lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCN1CCC(NC(=O)c2ccc(N/C(=C3\C(=O)Nc4cc(C(=O)OC)c(C)cc43)c3ccccc3)cc2)CC1
InChIKey: OPFGGEANRKOGEG-FLWNBWAVSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 Enzyme Inhibition: The inhibitory activities of compounds of the invention against FGFR1 (FGFR1 Kinase Enzyme System: Promega), were evaluated by mixing the FGFR1 protein (3.12 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was then initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision Multilabel Reader, Perkin Elmer).
Measurement: Ki = 48.5 nM (pChEMBL 7.309999942779541)
Descriptors: MW 568.67, ALogP 4.55, HBA 7, HBD 3, PSA 109, QED 0.26
Compound (SMILES): CC(CCCN)N[C@H]1CC[C@H](c2ccc(C(F)(F)F)c(S(N)(=O)=O)c2-c2nnn[nH]2)CC1
InChIKey: VWTAIIUSARJWJU-VLXSWZPNSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 44.1 nM (pChEMBL 7.360000133514404)
Descriptors: MW 475.54, ALogP 2.28, HBA 7, HBD 4, PSA 152.67, QED 0.46
Compound (SMILES): O=C(NNc1cnc(-c2ccc(OC(F)(F)F)cc2)cn1)C1(F)CC1
InChIKey: UDSSJRBQXYVEKV-UHFFFAOYSA-N
Target: Cytochrome P450 1A2 (SINGLE PROTEIN), Homo sapiens
UniProt: P05177
Assay: CYP1A2 Inhibition Assay: CYP1A2 Inhibition in Human Liver Microsomes. Pooled human liver microsomes were incubated with individual CYP, CYP1A2, isozyme-specific marker substrate (Phenacetin) in the presence of test compound at various concentrations (0.05, 0.15, 0.5, 1.5, 5, 15, 50 uM). The specific marker metabolites are measured with LC/MS/MS. The remaining enzymatic activities and inhibitory potency IC50 are determined. Procedure: Microsomes are removed out of the −80° C. freezer to thaw on ice and 20 μL of the substrates solution added to the corresponding wells. Then 20 μL PB was added to blank wells and 2 μL of the test compounds and positive control working solution added to the corresponding wells. 2 μL of solvent was added to No Inhibitor wells and Blank wells. Then 158 μL of the HLM working solution was added to all wells of the incubation plate. The plate was pre-warmed for about 10 min using a 37° C. water bath. Then 20 μL of the NADPH cofactor solution was added to all incubation wells, mixed and incubated for 10 minutes at 37° C. water bath. The reaction is terminated by adding 400 μL cold stop solution (200 ng/mL Tolbutamide and 200 ng/mL Labetalol in ACN). The samples were centrifuged at 4000 rpm for 20 minutes to precipitate protein. Finally 200 μL of the supernatant was transferred to 100 μL HPLC water, shaken for 10 min and analyzed by LC/MS/MS.
Measurement: IC50 = 8000 nM (pChEMBL 5.099999904632568)
Descriptors: MW 356.28, ALogP 2.99, HBA 5, HBD 2, PSA 76.14, QED 0.64
Compound (SMILES): Cc1ncoc1C(CNC(=O)c1c(F)cccc1Cl)N1CCC(F)(F)CC1
InChIKey: WKQHGTKGYIKDDD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO-1 Uptake Assay: YO-PRO®-1 is a fluorescent DNA-binding dye with a MW of 374 Da (Molecular Probes, cat. Y3603). This method is based upon the presumed ability of YO-PRO-1 to enter through the dilated or "large pore form" of P2X7 receptor and to bind to intracellular DNA whereupon it increases many fold its fluorescence intensity. The dye has an absorption spectrum compatible with excitation at 488 nm by argon laser sources and its emission wavelength is in the range of 515-575 nm. The aim of this assay was to validate the interaction of antagonists with P2X7 receptor using an alternative readout to Ca2+-sensitive fluorescent dyes.HEK-293 cells stably transfected with P2X7 receptor were seeded overnight in growth medium at 20,000 cells/well in 384-well plate. 24 hours later, the medium was removed, the cells were washed with Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+) and then pre-loaded with 20 μL/w of 5 μM YO-PRO-1 dye. FLIPRTETRA fluorescence measurement immediately started. Then, 10 μL/w of test compounds and reference antagonist A438079 at 3×-concentration were injected with the FLIPRTETRA and the kinetic response over a period of five minutes was monitored. A second injection of 10 μL/w of 3× Bz-ATP EC80 (30 μM) was performed with the FLIPRTETRA and the signal of the emitted fluorescence was recorded for additional 60 minutes. All the experiment was carried out with a Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+).The effect of the test compounds was measured as percent inhibition vs the reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 2067 nM (pChEMBL 5.679999828338623)
Descriptors: MW 401.82, ALogP 3.98, HBA 4, HBD 1, PSA 58.37, QED 0.82
Compound (SMILES): COc1cccc(C(=O)N2CC3CN(Cc4c(-c5ccc(Br)cc5)nc5ccccn45)CC3C2)n1
InChIKey: IUNZPGRRUGDWGV-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: In Vitro Electrophysiological Assay: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis OocytesSubsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique was used. The measurements were conducted at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 532.44, ALogP 4.37, HBA 6, HBD 0, PSA 62.97, QED 0.38
Compound (SMILES): CNc1cc(-c2cn([C@@H]3COC[C@H]3OC)c3ncccc23)nc2c(C(=O)NC3CC[C@@H]3F)cnn12
InChIKey: UVNSFQQBVLBJGY-RMGPQRKISA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 479.52, ALogP 2.6, HBA 9, HBD 2, PSA 107.6, QED 0.44
Compound (SMILES): CCn1cc(C(=O)NCc2cccc(Cl)c2)c2cc(OC)c(OC)cc2c1=O
InChIKey: NWYRMODHJOPADD-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Enzymatic Assay: The PDE enzymatic reaction was carried out in assay buffer (20 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1% bovine serum albumin) containing enzyme and substrate. The PDE enzymes concentration ranged from 10 pM 250 pM, depending on each enzyme's specific activity. The substrate cyclic nucleotide (cAMP or cGMP) concentration used in the assay was 20 nM for PDE10, and 100 nM for other PDEs. The inhibitory effect of compound was determined by incubating various concentration of inhibitor in the enzymatic assay. Typically, compound was serial diluted in DMSO then further diluted in assay buffer. Next, the compound at varying concentration was mixed with PDE enzyme. The reaction was initiated by addition of cyclic nucleotide substrate, and incubated for 60 minutes at 29 C. The reaction was stopped by addition of lysis buffer from assay kit. The cAMP-d2 and anti-cAMP cryptate in the lysis buffer detected the level of cAMP left from the PDE hydrolysis reaction. The PDE activity is reversely correlated with the amount of cAMP left in the reaction and can be converted to the percent activity of an uninhibited control (100%).
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 400.86, ALogP 3.62, HBA 5, HBD 1, PSA 69.56, QED 0.69
Compound (SMILES): CCO[C@H](C(=O)Nc1nnc(N[C@@H]2CCN(c3nccnn3)C2)s1)c1ccc(F)cc1
InChIKey: HIJNQGBERQOCKM-CABCVRRESA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: GLS Enzyme Potency Assay: A Glutamate Oxidase/AmplexRed coupled assay was used to measure the ability of compounds to bind to and inhibit the activity of GLS1 in vitro. 6His tagged GLS protein (amino acids 63-669) expressed in E. Coli was purified and stored at −80° C. in aliquots. GLS1 was diluted to 2× working concentration and incubated at r.t. to allow the tetrameric/dimeric forms to reach steady state. Assay measurements were performed in buffer comprising 50 mM TRIS pH 7.8, 100 mM NaPO4, pH 7.8, 0.001% v/v Tween20. Purified recombinant GLS1 protein was diluted in assay buffer to 12 nM and pre-incubated at r.t. for 30 minutes. Test compounds were prepared by dilution in 100% DMSO to give the correct dose range for 12 point concentration response and an appropriate volume (2.5-60 nl) dispensed into 384 well micro assay plates (Greiner product code 784900) using a Labcyte Echo 555 acoustic dispenser. DMSO concentration was maintained at 2% by back filling with DMSO solution. 3 μL of diluted GLS1 protein (12 nM) was then dispensed into each well using a BioRaptr automated dispenser (Beckman-Coulter) and incubated for 15 minutes at r.t. 3 μL of 100 mM glutamine diluted in assay buffer was then added and the reaction incubated at r.t. for 60 minutes. The reaction was then stopped by addition of 45 μM 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, 75 μM Amplex Red, 0.375 units/mL Horseradish Peroxidase, 0.12 units/mL Glutamate Oxidase in 100 mM TRIS pH7.5. After 30 minutes at room temp in the dark, plates were read on a Perkin Elmer EnVision using 535/590 nm optic filters and raw data analysed using Genedata to generate IC50 values. An artefact version of the assay where the 6His tagged GLS protein and glutamine were replaced with assay buffer was also used to rule out non specific effects on the assay components.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 444.5, ALogP 2.27, HBA 10, HBD 2, PSA 118.05, QED 0.54
Compound (SMILES): Cc1c(Cl)cc(C(=O)Nc2ccc(-c3cc(C4CCN(C(=O)C(C)C)CC4)n4ncnc(N)c34)cc2)c(=O)n1-c1cccnc1
InChIKey: HOODYPQSDBBQKD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 625.13, ALogP 5.1, HBA 9, HBD 2, PSA 140.51, QED 0.27
Compound (SMILES): C=CC(=O)N[C@@H]1CCCC[C@@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1C
InChIKey: ZWTAFPNHNSTUJJ-YADHBBJMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 567.67, ALogP 6.42, HBA 6, HBD 3, PSA 112.66, QED 0.22
Compound (SMILES): Cc1sc(C(=O)NC2CCOCC2)cc1[C@H]1C[C@@H]1NC1CCC1
InChIKey: GDYDCSMFSLCSII-CVEARBPZSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibitory Activity Assay: The MAO-A inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK.A test compound dissolved in DMSO was added to a reaction solution (100 mM HEPES (pH 7.5), 5% glycerol) containing MAO-A enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 15 min. MAO substrate (Promega KK) was added to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-A inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate (%)=(1-(test compound count-blank)/(control-blank))x100The count of the MAO-A enzyme reaction mixture under compound non-addition conditions is indicated as control, and the count under compound non-addition and MAO-A enzyme non-addition conditions is indicated as blank. A concentration necessary for achieving 50% inhibitory rate was taken as IC50 value.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 334.49, ALogP 2.96, HBA 4, HBD 2, PSA 50.36, QED 0.87
Compound (SMILES): CC(C)C[C@H](N)[C@](O)(Cc1nccc2c1CCC(OCc1ccccc1)C2)C(=O)O
InChIKey: KYXXNWQHUSVSGA-DGUFBMEBSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition Assay: The inventors have assumed that inhibition of nocturnal activity of placental leucine aminopeptidase (P-LAP), i.e. aminopeptidase that cleaves AVP, would maintain and/or increase an endogenous AVP level to enhance the antidiuretic effect, which would contribute to a decreased number of nocturnal voids, and have extensively studied compounds which inhibit P-LAP.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 412.53, ALogP 2.89, HBA 5, HBD 3, PSA 105.67, QED 0.58
Compound (SMILES): NS(=O)(=O)OC[C@H]1C[C@@H](Nc2ccnc3cc(-c4cc5ccccc5[nH]4)nn23)[C@H](O)[C@@H]1O
InChIKey: BVKKBZVZXKGDAW-KRVNLHHGSA-N
Target: Ubiquitin-conjugating enzyme E2 E1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51965
Assay: HTRF Assay : The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE 1050 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 458.5, ALogP 0.62, HBA 9, HBD 5, PSA 167.86, QED 0.28
Compound (SMILES): NC[C@]12CCCC[C@H]1CN(c1nc(N)c(-c3cccc(Cl)c3Cl)c(C(N)=O)n1)C2
InChIKey: IKMGLAPRJVWUEO-YBTHPKLGSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: The inhibition of SHP2 by compounds of the invention was monitored using the surrogate substrate DiFMUP after protein activation by a peptide bearing two appropriately spaced phosphotyrosine. Full length SHP2 protein (Recombinant HumanSHP-2, E. coli derived Ser2Arg593, N-terminal 6His tag from R&D systems; 0.0.24 nM) was incubated with activating peptide, IRSI_2pY (New England Peptide, 140 nM) and DiFMUP (molecular probes, 80 uM) at RT in buffer (HEPES pH 7.2 60 mM, DDT 5 mM, KCl 75 mM, NaCl 75 mM, EDTA 1 mM, Tween 20 0.05%) in presence of compound (10 concentrations range, top concentration 50 Î¼M) for 60 min. The generation of the DiFMU product by activated SHP2 was monitored through Fluorescence measurement with a PerkinElmer Envision reader.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 435.36, ALogP 3.09, HBA 6, HBD 3, PSA 124.15, QED 0.68
Compound (SMILES): C[C@H](Oc1cc(-n2nc3n(c2=O)CCCC3)c(F)cc1C(=O)NCC(C)(C)O)C(F)(F)F
InChIKey: MMALLKGIEOGPJM-NSHDSACASA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 460.43, ALogP 2.34, HBA 7, HBD 2, PSA 98.38, QED 0.65
Compound (SMILES): CSc1cnc(NC[C@H](CO)CNc2nc3ccc(S(=O)(=O)N4CCOCC4)cc3s2)nc1
InChIKey: JLGYTNBXQJBKBF-AWEZNQCLSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Surface Plasmon Resonance (SPR) Assay: Surface plasmon resonance data was collected on a Biacore T200 or 3000 system (GE Healthcare) at 25° C. Streptavidin was immobilized on a CMS (GE Healthcare) or CMD500d sensor chip (XanTec Bioanalytics) using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μL/min. For capture of streptavidin, protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Avi-tagged PCSK9 protein was captured on the streptavidin surface by injection of 150 μL of protein diluted to 16 μg/mL in HBS-N, 0.05% tween-20, 0.1 mM CaCl2. Typical surface densities obtained were 8000-10000 RU. SPR binding data were obtained using an appropriate dilution series of each compound at a flow rate of 30 μL/ min, with a capture time of 100 s and dissociation times of 300 s. Running buffer for compound binding studies was HBS-N, 0.05% tween-20, 0.1 mM CaCl2, 4% DMSO. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).
Measurement: Kd = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 510.67, ALogP 1.96, HBA 11, HBD 3, PSA 129.57, QED 0.35
Compound (SMILES): CCC(Cl)C(=O)Nc1cccc(-c2ccc(C(N)=O)c3[nH]c(C4=CCN(S(C)(=O)=O)CC4)cc23)c1C
InChIKey: VZWUOYXMQAIGIZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 529.06, ALogP 4.25, HBA 4, HBD 3, PSA 125.36, QED 0.4
Compound (SMILES): NC1=N[C@@]2(c3cc(/C=C(\F)c4ccc(C(F)(F)F)cn4)ccc3F)CO[C@H](C(F)(F)F)[C@H]2CS1
InChIKey: JZGAHRFJHBROMI-XKMNTDACSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: n Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assay: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about luL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 509.42, ALogP 5.54, HBA 5, HBD 1, PSA 60.5, QED 0.55
Compound (SMILES): N#CCC1(CS(=O)(=O)Nc2ccc(-c3cc(NC(=O)C4CC4)nc4[nH]ccc34)cc2F)CC1
InChIKey: BDZPZPUXXNKBJP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 467.53, ALogP 4.15, HBA 5, HBD 3, PSA 127.74, QED 0.46
Compound (SMILES): C=C(c1ccc(F)cc1)c1nc2nc(-c3noc(=O)[nH]3)nc(N[C@H](C)C3CCC3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: RUPOEXKYZXNCSL-BHIYHBOVSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 1.84 nM (pChEMBL 8.729999542236328)
Descriptors: MW 531.64, ALogP 5.8, HBA 8, HBD 2, PSA 114.52, QED 0.3
Compound (SMILES): COC[C@]12C[C@H]1[C@@](C)(c1cc(/C=C(\F)c3cnc(OCC4CCC4)cn3)ccc1F)N=C(N)S2
InChIKey: AHOCSRKOXSHDRM-SGSMAQGXSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 716 nM (pChEMBL 6.139999866485596)
Descriptors: MW 486.59, ALogP 4.94, HBA 7, HBD 1, PSA 82.62, QED 0.57
Compound (SMILES): N#Cc1cc(-c2ncnc(Nc3ccc(N4CCN(C5COC5)CC4)cc3)n2)ccc1O[C@H]1CCCN(C(=O)CO)C1
InChIKey: GRMNZGJJAHSVGX-SANMLTNESA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assays for TBK1 and IKKe: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the doseresponse curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 570.65, ALogP 2.04, HBA 11, HBD 2, PSA 139.97, QED 0.41
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(N2CCOCC2)nc1Cl)C3c1cc(C(=O)O)cs1
InChIKey: LHAMZXQRQUXKHH-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 575.07, ALogP 3.3, HBA 11, HBD 1, PSA 122.39, QED 0.35
Compound (SMILES): COc1ccc2cc(C(=O)C3CN(C4CCC4)CCN3Cc3ccccc3)ccc2c1
InChIKey: DYKQHRZFBFVIRV-UHFFFAOYSA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: KDM2B TR-FRET Assay: Full length KDM2B was cloned, expressed, and purified to homogeneity. Compound inhibition of KDM2B demethylase activity was assessed by monitoring the methylation status of a biotin-H3K36me2 peptide substrate (H2N-RKSAPATGGV(KMe2)KPHRYRPGTV-NTPEGBiot; New England Peptide) in the presence of α-keotglutarate (2-OG) and iron (Fe2+) using the TR-FRET assay technology (Cisbio). Specifically, in a 384 well ProxiPlate KDM2B (5 mM final), ascorbate (500 μM final) and DTT (2 mM final) were combined with the biotin-H3K36me2 peptide substrate (200 nM final), 2-OG (0.3 μM or 6 μM final; Sigma K2010) and Fe2+ (100 μM final; Sigma F1543) in 50 mM HEPES (pH 6.5) and 0.01% Triton-X 100 either in the presence of DMSO (final 0.25% DMSO) or compound dilution series in DMSO and mixed. After a two hour incubation at room temperature, a mixture of EU-anti-H3K36me1 antibody (2 nM final; Cisbio #64CUSKAZ), and Streptavidin-d2 (50 nM final; Cisbio #64CUS000) in 200 mM KF, 200 mM EDTA, 0.1% BSA and 50 mM HEPES (pH 6.5) was added. After 1 hour incubation, the plates were read on an Envision instrument, the readouts were transformed into % inhibition, and IC50 values were generated using a four parameter logistic model (XLFIT5).
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 414.55, ALogP 4.77, HBA 4, HBD 0, PSA 32.78, QED 0.54
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc4[nH]c(CCN5CCN(C)CC5)cc4c3N(c3ccc(F)cc3)C2=O)c1F
InChIKey: LQOBRJXLLSUOKJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 580.61, ALogP 5.07, HBA 6, HBD 1, PSA 77.17, QED 0.33
Compound (SMILES): CC1CN(c2cncnc2)Cc2cc(NC(=O)c3cc(CN4CCN(C)CC4)cc(C(F)(F)F)c3)ccc21
InChIKey: FXWGWJNBGHQODS-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Lantha Screen Eu Kinase Activity Assay: The effects of compounds on the kinases DDR1 and DDR2 were assessed by using a Lantha Screen Eu kinase activity assay technology (Invitrogen, USA). Kinase reactions are performed in a 10 uL volume in low-volume 384-well plates. The kinases in reaction buffer consist of 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgCl2, and 1 mM EGTA, the concentration of Fluorescein-Poly GAT substrate (Invitrogen, USA) in the assay is 100 nM. Kinase reactions were initiated with the addition of 100 nM ATP in the presence of serials of dilutions of compounds. The reactions were allowed to proceed for 1 h at room temperature before a 10 uL preparation of EDTA (20 mM) and Eu-labeled antibody (4 nM) in TR-FRET dilution buffer are added. The final concentration of antibody in the assay well is 2 nM, and the final concentration of EDTA is 10 mM. The plate is allowed to incubate at room temperature for one more hour before the TR-FRET emission ratios of 665 nm/340 nm were acquired on a PerkinElmer EnVision multilabel reader (Perkin-Elmer, Inc.). Data analysis and curve fitting were performed using GraphPad Prism4 software, resulting in the half maximal inhibitory concentration (IC50) shown in table 1. The functional assays of compounds on the kinase activities of c-kit and Abl were determined using the FRET-based Z'-Lyte assay system according to the manufacturer's instructions (Invitrogen, USA). Tyrosine 2 peptide was used as Abl substrate, and Ser/Thr 6 peptide was used as the substrate for c-kit. The reactions were carried out in 384-well plates in a 10 uL of reaction volume with appropriate amount of kinases in 50 mM HEPES (pH 7.5), 10 Mm MgCl2, 1 mM EGTA, and 0.01% Brij-35. The reactions were incubated 1 h at room temperature in the presence of 2 uM of substrate with 10 uM of ATP (for Abl1 assays) or 300 uM of ATP (kit assay) and in the presence of various concentrations of the compounds.
Measurement: IC50 = 444 nM (pChEMBL 6.349999904632568)
Descriptors: MW 524.59, ALogP 4.62, HBA 6, HBD 1, PSA 64.6, QED 0.53
Compound (SMILES): N#Cc1cnc2c(Cl)cc(N[C@H](C3=CN(C4COC4)NN3)c3cccnc3Cl)cc2c1Nc1ccc(F)c(Cl)c1
InChIKey: VLSSGFPGEVYDNS-SANMLTNESA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 597.87, ALogP 6.06, HBA 9, HBD 4, PSA 110.16, QED 0.19
Compound (SMILES): OC1CCC(C(O)C[C@H]2c3c(F)cccc3-c3cncn32)CC1
InChIKey: YGACXVRLDHEXKY-IQGLFJIHSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic IDO Assay: The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 200 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 200 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound. Nonlinear regressions and determination of the IC50 values were performed using the GraphPad Prism 4 software. Compounds with an IC50 of less than 500 μM were considered as active inhibitors in this assay.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 316.38, ALogP 2.89, HBA 4, HBD 2, PSA 58.28, QED 0.92
Compound (SMILES): O=C1CCc2ccc(CCN3CCC(c4cccc5sccc45)CC3)cc2N1
InChIKey: DMWOAPDORNWCNR-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: 5-HT1A Receptor Agonism Activity Test: The 5-HT1A receptor agonism activity test (The agonism activity of test compounds on 5-HT1A receptor expressing human recombinant 5-HT1A receptor in HEK293 cells) was performed using LANCE cAMP 384 Kit (Product of USA PerkinElmer Inc.). The 5-HT1A receptor agonism activity of test compounds was evaluated through their inhibition on cAMP production in HEK293 cells. cAMP concentration test was performed according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM, 8-OH-DPAT was used as a positive control, EC50 value was calculated by the Excelfit software.
Measurement: EC50 = 4.91 nM (pChEMBL 8.3100004196167)
Descriptors: MW 390.55, ALogP 5.21, HBA 3, HBD 1, PSA 32.34, QED 0.66
Compound (SMILES): C[C@@H](CN[C@@H](C(=O)Nc1ccc(-c2cnn(C)c2)cn1)c1ccccc1)c1ccc(C#N)c(F)c1
InChIKey: RMRQRQHCCOEUNN-HFJWLAOPSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 = 473 nM (pChEMBL 6.329999923706055)
Descriptors: MW 468.54, ALogP 4.57, HBA 6, HBD 2, PSA 95.63, QED 0.4