lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(F)cccc3C#N)=CC1=O)c1cc-2ccn1
InChIKey: HLWOCEXYCJLWGO-GDJIYFAZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 2.64 nM (pChEMBL 8.579999923706055)
Descriptors: MW 567.62, ALogP 6.05, HBA 6, HBD 2, PSA 124.42, QED 0.4
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2cc(F)ccc2NC1=O
InChIKey: RUOAINJRKCJLQF-CPJLOUKISA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 18.6 nM (pChEMBL 7.730000019073486)
Descriptors: MW 539.99, ALogP 6.93, HBA 3, HBD 1, PSA 62.3, QED 0.36
Compound (SMILES): Cc1ccnc(-n2nnc3c2CCN(C(=O)c2cccc(C(F)(F)F)c2Cl)[C@@H]3C)c1
InChIKey: YBKVGRQSYAWVKA-GFCCVEGCSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Ca2+ flux assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 19.5 nM (pChEMBL 7.710000038146973)
Descriptors: MW 435.84, ALogP 4.4, HBA 5, HBD 0, PSA 63.91, QED 0.6
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COC(=O)[C@@H](N)C(C)O)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: CLKIUKXZXLDKKB-GRWGPRNOSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Functional Assay: Functional [35S]GTPgammaS binding assays were conducted as follows. kippa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kippa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 2001 ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours.
Measurement: EC50 = 47.61 nM (pChEMBL 7.320000171661377)
Descriptors: MW 512.65, ALogP 2.17, HBA 8, HBD 2, PSA 103.48, QED 0.51
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(NC3O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]3O)cc2NC1=O
InChIKey: IKJOOTAXPJZGLI-ZXGJDRJESA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 7.22 nM (pChEMBL 8.140000343322754)
Descriptors: MW 699.15, ALogP 4.01, HBA 9, HBD 6, PSA 164.48, QED 0.22
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4CSc4nncs4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: CEBOEHUQMKNZOK-KOEYQPMUSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Functional Assay: Functional [35S]GTPgammaS binding assays were conducted as follows. kippa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kippa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 2001 ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours.
Measurement: EC50 = 191.5 nM (pChEMBL 6.71999979019165)
Descriptors: MW 511.71, ALogP 4.56, HBA 8, HBD 0, PSA 56.71, QED 0.51
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3cccc(Cl)n3)=CC1=O)c1cc-2ccn1
InChIKey: MXTYQFYCUOGQNN-DWXRJYCRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 74.9 nM (pChEMBL 7.130000114440918)
Descriptors: MW 560.05, ALogP 6.09, HBA 6, HBD 2, PSA 113.52, QED 0.38
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(N)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccccc2NC1=O
InChIKey: RBNWLYPYPBBIBI-NSYGIPOTSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 66.6 nM (pChEMBL 7.179999828338623)
Descriptors: MW 519.02, ALogP 6.24, HBA 4, HBD 2, PSA 88.32, QED 0.39
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n([C@@H]2CCOC2)c(=O)n4C)c(F)n1
InChIKey: YEFCFNFQLXFZGI-QGZVFWFLSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 4480 nM (pChEMBL 5.349999904632568)
Descriptors: MW 465.53, ALogP 3.38, HBA 8, HBD 0, PSA 74.41, QED 0.31
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4C(NC(C(=O)O)c4ccccc4)c4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: CWRICNOKPCBBSF-UJVLHDJOSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: GTPgammaS Functional Assay (kappa): Membranes from recombinant HEK-293 cells, CHO cells expressing the recombinant human kappa opioid receptor (kappa) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Functional [35S]GTPgammaS binding assays were conducted as follows. kappa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kappa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2,20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B.
Measurement: EC50 = 1769.34 nM (pChEMBL 5.75)
Descriptors: MW 620.79, ALogP 6.08, HBA 6, HBD 2, PSA 80.26, QED 0.28
Compound (SMILES): COC(=O)c1ccc2c(c1)-c1cccc(c1)[C@H](N1CCC(c3c(Br)ccc(Cl)c3F)=CC1=O)CCC[C@@H](C)C(=O)N2
InChIKey: BSNSKODJGQKGRG-DNOBIOAJSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 639.95, ALogP 7.81, HBA 4, HBD 1, PSA 75.71, QED 0.23
Compound (SMILES): Cc1cc(C(O)P(=O)(O)CC[C@@H](N)C(=O)O)cc(C)c1OCC(=O)O
InChIKey: HECCCCVGDLWFAY-ZRKZCGFPSA-N
Target: Metabotropic glutamate receptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P35400
Assay: Pharmacological Assay: Metabotropic glutamate receptors were transiently transfected in HEK293 cells by electroporation as described elsewhere (Brabet I. et al., 1998) and plated in 96-well microplates. The high affinity glutamate transporter EAAC1 was co-transfected with the receptor in order to avoid any influence of glutamate released by the cells in the assay medium. In the experiments carried out by the inventors, Group-III mGluRs were co-transfected with a chimeric G-protein which couples the activation of the receptor to the phospholipase-C (PLC) pathway. Thus receptor activation induces production of inositol phosphate (IP) which in turn induces intracellular Ca2+ release. Receptor activity was then determined by measurement of the IP production or Ca release as already described (Goudet C. et al., PNAS 2004). For intracellular calcium measurements, cells expressing mGluRs were loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM (Invitrogen, Cergy-Pontoise, France) dissolved in Hanks' balanced Salt Solution.
Measurement: EC50 = 51600 nM (pChEMBL 4.289999961853027)
Descriptors: MW 375.31, ALogP 0.83, HBA 6, HBD 5, PSA 167.38, QED 0.39
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2cccc(OC(F)F)c2)no1
InChIKey: ZTGPIJZQTRQNGI-MDLDFJCZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 462.41, ALogP 3.01, HBA 10, HBD 2, PSA 135.73, QED 0.51
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(C#N)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccccc2NC1=O
InChIKey: BYCWLOQDZYMODR-DWXRJYCRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 1.06 nM (pChEMBL 8.970000267028809)
Descriptors: MW 529.02, ALogP 6.53, HBA 4, HBD 1, PSA 86.09, QED 0.41
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccc(C)cc4)[C@@H](C2)[N+](C)(CC2CC2)CC[C@]315
InChIKey: DNUUSHHCUFRDIJ-XODAALMBSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: Radioligand Dose-Displacement Binding Assay: Membranes from recombinant HEK-293 cells expressing the human opioid receptor-like receptor (ORL-1) (Perkin Elmer, Shelton, Conn.) were prepared by lysing cells in ice-cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 ml/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Membranes were collected by centrifugation at 30,000xg for 15 min at 4 C. and pellets resuspended in hypotonic buffer to a final concentration of 1-3 mg/ml. Protein concentrations were determined using the BioRad protein assay reagent with bovine serum albumen as standard. Aliquots of the ORL-1 receptor membranes were stored at -80 C.Radioligand binding assays (screening and dose-displacement) used 0.1 nM [3H]-nociceptin (Perkin Elmer, Shelton, Conn.; 87.7 Ci/mmole) with 12 ug membrane protein in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4).
Measurement: Ki > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 530.73, ALogP 5.59, HBA 4, HBD 0, PSA 36.92, QED 0.42
Compound (SMILES): CO[C@@H]1CC[C@@H](n2c(=O)n(C)c3cnc4ccc(-c5ccc(OCCCN6CCCC6)nc5)cc4c32)C1
InChIKey: QEOJMXMERFKIIJ-DHIUTWEWSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 996 nM (pChEMBL 6)
Descriptors: MW 501.63, ALogP 4.55, HBA 8, HBD 0, PSA 74.41, QED 0.33
Compound (SMILES): CCC(O)c1cc(C(=O)N[C@H](C)c2ccccc2)ccc1-c1cn[nH]c1
InChIKey: SAUMCUQQDUAVFI-QMRFKDRMSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 349.43, ALogP 4.01, HBA 3, HBD 3, PSA 78.01, QED 0.63
Compound (SMILES): COCCOCCOCCOCCOCCOCCOCCOCCOCCO[C@H]1CC[C@@]2(O)C3Cc4ccc(O)c5c4[C@@]2(CCN3C)[C@H]1O5
InChIKey: KRDZWSNJWKZJMK-WFNHTRGDSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Measurement: EC50 = 1061 nM (pChEMBL 5.96999979019165)
Descriptors: MW 713.86, ALogP 1.34, HBA 14, HBD 2, PSA 145.23, QED 0.12
Compound (SMILES): CC(=O)Nc1ccc(Cl)c(F)c1C1=CC(=O)N([C@H]2CCC[C@@H](C)C(=O)Nc3ccccc3-c3ccnc2c3)CC1
InChIKey: WTYSEXAPGLRBCV-CLYVBNDRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 75.1 nM (pChEMBL 7.119999885559082)
Descriptors: MW 561.06, ALogP 6.62, HBA 4, HBD 2, PSA 91.4, QED 0.38
Compound (SMILES): N#C[C@H]1CCOC[C@@H]1n1cc(C(N)=O)c(Nc2ccc(OC(F)(F)F)cc2)n1
InChIKey: CLKADAOEZIONGA-YGRLFVJLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format 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 μL of 1.11× enzyme and 1.11× substrate in 1× 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 ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× 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. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 395.34, ALogP 2.73, HBA 7, HBD 2, PSA 115.19, QED 0.8
Compound (SMILES): Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)[C@@H]3C)co1
InChIKey: HNHPUPCBAQSHTI-CYBMUJFWSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK2 receptor was evaluated in CHO recombinant cells which express the human NK2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 405.48, ALogP 4.19, HBA 7, HBD 0, PSA 77.05, QED 0.51
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(Cl)c(Cl)c2)[C@@H]3C)n1
InChIKey: ZMVUVIKWZWFBIB-MRVPVSSYSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki = 7500 nM (pChEMBL 5.119999885559082)
Descriptors: MW 409.3, ALogP 3.63, HBA 7, HBD 0, PSA 76.8, QED 0.65
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(Br)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2cc(F)c(F)cc2NC1=O
InChIKey: VTBXUUQDJAJGJV-BZQUYTCOSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 11.8 nM (pChEMBL 7.929999828338623)
Descriptors: MW 618.88, ALogP 7.7, HBA 3, HBD 1, PSA 62.3, QED 0.3
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3cc(CN4CC[C@@H](O)C4)c4ccccc4n3)c(C)c12
InChIKey: UXOLHRWLSKHKNL-HXUWFJFHSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 503.62, ALogP 4.93, HBA 7, HBD 2, PSA 91.76, QED 0.35
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3ccnc(Cl)c3)=CC1=O)c1cc-2ccn1
InChIKey: RVFWLEJQHJVOBX-DWXRJYCRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 446 nM (pChEMBL 6.35099983215332)
Descriptors: MW 560.05, ALogP 6.09, HBA 6, HBD 2, PSA 113.52, QED 0.38
Compound (SMILES): CCc1noc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)[C@@H]3C)n1
InChIKey: DOEVSBYTUDFXBA-CYBMUJFWSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki = 32000 nM (pChEMBL 4.5)
Descriptors: MW 420.5, ALogP 3.84, HBA 8, HBD 0, PSA 89.94, QED 0.5
Compound (SMILES): C[C@]1(c2nc(NC(=O)c3ncc(OCC(F)(F)F)nc3N)ccc2F)N=C(N)OCC1(F)F
InChIKey: LTIOGWHOZDPVCK-OAHLLOKOSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition Assay: Chinese hamster ovary cells are transfected with the human gene for amyloid precursor protein. The cells are plated at a density of 8000 cells/well into 96-well microtiter plates and cultivated for 24 hours in DMEM cell culture medium containing 10% FCS. The test compound is added to the cells at various concentrations, and the cells are cultivated for 24 hours in the presence of the test compound. The supernatants are collected, and the concentration of amyloid peptide 1-40 is determined using state of the art immunoassay techniques, for example sandwich ELISA, homogenous time-resolved fluorescence (HTRF) immunoassay, or electro-chemiluminescence immunoassay. The potency of the compound is calculated from the percentage of inhibition of amyloid peptide release as a function of the test compound concentration.
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 479.34, ALogP 1.98, HBA 9, HBD 3, PSA 150.63, QED 0.55
Compound (SMILES): COCCOCCOCCOCCOCCO[C@H]1CC[C@@]2(O)C3Cc4ccc(O)c5c4[C@@]2(CCN3C)[C@H]1O5
InChIKey: VQTHZCXPQJPNIU-WNSMAMQXSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Measurement: EC50 = 157 nM (pChEMBL 6.800000190734863)
Descriptors: MW 537.65, ALogP 1.27, HBA 10, HBD 2, PSA 108.31, QED 0.3
Compound (SMILES): O=C(Nc1ccc(C(F)(F)C(F)(F)Cl)cc1)c1cnc(N2CC[C@@H](O)C2)c(-c2cncnc2)c1
InChIKey: RGTJWXUAMAEEIP-QGZVFWFLSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Cellular Proliferation Assay: Test compounds were resuspended in DMSO at a concentration of 10 mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser (PerkinElmer). Compound was delivered to the assay plates containing 2500 cells in a 50 μL volume via Acoustic delivery from an ATS-100 (EDC). For Ba/F3-BCR-ABL1-WT cell assays, 2 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 0.4 μM, 0.13 μM, 0.044 μM, 0.015 μM, 0.005 μM, 0.001 μM, 0.00033 μM, 0.00011 μM, 0.000037 μM, 0.000012 μM. For Ba/F3-WT and Ba/F3-BCR-ABL1-T315I cell assays, 50 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.041 μM, 0.014 μM, 0.0046 μM, 0.0015 μM, 0.00051 μM.Cells were incubated at 37° C. in a humidified environment with 5% car
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 495.86, ALogP 4.29, HBA 6, HBD 2, PSA 91.24, QED 0.39
Compound (SMILES): CC1(C)OC(=O)N2C[C@H](c3nc(-c4ccc(C(=O)Nc5cc(C(F)(F)F)ccn5)cc4)c4c(N)nccn34)CC[C@H]21
InChIKey: FUZSHLVNKZZEKR-MJGOQNOKSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human whole blood assessed as reduction in cell surface CD69 expression preincubated for 1 hr followed by stimulation with anti-CD79b for 3 hrs by immunostaining analysis
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 565.56, ALogP 5.12, HBA 8, HBD 2, PSA 127.74, QED 0.35
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2c(cccc2C(=O)O)NC1=O
InChIKey: OROOHDNCDLETBU-GYCJOSAFSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 423 nM (pChEMBL 6.374000072479248)
Descriptors: MW 566, ALogP 6.49, HBA 4, HBD 2, PSA 99.6, QED 0.35
Compound (SMILES): Cc1ccccc1-c1nnc(C(C)Nc2nccc(N3C(=O)OC[C@@H]3[C@@H](C)O)n2)o1
InChIKey: CCYGBRQOYRJHRW-JYRZLJSNSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 32926 nM (pChEMBL 4.480000019073486)
Descriptors: MW 410.43, ALogP 2.71, HBA 9, HBD 2, PSA 126.5, QED 0.63
Compound (SMILES): CCC(=O)OC(C)n1nnc(-c2c(-c3ccc(C(=O)N(c4ncccc4C)[C@@H]4CCCNC4)cc3)cnn2C)n1
InChIKey: UTFQKCKDAOKSGF-AVKWCDSFSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Cell Free Assay: In order to eliminate the permeability barrier inherent to the WT7 and SCHH cell-based assays a cell-free system was also established to access compound activity. A sequence containing the full length human PCSK9 (NCBI reference identifier, NM-174936.3, where coding sequence start annotated at position 363) along with 84 additional 3â¿² nucleotides, comprising a V5 tag and polylinkinker followed by an in frame modified firefly luciferase reporter (corresponding to nucleotide positions 283-1929 of pGL3, Gen Bank reference identifier JN542721.1) was cloned into the pT7CFE1 expression vector (ThermoScientific). The construct was then in-vitro transcribed using the MEGAscript T7 Kit (Life Technologies) and RNA subsequently purified incorporating the MEGAclear Kit (Life Technologies) according to manufacturer's protocols. HeLa cell lysates were prepared following the protocols described by Mikami (reference is Cell-Free Protein Synthesis Systems with Extracts from Cultured Human Cells, S. Mikami, T. Kobayashi and H. Imataka; from Methods in Molecular Biology, vol. 607, pages 43-52, Y. Endo et al. (eds.), Humana Press, 2010) with the following modifications.
Measurement: IC50 = 25100 nM (pChEMBL 4.599999904632568)
Descriptors: MW 543.63, ALogP 3.31, HBA 11, HBD 1, PSA 132.95, QED 0.33
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc(Cl)c(OC(F)(F)F)c2)no1
InChIKey: NXPYTJOUNUPUMU-SVLLLCHKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 514.85, ALogP 3.96, HBA 10, HBD 2, PSA 135.73, QED 0.48
Compound (SMILES): CNC1=N[C@@H]2[C@@H](O)[C@H](O)[C@@H](CF)O[C@@H]2S1
InChIKey: DJQLZCGDQCJIBK-NYMZXIIRSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Inhibition of recombinant human OGA
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 236.27, ALogP -0.91, HBA 6, HBD 3, PSA 74.08, QED 0.55
Compound (SMILES): COC(=O)c1ccc2c(c1)-c1ccnc(c1)[C@@H](N1CCC(c3c(C(F)(F)F)ccc(Cl)c3F)=CC1=O)CCC[C@@H](C)C(=O)N2
InChIKey: KGBUFRIXAWSHKU-QUGAMOGWSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 57.9 nM (pChEMBL 7.239999771118164)
Descriptors: MW 630.04, ALogP 7.46, HBA 5, HBD 1, PSA 88.6, QED 0.24
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1ccccc1)[C@H]1Cc4ccc(-c5nn[nH]n5)c5c4[C@@]3(CCN1CC1CC1)[C@H]2O5
InChIKey: KJEGDIQWAMKKMD-ZZVPPEEFSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Functional Assay: Functional [35S]GTPgammaS binding assays were conducted as follows. kippa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kippa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 2001 ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours.
Measurement: EC50 = 367.28 nM (pChEMBL 6.429999828338623)
Descriptors: MW 539.68, ALogP 4.31, HBA 7, HBD 1, PSA 85.39, QED 0.46
Compound (SMILES): Nc1nccn2c([C@@H]3CC[C@H]4COCC(=O)N4C3)nc(-c3ccc(C(=O)Nc4cc(C(F)(F)F)ccn4)cc3F)c12
InChIKey: IKMPSGMVJMNBLT-WBVHZDCISA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human whole blood assessed as reduction in cell surface CD69 expression preincubated for 1 hr followed by stimulation with anti-CD79b for 3 hrs by immunostaining analysis
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 569.52, ALogP 3.89, HBA 8, HBD 2, PSA 127.74, QED 0.36