lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CO[C@H]1C[C@@H](n2c(=O)n(C)c3cnc4ccc(-c5ccc(OCCCN6CCCC6)nc5)cc4c32)C1
InChIKey: OPNZWUZVFPAJIO-SZPZYZBQSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cellular Assay: Assay c) BT474 cells (human breast ductal carcinoma, ATCC HTB-20) were seeded into black 384 well plates (Costar, #3712) at a density of 5600 cells/well in DMEM containing 10% FBS and 1% glutamine and allowed to adhere overnight. The following morning compounds in 100% DMSO were added to assay plates by acoustic dispensing. After a 2 h incubation at 37° C. and 5% CO2, the medium was aspirated and the cells were lysed with a buffer containing 25 mM Tris, 3 mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mM Sodium orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail tablets (Roche #04 693 116 001, used 1 tab per 50 ml lysis buffer). After 20 minutes, the cell lysates were transferred into ELISA plates (Greiner #781077) which had been pre-coated with an anti total-AKT antibody in PBS buffer and non-specific binding was blocked with 1% BSA in PBS containing 0.05% Tween 20. Plates were incubated over night at 4° C. The next day the plates were washed with PBS buffer containing 0.05% Tween 20 and further incubated with a mouse monoclonal anti-phospho AKT T308 for 2 h. Plates were washed again as above before addition of a horse anti-mouse-HRP conjugated secondary antibody. Following a 2 h incubation at r.t., plates were washed and QuantaBlu substrate working solution (Thermo Scientific #15169, prepared according to provider's instructions) was added to each well. The developed fluorescent product was stopped after 60 minutes by addition of Stop solution to the wells. Plates were read using a Tecan Satire plate reader using 325 nm excitation and 420 nm emission wavelengths respectively. Except where specified, reagents contained in the Path Scan Phospho AKT (Thr308) sandwich ELISA kit from Cell Signalling (#7144) were used in this ELISA assay.
Measurement: IC50 = 2030 nM (pChEMBL 5.690000057220459)
Descriptors: MW 487.6, ALogP 4.16, HBA 8, HBD 0, PSA 74.41, QED 0.35
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(C#CC3CC3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: BVZRDCPNICTPGQ-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 460.54, ALogP 4.14, HBA 7, HBD 1, PSA 91.62, QED 0.41
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3cc(Br)cc(C#N)c3)cc1C21N=C(C)C(N)=N1
InChIKey: BCPJXJSUDZCZSL-TYRCWPPYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 4950 nM (pChEMBL 5.300000190734863)
Descriptors: MW 477.41, ALogP 5.1, HBA 5, HBD 1, PSA 83.76, QED 0.65
Compound (SMILES): CCC(C)Oc1c(O)ccc2c1CN1CCc3cc(OC)c(O)cc3[C@@H]1C2
InChIKey: BTEHEQVRGFEGCF-UWBLVGDVSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]N-methylspiperone from human D3 receptor expressed in HEKT cell membranes after 90 mins by microbeta scintillation counting method
Measurement: Ki = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 369.46, ALogP 3.94, HBA 5, HBD 2, PSA 62.16, QED 0.85
Compound (SMILES): CCOC[C@@H]1[C@@H](c2ccc(Cl)c(Cl)c2)CC2CC[C@H]1N2C
InChIKey: VCVWXKKWDOJNIT-XOLBZYKYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31652
Assay: Inhibition of re-uptake of [3H]5-hydroxytryptamine at SERT in Wistar rat cortical synaptosomes by liquid scintillation counting
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 328.28, ALogP 4.6, HBA 2, HBD 0, PSA 12.47, QED 0.8
Compound (SMILES): COc1ccc(/C=C/C(=O)Nc2ccc(CN(C)Cc3ccccc3)cc2)c(OC)c1
InChIKey: BVGVBFYFLUSQMK-FOWTUZBSSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of human recombinant MAOA using p-tyramine as substrate incubated for 15 mins by fluorimetric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 416.52, ALogP 4.99, HBA 4, HBD 1, PSA 50.8, QED 0.5
Compound (SMILES): C[C@H]1CN(c2ccncc2Nc2ncc3ccc(-c4nccs4)nn23)C[C@@H](N)[C@@H]1O
InChIKey: KBIGBJJRXNTKSO-WPKBUWHJSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: PIM Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 422.52, ALogP 2.14, HBA 10, HBD 3, PSA 117.49, QED 0.46
Compound (SMILES): C[C@H]1COCCN1c1ccnc(-c2c[nH]c3ccc(-c4nnc(N)o4)cc23)n1
InChIKey: DETSRRVOAOWTKK-NSHDSACASA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 2.211 nM (pChEMBL 8.649999618530273)
Descriptors: MW 377.41, ALogP 2.48, HBA 8, HBD 2, PSA 118.98, QED 0.56
Compound (SMILES): O[C@H]1CC[C@H](Nc2ncc(-c3ccc4ccccc4c3)c(-c3ccccc3)n2)CC1
InChIKey: ZQDPBVRIRZFYKQ-YHBQERECSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Inverse agonist activity at recombinant human adenosine A1 receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding preincubated for 30 mins followed by [35S]GTPgammaS addition measured after 90 mins by beta scintillation counting method
Measurement: EC50 = 166 nM (pChEMBL 6.78000020980835)
Descriptors: MW 395.51, ALogP 5.68, HBA 4, HBD 2, PSA 58.04, QED 0.47
Compound (SMILES): c1ccc2c3c([nH]c2c1)CN1CCc2cc4c(cc2[C@@H]1C3)OCO4
InChIKey: KROMIZUJAJKGLD-SFHVURJKSA-N
Target: Alpha-1D adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25100
Assay: Antagonist activity at human full-length N-terminal SNAP-tagged alpha1D adrenoceptor expressed in HEK293 cells assessed as inhibition of agonist-induced calcium mobilization preincubated for 10 mins followed by agonist addition by fluo-4 AM dye-based assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 318.38, ALogP 3.55, HBA 3, HBD 1, PSA 37.49, QED 0.69
Compound (SMILES): Cn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCNCC(O)C3)n2)cn1
InChIKey: WHGGAJVOOZRMSO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 9.39 nM (pChEMBL 8.029999732971191)
Descriptors: MW 390.45, ALogP 0.99, HBA 9, HBD 2, PSA 96.92, QED 0.54
Compound (SMILES): O=S(=O)(Nc1nncs1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1Cl
InChIKey: CIYPGEZJBSEFKR-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 476.89, ALogP 4.54, HBA 7, HBD 1, PSA 84.42, QED 0.59
Compound (SMILES): COc1cc2c(Nc3cccc(Cl)c3F)ncnc2cc1O[C@H]1CO[C@H]2[C@@H]1OC[C@@H]2F
InChIKey: GBVHHGINCYVVRZ-TVJWEXTRSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 449.84, ALogP 4.06, HBA 7, HBD 1, PSA 74.73, QED 0.63
Compound (SMILES): N#Cc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)s1
InChIKey: GLTHTYRWIILEGV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 363.45, ALogP 2.92, HBA 7, HBD 1, PSA 85.9, QED 0.6
Compound (SMILES): C#CC(=O)Nc1cccc(-c2cnc3[nH]cc(Cl)c3c2)n1
InChIKey: DZAHHFCGFLVKIL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 296.72, ALogP 2.85, HBA 3, HBD 2, PSA 70.67, QED 0.71
Compound (SMILES): Cc1ccc(C(=O)Nc2cccc(C(F)(F)F)c2)cc1-c1nc(N2CCOCC2)cc(N2CCOCC2)n1
InChIKey: HKDMNDSYJYCJAJ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.616 nM (pChEMBL 9.210000038146973)
Descriptors: MW 527.55, ALogP 4.4, HBA 7, HBD 1, PSA 79.82, QED 0.53
Compound (SMILES): CCN1CCN(CC)C(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)C1
InChIKey: SHQDTJIUTBSGPO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 490.44, ALogP 5.09, HBA 7, HBD 1, PSA 62.75, QED 0.47
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCNC(C)C3)n2)n1
InChIKey: BVSPZNRNBYKDLE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.046 nM (pChEMBL 10.34000015258789)
Descriptors: MW 386.46, ALogP 2.38, HBA 8, HBD 1, PSA 84.65, QED 0.58
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc(O)c(Cl)c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: GFOYBQMPWPBWIV-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 523, ALogP 5.4, HBA 8, HBD 2, PSA 111.85, QED 0.32
Compound (SMILES): Cc1ccc(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cccc(-c3ncccn3)c2)c(-n2nccn2)c1C
InChIKey: IEHBRXSPEXUDES-AUSIDOKSSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 468.56, ALogP 3.81, HBA 7, HBD 0, PSA 86.03, QED 0.44
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCc2ccc(F)cc2)CC1
InChIKey: YSJPLNKYRLBRBB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 465.51, ALogP 3.74, HBA 7, HBD 3, PSA 121.65, QED 0.44
Compound (SMILES): COc1cc2c(ccn2CCn2cc(NC(=O)c3ncoc3-c3cccc(C)c3)cn2)cn1
InChIKey: XJRKTUDQYITKPM-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonist activity at human OX1R expressed in CHO cells assessed as orexin A-induced intracellular Ca2+ mobilization preincubated for 120 mins followed by orexin A addition by FLIPR assay
Measurement: IC50 = 1253 nM (pChEMBL 5.900000095367432)
Descriptors: MW 442.48, ALogP 4.16, HBA 8, HBD 1, PSA 100, QED 0.41
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccccc3Cl)CC2)C1
InChIKey: OSKNEJAXRNEFRX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 516.01, ALogP 4.1, HBA 7, HBD 2, PSA 118.76, QED 0.41
Compound (SMILES): CC(C)C1=NN(c2ccc(C(=O)O)cc2)C(c2cccc([N+](=O)[O-])c2)C1
InChIKey: FKFGESLBLYGMRL-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: Colorimetric Assay: The activities of COX-1 and COX-2 were measured after the addition of arachidonic acid and incubation at 25° C. for 5 min by absorbance at 590 nm as specified by the manufacturer.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 353.38, ALogP 4.26, HBA 5, HBD 1, PSA 96.04, QED 0.64
Compound (SMILES): COc1cc(N2CCN(C3CCN(CCF)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: WROBOUZJBUWDDO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 761.85, ALogP 7.3, HBA 10, HBD 2, PSA 103.16, QED 0.14
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(CO)(CCCCc2ccccc2)CC1
InChIKey: BROMZAHMHRNILO-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: The affinity to the human μ-opiate receptor was determined in a homogeneous preparation in microtiter plates. For this, dilution series of the respective compound to be tested were incubated for 90 minutes at room temperature with a receptor membrane preparation (15-40 mg of protein per 250 μl of incubation batch) of CHO-K 1 cells, which express the human μ-opiate receptor (RB-HOM receptor membrane preparation of NEN, Zaventem, Belgium), in the presence of 1 nmol/l of the radioactive ligand [3H'-naloxone (NET719, NEN, Zaventem, Belgium) and of 1 mg WGA-SPA beads (wheat germ agglutinin SPA beads from Amersham/Pharmacia, Freiburg, Germany) in a total volume of 250 μl. 50 mmol/l of tris-HCl supplemented by 0.05% by wt. of sodium azide and 0.06% by wt. of bovine serum albumin was used as incubation buffer. 25 μmol/l of naloxone were additionally added to determine the non-specific bond. After the ninety-minute incubation time had ended, the microtiter plates were centrifuged for 20 minutes at 1000 g and the radioactivity measured in a β-counter (Microbeta-Trilux, PerkinElmer Wallac, Freiburg, Germany).
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 365.56, ALogP 5.41, HBA 2, HBD 1, PSA 23.47, QED 0.63
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCCC(N)C3)n2)n1
InChIKey: CRQPGRMAMXKTSC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.067 nM (pChEMBL 10.170000076293945)
Descriptors: MW 400.49, ALogP 2.9, HBA 8, HBD 1, PSA 98.64, QED 0.56
Compound (SMILES): COc1cc(OC)cc(N(CCNC(C)C)c2ccc3ncc(-c4cnn(C)c4CN(C)C)nc3c2)c1
InChIKey: CCDNYIWGTYFDSI-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 7762.47 nM (pChEMBL 5.110000133514404)
Descriptors: MW 503.65, ALogP 4.25, HBA 9, HBD 1, PSA 80.57, QED 0.33
Compound (SMILES): Cc1ccc(OCc2nnc(NC(=O)Nc3cccc(Cl)c3)s2)cc1C
InChIKey: PKUMGHOUAGUDQS-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Bos taurus
UniProt: P00735
Assay: High-Throughput Screening Assay: To identify novel inhibitors of hepsin, the Chembridge DIVERSet 10,000 compound high-diversity library was screened using an assay based on the cleavage of the chromogenic peptide. In addition, the NINDS II library of 1040 compounds (MicroSource Discovery) was screened to identify hepsin inhibitors among established drugs and known bioactive molecules. Screens were performed in a 96-well format at a final compound concentration of 2 uM. To minimize false positives, positions 1 and 12 of each row contained DMSO/buffer controls. As a measure of reproducibility, the Z' score for this assay was 0.78 (Zhang et al., 1999 J Biomol Screen 4:67).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 388.88, ALogP 5.03, HBA 5, HBD 2, PSA 76.14, QED 0.64
Compound (SMILES): Cc1nc(C(C)(CS(C)(=O)=O)NC(=O)c2cc(OCC(F)(F)F)c(C3CCC3)cn2)no1
InChIKey: VELIFIGTMFBRPH-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 476.48, ALogP 2.67, HBA 8, HBD 1, PSA 124.28, QED 0.62
Compound (SMILES): CCC1(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CCS(=O)(=O)CC1
InChIKey: HQZCXDWOMVTYLT-DEOSSOPVSA-N
Target: Nuclear receptor subfamily 1 group I member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75469
Assay: Transactivation of PXR (unknown origin) expressed in human HepG2 cells
Measurement: EC50 = 5200 nM (pChEMBL 5.28000020980835)
Descriptors: MW 661.63, ALogP 5.62, HBA 5, HBD 0, PSA 88.59, QED 0.3
Compound (SMILES): COc1nc2c(C)cc(C(O)(c3ccc(C(F)(F)F)nc3)c3cncn3C)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: NTMKNDJXPHULDI-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 619.05, ALogP 6.41, HBA 8, HBD 1, PSA 90.88, QED 0.22
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3ccc(N4CCN(CCOC)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: YFQKRLUWZVQGQS-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 734.81, ALogP 6.91, HBA 11, HBD 2, PSA 118.38, QED 0.13
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1N1CCC1
InChIKey: ZQGMAEVBUDWZSM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 24.2 nM (pChEMBL 7.619999885559082)
Descriptors: MW 333.4, ALogP 2.43, HBA 4, HBD 2, PSA 73.91, QED 0.76
Compound (SMILES): O=C(c1ccc(F)cc1)C1CCN(CC(=O)N(Cc2cccc(F)c2)Cc2nc3c(c(=O)[nH]2)COCC3)CC1
InChIKey: QIPLEHXSZJJEQF-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 > 19000 nM (pChEMBL 4.7210001945495605)
Descriptors: MW 536.58, ALogP 3.24, HBA 6, HBD 1, PSA 95.6, QED 0.44
Compound (SMILES): Nc1cnc(-n2c([C@@H](Nc3nc(N)nc(N)c3Cl)C3CC3)nc3cccc(Cl)c3c2=O)cn1
InChIKey: GKLFMMWEERPOPG-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The activities were measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (75-500 uM), 2 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 uM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) and Compound Y ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window). The results were normalized based on positive (1 uM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ and γ were calculated from the fit of the dose-response curves to a four-parameter equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 485.34, ALogP 2.58, HBA 11, HBD 4, PSA 176.54, QED 0.33
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(-c2nc3cc(NS(=O)(=O)CCC)ccc3o2)cc1
InChIKey: MUSNXRCOWVRSMH-UHFFFAOYSA-N
Target: Phosphotyrosine protein phosphatase (SINGLE PROTEIN), Mycobacterium tuberculosis
UniProt: A0A045ISB3
Assay: Inhibition of Mycobacterium tuberculosis His-tagged PtpB expressed in Escherichia coli Rosetta 2 (DE3) using pNPP as substrate after 30 mins by spectrophotometric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 437.54, ALogP 3.8, HBA 6, HBD 2, PSA 118.37, QED 0.53
Compound (SMILES): NCCOc1c(-c2ccc3c(c2)OCO3)[nH]c(=O)c2cc(F)ccc12
InChIKey: XVJKJDSTWKHYJD-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PAR Assay: Studies were performed as follows: 6000 cells/well were seeded in 96 well plates (Perkin Elmer) in MEM/10% FCS and incubated for 24 hs at 37° C., 5% carbon dioxide. Test compounds were then added at the required concentration for 30'. DNA damage was then induced adding hydrogen peroxide at the concentration of 0.1 mM for 15 min. Concentration curves were prepared in MEM/10% FCS from compound stocks in dimethylsulfoxide (DMSO), and final DMSO concentration was 0.002% (v/v). Duplicate wells for each concentration point were prepared with a typical highest compound concentration of 20 μM and serial dilution 1:3. Plates were dried and fixed adding a cold methanol-acetone (70:30) solution for 15 min at RT; fixing solution was aspired and wells were air dried for 5 min and then dehydrated in PBS. Non-specific binding sites were blocked by incubating wells for 30 min in PBS containing 5% (w/v) FBS 0.05% Tween20. Wells were then incubated for 1 h at RT in PBS containing anti PAR mouse monoclonal antibody (Anti-PAR, Mouse mAb 10H, Tulip Cat No 1020) diluted 1:200 in blocking solution. After 3 washes in PBS, wells were incubated in PBS (w/v) 5% FBS 0.05% Tween20 containing 2 μg/mL Cy2-conjugated Goat anti mouse secondary antibody (Amersham Pharmacia Biotech cat. No PA 42002) (Absorption maximum 489 nm, fluorescence maximum 506 nm) and 1 μg/mL DAPI (Absorption maximum 359 nm, fluorescence maximum 461 nm) (4',6-diamidino-2-phenylindole dilactate) (Sigma cat. No D9564), a high sensitivity dye for nucleic acid staining. After washing further 3 times in PBS, cellular PAR immunoreactivity was assessed using the ArrayScan vTi instrument, with a Zeiss 10×0.5 N.A. objective, and applying the Cytotoxicity.V3 algorithm (Cellomics/Thermo Fisher) with a XF100 filter. At least 10 fields, corresponding to at least 900 cells, were read for each well.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 342.33, ALogP 2.4, HBA 5, HBD 2, PSA 86.57, QED 0.76
Compound (SMILES): CC(C)Oc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C)CCO1
InChIKey: HPSOKGKCQBAFCF-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 477.39, ALogP 5.18, HBA 7, HBD 1, PSA 68.74, QED 0.5
Compound (SMILES): CCCN1CCC(c2cc(OC)c(Nc3nccc(-c4c(-c5ccc(OC)c(C(=O)Nc6c(F)cccc6F)c5)nc5ccccn45)n3)cc2C)CC1
InChIKey: YXAQENFRPVZZLS-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 717.82, ALogP 8.65, HBA 9, HBD 2, PSA 105.91, QED 0.14
Compound (SMILES): N#CCc1ccc(Nc2ncnc3ccc(-c4ccccc4)cc23)cc1
InChIKey: FSQLCPIRMDMFRQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3Kalpha (unknown origin) using diC8 PIP2 as substrate after 80 mins by ADP-glo assay
Measurement: IC50 = 475 nM (pChEMBL 6.320000171661377)
Descriptors: MW 336.4, ALogP 5.11, HBA 4, HBD 1, PSA 61.6, QED 0.56
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCN(c3cnc(C(=O)NCC(C)C)cn3)[C@H](C)C2)c1C
InChIKey: KNIRQAJTBSVZFK-LJQANCHMSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 473.63, ALogP 3.58, HBA 7, HBD 1, PSA 87.14, QED 0.56
Compound (SMILES): Fc1cc(NC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc(F)c1F
InChIKey: IRNWELPJOWEMGB-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 400.41, ALogP 4.41, HBA 5, HBD 2, PSA 70.9, QED 0.5