cells pre-incubated with enzyme for 30 mins prior to addition of ATP using (Glu4Tyr) or Met2 peptide substrate by coupled enzymatic assay
Measurement: Ki = 211 nM (pChEMBL 6.679999828338623)
Descriptors: MW 473.94, ALogP 4.16, HBA 8, HBD 2, PSA 96.45, QED 0.57
Compound (SMILES): CN(CCN)Cc1c[nH]cc1-c1cccc(C(F)(F)F)c1
InChIKey: VZDPLMKEFMWBMM-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human PRMT6 using (3H) methylated biotin labelled peptide as substrate in presence of 3H-SAM by scintillation proximity assay
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 297.32, ALogP 3.09, HBA 2, HBD 2, PSA 45.05, QED 0.89
Compound (SMILES): Cc1ccc2[nH]c(CNCCC[C@H]3CCOC3)cc(=O)c2c1
InChIKey: IZKUHPROPBOLIW-AWEZNQCLSA-N
Target: D(4) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21917
Assay: Agonist activity at DRD4 expressed in human HEK293T cells assessed as cAMP accumulation incubated for 15 mins by split-luciferase-based biosensor analysis
Measurement: EC50 = 68.3 nM (pChEMBL 7.170000076293945)
Descriptors: MW 300.4, ALogP 2.74, HBA 3, HBD 2, PSA 54.12, QED 0.81
Compound (SMILES): CN(CCN)Cc1n[nH]nc1-c1cccc(C(F)(F)F)c1
InChIKey: KXMOTUWXUMLQRY-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human PRMT6 using (3H) methylated biotin labelled peptide as substrate in presence of 3H-SAM by scintillation proximity assay
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 299.3, ALogP 1.88, HBA 4, HBD 2, PSA 70.83, QED 0.88
Compound (SMILES): CCC(=O)N1CCN(c2ccc(N3C(=O)CCc4cnc5ccc(-c6cnc7ccccc7c6)cc5c43)cc2C(F)(F)F)CC1
InChIKey: ZFSIZIIEXBCBTN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: O00443
Assay: Inhibition of PIK3C2A (unknown origin) by drug competition for substrate binding based assay
Measurement: EC50 = 176 nM (pChEMBL 6.75)
Descriptors: MW 609.65, ALogP 7.14, HBA 5, HBD 0, PSA 69.64, QED 0.22
Compound (SMILES): OCCn1cc(-c2cnc3nnn(Cc4cc5cccnc5cc4F)c3n2)cn1
InChIKey: RSZZWPDCCYKXGM-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition of His6-tagged human c-Met Y1230H mutant expressed in Sf9 cells pre-incubated with enzyme for 30 mins prior to addition of ATP using (Glu4Tyr) or Met2 peptide substrate by coupled enzymatic assay
Measurement: Ki = 3500 nM (pChEMBL 5.460000038146973)
Descriptors: MW 390.38, ALogP 1.81, HBA 9, HBD 1, PSA 107.43, QED 0.49
Compound (SMILES): CC(Oc1cc(-c2cnn(CCCC#N)c2)cnc1N)c1c(Cl)ccc(F)c1Cl
InChIKey: OJRVINDNYRRSAQ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition of His6-tagged human c-Met Y1230H mutant expressed in Sf9 cells pre-incubated with enzyme for 30 mins prior to addition of ATP using (Glu4Tyr) or Met2 peptide substrate by coupled enzymatic assay
Measurement: Ki = 336 nM (pChEMBL 6.46999979019165)
Descriptors: MW 434.3, ALogP 5.42, HBA 6, HBD 1, PSA 89.75, QED 0.4
Compound (SMILES): CC(C)[C@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C)NC(=O)OC(C)(C)C)C(=O)N[C@H](C=O)CC(=O)O.O=C(O)C(F)(F)F
InChIKey: YLSRAFBNEHVRLB-PGKYVIAHSA-N
Target: Caspase-8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14790
Assay: Affinity Biochemical interaction: (enzymatic assay (fluorogenic substrate cleavage)) EUB0002190aAD CASP8
Measurement: Ki = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 516.55, ALogP -0.45, HBA 8, HBD 6, PSA 217.3, QED 0.16
Compound (SMILES): COc1cccc(OC)c1S(=O)(=O)Nc1noc2cc(Cn3cccn3)cc(OC)c12
InChIKey: VNSQFPFWUAZOTQ-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: Kat6A Assay 2: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT6A 15 nM, 10 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction   2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 0.83 nM (pChEMBL 9.079999923706055)
Descriptors: MW 444.47, ALogP 2.9, HBA 9, HBD 1, PSA 117.71, QED 0.44
Compound (SMILES): CN(C)CC(c1ccc(O)cc1)C1(O)CCCCC1.Cl
InChIKey: IMWPSXHIEURNKZ-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Affinity On-target Cellular interaction: (Inhibition of neurotransmitter uptake, [3H]5-HT, MDCK-hNWET6 cells) EUB0002556aCl SLC6A4
Measurement: IC50 = 47.3 nM (pChEMBL 7.329999923706055)
Descriptors: MW 263.38, ALogP 2.73, HBA 3, HBD 2, PSA 43.7, QED 0.88
Compound (SMILES): CCOCC1(COCC)CCC(c2n[nH]cc2CN(C)CCNC)CC1.Cl
InChIKey: WRVJUOWIQSTJPE-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99873
Assay: Affinity Biochemical interaction: (enzymatic assay (inhibition of methyl transfer from radiolabelled substrate [3H]SAM to peptide)) EUB0002245aCl PRMT1
Measurement: Ki = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 366.55, ALogP 2.78, HBA 5, HBD 2, PSA 62.41, QED 0.6
Compound (SMILES): CS(=O)(=O)c1ccc(-c2nnc(C=Cc3nnc(-c4ccc(C#N)cc4)o3)n2-c2ccccc2Cl)nc1
InChIKey: HIWVLHPKZNBSBE-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Affinity On-target Cellular interaction: (SuperTOP luciferase assay, HEK293 cells) EUB0002580a TNKS2
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 529.97, ALogP 4.48, HBA 10, HBD 0, PSA 140.45, QED 0.31
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)c1ccc(NC=O)cc1
InChIKey: KKJBUZQVEAFPRN-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Affinity On-target Cellular interaction: (Inhibition of enzyme activity in 293T cells transfected with vector alone) EUB0002519a HDAC2
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 337.4, ALogP 3.82, HBA 4, HBD 3, PSA 84.22, QED 0.49
Compound (SMILES): Cc1cc(-c2ccccc2)cc(C(=O)NNS(=O)(=O)c2cccc(O)c2)c1F
InChIKey: KVJFJJXCBRSCDY-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: Affinity Biochemical interaction: (SPR) EUB0001094a MYST3
Measurement: Kd = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 400.43, ALogP 3.13, HBA 4, HBD 3, PSA 95.5, QED 0.57
Compound (SMILES): CC(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C=O)CC(=O)O)C(C)C.O=C(O)C(F)(F)F
InChIKey: PAIYHRXEVLCTGC-XVQUUFRESA-N
Target: Caspase-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P55210
Assay: Affinity Biochemical interaction: (enzymatic assay (fluorogenic substrate cleavage)) EUB0002173aAD CASP7
Measurement: Ki = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 502.48, ALogP -2.39, HBA 8, HBD 7, PSA 245.37, QED 0.11
Compound (SMILES): Cl.Cl.N[C@@H](CCNCc1ccc(Cl)cc1)C(=O)N1CCCCC1
InChIKey: IWXMOQGMIWZNPR-CKUXDGONSA-N
Target: Dipeptidyl peptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHL4
Assay: Affinity On-target Cellular interaction: (inhibition of fluorogenic substrate cleavage, U937 cell) EUB0002750aCl DPP7
Measurement: IC50 = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 309.84, ALogP 2.16, HBA 3, HBD 2, PSA 58.36, QED 0.79
Compound (SMILES): CC(C)(OCc1cc(-c2cccc(OC3CC3)c2)n(-c2ccccc2Cl)n1)C(=O)O
InChIKey: PZHRLEZWTVYGMP-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: MCT1-Mediated Lactate Transport in BT20 Breast Cancer Cells: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 426.9, ALogP 5.11, HBA 5, HBD 1, PSA 73.58, QED 0.54
Compound (SMILES): CCCCC(=O)N1CC2(C1)CN(c1ccccc1-c1nnn[nH]1)C2
InChIKey: RIEBAXQZOMUWKX-UHFFFAOYSA-N
Target: Leukotriene B4 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC1
Assay: Displacement of FITC-labeled tracer from Lumi4 Tb-labeled BLT2 (unknown origin) expressed in CHO-K1 cells incubated for 1 hrs by Tag-lite displacement based HTRF assay
Measurement: IC50 = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 326.4, ALogP 1.71, HBA 5, HBD 1, PSA 78.01, QED 0.9
Compound (SMILES): Cc1cc(C(=O)N2CC[C@@H](C)[C@H](c3cc(C(F)F)nc4ncnn34)C2)cc(C)n1
InChIKey: OKZITNMVZCTKEZ-IAQYHMDHSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: In Vitro Assay PDE3A: Human recombinant PDE3A (hPDE3A) was supplied as a partially purified insect cell lysate by Scottish Biomedical, it was cloned from human brain and expressed in Sf9 cells. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 μl) were added in 384 well plates to 20 μl of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 μl of hPDE3A enzyme in incubation buffer was added and the reaction was started by addition of 10 μl substrate to a final concentration of 0.4 μM cAMP and 2.4 μCi/ml [3H]-cAMP. The reaction was incubated for 60 min at room temperature. After incubation, the reaction was stopped with 20 μl of stop solution consisting of 17.8 mg/ml PDE SPA (scintillation proximity assay) beads supplemented with 200 mM ZnCl2. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blanc values the enzyme was omitted from the reaction and replaced by incubation buffer. Control values were obtained by addition of a final concentration of 1% DMSO instead of compound. A best fit curve is fitted by a minimum sum of squares method to the plot of % of control value substracted with blanc value versus compound concentration and the half maximal inhibitory concentration (IC50) value is derived from this curve.
Measurement: IC50 = 6166 nM (pChEMBL 5.210000038146973)
Descriptors: MW 400.43, ALogP 3.34, HBA 6, HBD 0, PSA 76.28, QED 0.67
Compound (SMILES): CC#CC(=O)N1CC(N2CCC(c3cc(C)c4c(c3)Nc3ncnc(N5CCS(=O)(=O)CC5)c3[C@@H](C)O4)CC2)C1
InChIKey: OLEFHQBTXFCZON-HXUWFJFHSA-N
Target: Cyclin-dependent kinase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P50613
Assay: In vitro pharmacodynamics: p-RNA pol II Ser-5 (CDK7 C312S) ( QuantiGene Plex Gene Expression Assay: downregulation at the messenger level of superenhancer driven genes. The level of phosphorylation of Serine 5 on RNA pol II Vs totalRNA pol II is determined. The level of p-RNA pol II Ser-5 detected and stained with primary (a specific rabbit Phospho-Rpb1 CTD (Ser5) antibody) and secondary antibodies (a goat anti rabbit Alexa Fluor 488 conjugated secondary antibody)  after 3 hrs incubation with probe.)
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 564.71, ALogP 2.63, HBA 9, HBD 1, PSA 107.97, QED 0.56
Compound (SMILES): CNC(=O)c1nn2c(c1-c1cc(F)c(Cl)cc1C)OCCC2
InChIKey: XWLDNGLNMIBYMP-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4A (SINGLE PROTEIN), Homo sapiens
UniProt: P27815
Assay: Scintillation Proximity Assay (SPA): Human PDE4A3 coding sequence (amino acids 2 to 825 from the sequence with accession number NP_001104779) was cloned into the baculovirus expression vector pFastBac (Invitrogen) engineered to include an N-terminal His6 affinity tag and a C-terminal FLAG affinity tag to aid in purification. The recombinant Bacmid was isolated and used to transfect insect cells to generate a viral stock. To generate cell paste for purification, insect cells were infected with the virus stock and cells were harvested 72 hours after infection. Insect cell paste was lysed and after centrifugation, the supernatant was batch bound to Ni-NTA agarose (GE Healthcare) and eluted with 250 mM imidazole. This eluate was diluted with FLAG buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM TCEP with protease inhibitors) and batch bound to ant-FLAG M2 agarose (Sigma) overnight at 4° C. The agarose was packed into a column, washed with buffer and eluted with buffer containing elute using 250 μg/mL Flag-peptide. Fractions were analyzed using SDS-PAGE Coomassie blue staining and pooled based on purity. Pooled fractions were chromatographed on a S200 120 mL column (GE Healthcare) in 50 mM Tris HCl pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM TCEP with protease inhibitors. PDE4A3 fractions were analyzed by SDS-PAGE Coomassie blue staining, pooled based on purity, dialyzed against 50 mM Tris HCl pH 7.5, 100 mM NaCl, 20% glycerol, 2 mM TCEP, frozen and stored at −80° C.
Measurement: IC50 = 363 nM (pChEMBL 6.440000057220459)
Descriptors: MW 323.75, ALogP 2.79, HBA 4, HBD 1, PSA 56.15, QED 0.92
Compound (SMILES): O=C(Nc1ccc2cnccc2c1)[C@@H]1CC1c1ccc(S(=O)(=O)NC[C@@H]2CCCNC2)cc1
InChIKey: ZUJRYKFJARKTIO-DORBDNKASA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 464.59, ALogP 3.25, HBA 5, HBD 3, PSA 100.19, QED 0.5
Compound (SMILES): Clc1ccc2c(c1)CCCC21CC1NCC1CCNCC1
InChIKey: NLONPTMXQRSZPT-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (50 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 50 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 556 nM (pChEMBL 6.25)
Descriptors: MW 304.87, ALogP 3.28, HBA 2, HBD 2, PSA 24.06, QED 0.9
Compound (SMILES): CCC(CC)CN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C(C)C)c4)c3)ccc21
InChIKey: FCKKBQZIGVYODN-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: In Vitro Enzyme Inhibition Using a Biochemical Peptide Phosphorylation Assay- Caliper Assay: Example 54:The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 uM of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 uM ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reactions were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 429.56, ALogP 5.48, HBA 4, HBD 0, PSA 55.2, QED 0.5
Compound (SMILES): CC[S@](C)(=O)=Nc1cc(N2CCOC[C@H]2C)nc(-c2ccnc3[nH]ccc23)n1
InChIKey: MFKMYMWETJVOSR-QYSRSOONSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition of ATR in 4-NQO treated human HT-29 cells assessed as inhibition of Chk1 phosphorylation at Ser345 residue incubated for 90 mins followed by incubation with 4-NQO for 120 mins by ALPHASCREEN SUREFIRE assay
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 400.51, ALogP 2.99, HBA 7, HBD 1, PSA 96.36, QED 0.72
Compound (SMILES): CNC[C@H](c1ccc(Cl)c(Cl)c1)[C@H](O)c1cccc([N+](=O)[O-])c1
InChIKey: UYAUOAOUUKBHEX-CZUORRHYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition Assay: Cytochrome P450 3A4 and 2D6:Recombinant enzymes, 3A4 and 2D6, generated using the ABL yeast expression system were used. For CYP3A4, the enzyme amount was 2 pmol per well, and the substrate (7-benzyloxy-4-(trifluoromethyl) coumarin; BFC) was used at 15 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 1 mM). The incubation time was 30 minutes, and the reference inhibitor was A-naphthoflavone. For CYP2D6, the enzyme amount was 2 pmol per well, and the substrate (7-methoxy-4-(aminomethyl)-coumarin; MAMC) was used at 20 μM. The assay mixture also included K2HPO4 (pH 7.4; conc. 0.1 M) and NADPH (conc. 0.06 mM). The incubation time was 35 minutes, and the reference inhibitor was quinidine.Briefly, 0.6 μL of serially diluted compound was added to 75 μL water. 10 μL of diluted compound in water was then added to each assay plate. 20 μL of a K2HPO4, enzyme, and substrate mixture was added. After 10 minutes of room temperature pre-incubation, 10 μL of NADPH was added, and the plates were placed at 37° C. for 30 or 35 minutes depending on the enzyme being tested. After the incubation was complete, 20 μL of 0.1% Tris/ACN was added to the plate. The plate was read on a FluorStar fluorescence plate reader.
Measurement: IC50 = 5700 nM (pChEMBL 5.239999771118164)
Descriptors: MW 355.22, ALogP 3.94, HBA 4, HBD 2, PSA 75.4, QED 0.61
Compound (SMILES): C=CC(=O)N1CC(Nc2nc(Nc3ccc(OC)nc3)ncc2C(C)=O)C1
InChIKey: GZRAWWJVYUECSE-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 368.4, ALogP 1.64, HBA 8, HBD 2, PSA 109.34, QED 0.56
Compound (SMILES): C=CC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c(C(N)=O)c2NCCO)C1
InChIKey: KNMRAYALGUIBCV-INIZCTEOSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 Activity Inhibition Test: The reaction buffer contains the following components: 5-fold diluted enzymatic buffer/kinase 5x(Cisbio, Cat No. 62EZBFDD)(its main component is 50nM HEPES, pH 7.0), 5mM MgCl2 and 1 mM DTT, a human recombinant FGFR4 catalytic domain protein (amino acid 460-802) which was purchased from Tsinghua University Protein Research Technology Center, a 0.5 kinase solution diluted by the reaction buffer; a substrate reaction solution containing 500 nM biotinylated tyrosine kinase substrate diluted by the reaction buffer (Cisbio, Cat. No. 62TKOPEC) and 90 uM ATP; a test solution containing 0.125 ng/uL Eu3+ labeled cage antibody (Cisbio, Cat. No 61T66KLB) diluted by a test buffer (Cisbio, Cat No 62SDBRDF), and 31.25nM streptavidin labeled XL665 (Cisbio, Cat. No. 610SAXLB).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 453.5, ALogP 0.76, HBA 8, HBD 3, PSA 131.94, QED 0.4
Compound (SMILES): CNC(=O)c1ccc(-c2cc3c(cn2)CN(c2c(F)c(OC)cc(OC)c2F)C(=O)C32CC2)cc1
InChIKey: FUSJNJRFUIEODK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 479.48, ALogP 3.98, HBA 5, HBD 1, PSA 80.76, QED 0.6
Compound (SMILES): COc1cc(O[C@H]2CCN(C)CC2(F)F)c2c(Nc3ccc(Oc4ccn5ncnc5c4)c(C)c3)ncnc2c1
InChIKey: IDQNCHCNBVCUHC-DEOSSOPVSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition Assay: EGFR: The compounds' inhibition of target modulation were determined as follows: NCI-H838 cells were sorted in 96 well plates (20000 cells/well) with the DMEM medium containing 1% FBS overnight and then treated with tested compounds at a series of concentrations (3 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, 0.0001 μM). The plates were incubated for 4 h at 37° C. with 5% CO2 followed by the stimulation of recombinant hEGF (100 ng/ml for 10 Min, RD, Cat #236-EG) and then the EGFR (Y1068) phosphorylation level of cells in each well were measured with MSD Kit (MULTI-SPOT®96 4-Spot HB Prototype EGFR Triplex ANALYTES: pEGFR(Tyr1068), pEGFR(Tyr1173), Total EGFR (Cat # N45ZB-1). The assay is a electrochemiluminescent method (MESO SCALE DISCOVERY) for determining both phosphorylated and total EGFR of cells with an MSD SECTOR® Imager and then the ratio of p-EGFR/total EGFR can be generated by the machine. The percentage of inhibition was used the formula: % inhibition=100×[1−(ratio of sample well−ratio of Min ctrl well)/ (ratio of MaxRatio of Min ctrl well)]. The IC50 values were further calculated as the compounds concentration required for 50% inhibition in best-fit curves using Prism GraphPad 7.0 or Microsoft Xlfit software.
Measurement: IC50 = 5222 nM (pChEMBL 5.28000020980835)
Descriptors: MW 547.57, ALogP 5.24, HBA 10, HBD 1, PSA 98.93, QED 0.29
Compound (SMILES): Nc1ccc(-c2cc(F)cc(F)c2)cc1NC(=O)c1ccc(N2CCNCC2)nc1
InChIKey: NLXPFKGEXHSNCX-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: HDAC Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten-point three-fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6-fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5-fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 μM final concentration were diluted in assay buffer at 6-fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2) and 0.08 ng/ml (HDAC3). The final substrate concentrations used were 16 μM (HDAC1), 10 μM (HDAC2) and 17 μM (HDAC3).Five μl of compounds and 20 μl of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five μl of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 255 nM (pChEMBL 6.590000152587891)
Descriptors: MW 409.44, ALogP 3.27, HBA 5, HBD 3, PSA 83.28, QED 0.58
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)C1=CCCN(C1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)C1(F)CCN(C(=O)C#CC(C)(C)N(C)C)CC1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: JFXKFMBIUWMFNA-DERPSJHNSA-N
Target: GTPase NRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01111
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 1008.29
Compound (SMILES): COc1ccc(-n2nc(C(F)(F)F)c3c2C(=O)N(C2CCN(N4CCCCC4=O)CC2)CC3)cc1
InChIKey: YFPMFJCXOLJIOD-UHFFFAOYSA-N
Target: Coagulation factor V/Coagulation factor X/Serine/threonine-protein kinase BRSK2 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q8IWQ3
Assay: In Vitro Assay: Prothrombinase Assay:The effect of test compounds on prothrombinase activity is measured via thrombin generation. Briefly, 12.5 μL human factor Xa is incubated in 10 mM HEPES buffer, pH 7.4, 2 mM CaCl2, the final concentration is 0.5 nM, and add 12.5 μL human platelets (1×107 mL−1) for 10 minutes at 37° C. The reaction is initiated by adding 25 μL prothrombin, the final concentration is 0.5 μM and 2 μL the assay buffer containing 2% (V/V) DMSO (uninhibited control) or various concentrations of the compounds to be tested diluted in the assay buffer containing 2% (V/V) DMSO. After 20 minutes, thrombin activity is measured by adding 48 μL substrate of S-2238 (Chromogenix) at the final concentration is 50 μM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 491.51, ALogP 3.29, HBA 6, HBD 0, PSA 70.91, QED 0.66
Compound (SMILES): COc1ncc(CN2CCN(c3ccc(-c4cc(-c5cnn(C)c5)cn5ncc(C#N)c45)cn3)CC2)cc1F
InChIKey: SCRDPVUHWJYHPU-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 30.9 nM (pChEMBL 7.510000228881836)
Descriptors: MW 523.58, ALogP 3.53, HBA 10, HBD 0, PSA 100.4, QED 0.33
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(N5CC(C)(O)C5)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: SKZSGEWYZRDIAG-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3595 nM (pChEMBL 5.440000057220459)
Descriptors: MW 472.64, ALogP 5.11, HBA 6, HBD 2, PSA 82.34, QED 0.43
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(OCc4ccccc4)c(F)c3)n2n1)C1CC1
InChIKey: YOOYWGYDXVABCG-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 Inhibition Assay: Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalog number 08-147) was purchased from Carna biosciences. 5 ng of TYK2 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (25 mM Hepes pH 7.5, 100 mM NaCl, 0.2 mM Na3VO4, 0.1% NP-40, 0.1 μM non-radioactive ATP, 0.125 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 402.43, ALogP 4.46, HBA 5, HBD 1, PSA 68.52, QED 0.52
Compound (SMILES): CC1(C(=O)N2Cc3cc(S(C)(=O)=O)ccc3[C@@H]2C(=O)Nc2ccc(C(O)(C(F)(F)F)C(F)(F)F)cc2)CCO1
InChIKey: OLNRSHJMSMRVKO-ITUIMRKVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microlitres DMSO were dispensed to assay plates using an Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 h at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORÎ³ (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer. Five microlitres of pre-mix 2 was dispensed to assay plates containing 0.1 microlitres of test compound and was incubated for 15 minutes prior to adding five microlitres of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nm was divided by the signal at 612 nm and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(Xâmin)/(maxâmin)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 580.5, ALogP 3.6, HBA 6, HBD 2, PSA 113.01, QED 0.52
Compound (SMILES): N=C(N)NS(=O)(=O)c1ccc(C2C[C@H]2C(=O)Nc2ccc3cnccc3c2)cc1
InChIKey: ZTBJDQDNHPHYGS-QRWMCTBCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 409.47, ALogP 2.15, HBA 5, HBD 4, PSA 138.03, QED 0.38
Compound (SMILES): O=C1NCC2(CCCCC2)n2c1cc1cnc(Nc3ccc(C(=O)N4CCNCC4)cc3)nc12
InChIKey: BKCOGSHTNWTZNI-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 459.55, ALogP 2.62, HBA 7, HBD 3, PSA 104.18, QED 0.56