lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(C1CC1)N(Cc1nc(-c2nnc(C(F)F)o2)cs1)c1cccc(Cl)c1
InChIKey: AUBMZYBLRGPCFH-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Biochemical Assay: The compounds disclosed herein were tested for potency against HDAC6 and selectivity against HDAC1 in a biochemical assay. A biochemical assay was adopted using a luminescent HDAC-Glo I/II assay (Promega) and measured the relative activity of HDAC6 and HDAC1 recombinant proteins. Compounds were first incubated in the presence of HDAC6 or HDAC1 separately, followed by addition of the luminescent substrate. The data was acquired using a plate reader and the biochemical IC50 were calculated from the data accordingly.
Measurement: IC50 = 7500 nM (pChEMBL 5.119999885559082)
Descriptors: MW 410.83, ALogP 4.73, HBA 6, HBD 0, PSA 72.12, QED 0.59
Compound (SMILES): CC1CC(N2CCOCC2)CCC1Nc1ncnc2ccc(C#N)cc12
InChIKey: PBPWOBUNCFGVTO-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 351.45, ALogP 2.8, HBA 6, HBD 1, PSA 74.07, QED 0.92
Compound (SMILES): CN1CCC(NC(=O)C(C)(C)COc2ncccc2C(F)(F)F)C(Cl)C1
InChIKey: ZBIPLRAZPOJZTK-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 1259 nM (pChEMBL 5.900000095367432)
Descriptors: MW 393.84, ALogP 2.93, HBA 4, HBD 1, PSA 54.46, QED 0.78
Compound (SMILES): NC(=O)c1cnc2ccc(/C=C\c3cccc(C(F)(F)F)n3)cn12
InChIKey: MZZXKMCZCKETPE-XQRVVYSFSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Enzyme Activity Inhibition: 1. Preparation of a 1× kinase buffer: 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.10% BSA, 1 mM DTT.2. Compound preparation: The final detection concentration of the compound was 10 μM, which was prepared to a 100-fold concentration, i.e., 1 mM. 100 μL of the 100-fold compound was added in the second well of the 384-well plate, and 60 μL of 100% DMSO was added to the other wells. 30 μL of compound from the second well was added to the third well, which was made a 3-fold dilution in sequence, diluting a total of 10 concentrations. 50 nL of the compound was transferred to the reaction plate with echo.3. Kinase reaction: The kinase was added to a 1× kinase buffer to form a 2× enzyme solution. The final concentration of the kinase solution was ALK5: 25 nM. The polypeptide TGFbR1 (purchased from Signal Chem, catalog number T36-58) and ATP were added to a 1× kinase buffer to form a 2× substrate solution. The final concentration of the substrate solution was peptide TGFbR1 0.1 mg/mL, ATP 7 μM. 2.5 μL of the 2× enzyme solution was added to the 384-well reaction plate (there was already 50 nL of 100% DMSO dissolved compound), and a 1× kinase buffer was added to a negative control well. The reaction solution was incubated at room temperature for 10 minutes. 2.5 μL of 2× substrate solution was added to the 384-well reaction plate. The 384-well plate was covered and incubated at 30° C. for 1 hour. ADP-Glo reagent (purchased from Promege, catalog number v9102) was equilibrated to room temperature. 5 μL of ADP-Glo reagent was transferred to the reaction well of the 384-well plate to stop the reaction.4. Detection of reaction results: 10 μL of kinase detection reagent was transferred to each well, shaken for 1 minute, and let stand at room temperature for 30 minutes. The sample luminescence value was read at Synegy.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 332.29, ALogP 3.02, HBA 4, HBD 1, PSA 73.28, QED 0.8
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(-c3cnc4[nH]c(C)nc4c3)cc12
InChIKey: UXAKJTOYWZKDLW-GCJKJVERSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 Î¼M each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 619.45, ALogP 4.22, HBA 8, HBD 2, PSA 138.76, QED 0.21
Compound (SMILES): CCOCCS(=O)(=O)NC(=O)c1c2c(nn1CCC1CC1)C[C@]1(CCc3cc(OCC(F)(F)F)ccc31)NC2=O
InChIKey: MPURMGPSNRUPJG-VWLOTQADSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 584.62, ALogP 2.85, HBA 8, HBD 2, PSA 128.62, QED 0.39
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(-c2cnc(N)c(C(=N)OC(=N)NC(=O)[C@H]3N[C@H]4CCC3C4)n2)cc1
InChIKey: PIKCQLMLOSWODT-HUUYTVAOSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 485.57, ALogP 1.44, HBA 10, HBD 5, PSA 184, QED 0.31
Compound (SMILES): C[C@@H]1CCN(c2n[nH]c3nc(N4CCC5(CC4)CO[C@@H](C)[C@H]5N)cnc23)c2ccc(-c3ncon3)nc21
InChIKey: RAXXROHNPNNUDK-LRAJWGHMSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 502.58, ALogP 2.78, HBA 11, HBD 2, PSA 148, QED 0.43
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H]3C[C@@]4(CNC(=O)C(C)(C)C/C=C\COCc5ccc(Br)nc5NC3=O)C[C@@H]24)c2ccc(-c3cnc(C)nc3)cc12
InChIKey: ORTINKITCFIDCA-MHNIQBHBSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 755.67, ALogP 4.78, HBA 10, HBD 2, PSA 161.3, QED 0.17
Compound (SMILES): Cc1cc(C2CN(C(=O)c3cn(C)c4ccccc34)CC(F)(F)C2)n2ncnc2n1
InChIKey: RDLKJZJNIPCIKQ-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 410.43, ALogP 3.19, HBA 6, HBD 0, PSA 68.32, QED 0.51
Compound (SMILES): CCn1c(-c2cccnc2COC)c2c3cc(ccc31)-c1cc(C)cc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)C#CCN(C)C)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: XMVIOQFGENLLNO-YIYRQFNASA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 943.2, ALogP 5.39, HBA 11, HBD 2, PSA 158.65, QED 0.16
Compound (SMILES): N[C@@H]1CCCC12CCN(c1ncc(Sc3cc(=O)[nH]c4ccccc34)c3nccn13)CC2
InChIKey: YYEBKEDQHHYBGQ-HXUWFJFHSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: Inhibition of SHP-2 activity by small molecule inhibitors was monitored by a biochemical DiFMUP pseudosubstrate-fluorogenic assay. SHP-2 enzyme at final concentration of 0.2 nM was incubated with an IRS-1 activator peptide at a final concentration of 1 μM in SHP-2 assay buffer (60 mM HEPES, pH 7.2-7.5, 0.05% Tween-20, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, and 5 mM DTT) for 75 min at room temperature. The reaction mix was added to the 384-well non-binding plate which had been pre-spotted with 1:3 serial diluted compounds from 10,000-0.005 nM, and incubated at room temperature for 90 min. DiFMUP substrate at final concentration of 50 μM was then added to the wells and incubated for additional 30 min at room temperature. The final reaction volume was 20 μl/well. The fluorescence intensity of the reaction product was measured by Envision at excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were generated using normalized IC50 regression curve fitting by in house dose response analysis tools. The wells with DMSO vehicle were used as controls (100% signal). The percent (%) control of SHP-2 compound was calculated as: (Compound well fluorescence counts/DMSO fluorescence counts)×100%.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 446.58, ALogP 3.82, HBA 7, HBD 2, PSA 92.31, QED 0.5
Compound (SMILES): Cc1cccc(Cl)c1-c1noc(C2CC2)c1CO[C@@H]1C[C@@H]2C[C@H]1CN2c1nc2c(C)cc(C(=O)O)cc2s1
InChIKey: UBDBTIZMOXBWLI-CNNODRBYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Binding Assay: The affinity of FXR ligands for the ligand binding domain of FXR was determined using a commercially available human FXR ligand binding assay (LanthaScreen, Thermofisher Cat # PV4833). The purified ligand binding domain of human FXR tagged with GST (glutathiones-S-transferase) is incubated with a terbium labelled anti-GLT antibody and a fluorescein-labelled SRC2-2 peptide (LKEKHKILHRLLQDSSSPV (SEQ ID NO.: 1)). Binding of FXR ligands to the FXR ligand binding domain promotes binding of the fluorescein-labelled SRC2-2 peptide. This causes a FRET signal between the terbium-labelled anti-GST antibody and the fluorescein-labelled SRC peptide which are both bound to the FXR ligand binding domain.Test compounds are dissolved in DMSO and a 3-fold serial dilution series is generated, then further diluted into assay buffer. The compounds are mixed with 5 nM GST-tagged FXR ligand binding domain, 5 nM Tb-labelled anti-GST antibody and 500 nM fluorescein-labelled SRC2-2 peptide in a pH7.4 buffer. The reaction is incubated at room temperature for 1 hour, then the FRET signal is measured as the ratio of the 520 nm/495 nm emission following excitation at 340 nm. The change in FRET signal is plotted against the test article concentration and fit to a 3-parameter logistical equation.
Measurement: EC50 = 3.16 nM (pChEMBL 8.5)
Descriptors: MW 550.08, ALogP 6.98, HBA 7, HBD 1, PSA 88.69, QED 0.27
Compound (SMILES): COCC#Cc1cnc2onc(N)c2c1
InChIKey: PFYYQOZVOGLKEV-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic Assay: Recombinant human IDO1 (rhIDO1) was expressed and purified from cultures of EC538 strain of E. coli transformed with pREP4 and pQE9-IDO plasmid. Reaction mixes were set up in 384-well microplates containing 50 mM phosphate buffer, 10 mM ascorbic, 10 μM methylene blue, 100 μg/mL catalase, 80 μM TRP, 0.01% Tween 20 (v/v) mixed with rhIDO1 (15 μL) at a final concentration of 9 nM in a total volume of 30 μL assay medium. The plates were incubated at 37° C. for 30 min, and the enzymatic reaction was terminated by adding piperidine (200 mM) and heated at 65° C. for 20 min. Fluorescence intensity was read at λex 400 nm and λem 500 nm. Test compounds were dissolved in 100% DMSO and pre-diluted in assay medium prior to adding rhIDO1.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 203.2, ALogP 0.8, HBA 5, HBD 1, PSA 74.17, QED 0.69
Compound (SMILES): C[C@H]1Cn2c(-c3cccnn3)nnc2-c2nnc(-c3cccc(C(F)(F)F)c3Cl)n21
InChIKey: SHWMMGPNRYDPOD-VIFPVBQESA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR 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 = 1047 nM (pChEMBL 5.980000019073486)
Descriptors: MW 432.8, ALogP 3.91, HBA 8, HBD 0, PSA 87.2, QED 0.48
Compound (SMILES): C[C@@H]1OC(=O)NN=C1c1ccc(-n2ccc(C(F)(F)F)n2)c(C(F)(F)F)c1
InChIKey: YRYSNJRREQGBOS-ZETCQYMHSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: PDE3A Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3A reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration.
Measurement: IC50 = 657 nM (pChEMBL 6.179999828338623)
Descriptors: MW 392.26, ALogP 3.74, HBA 5, HBD 1, PSA 68.51, QED 0.79
Compound (SMILES): CCOC(=O)N1CC(c2nc3cnc4[nH]ccc4c3n2[C@H]2CC[C@H](CC#N)CC2)C1
InChIKey: SFTKIPQQWBBBPX-KOMQPUFPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 406.49, ALogP 4.11, HBA 6, HBD 1, PSA 99.83, QED 0.7
Compound (SMILES): O=C(NC(CCO[C@H]1C[C@H](CCc2ccc3c(n2)NCCC3)C1)C(=O)O)c1cccc(F)c1
InChIKey: PMLIVDCUMZWOAH-SOVYMCNHSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 455.53, ALogP 3.58, HBA 5, HBD 3, PSA 100.55, QED 0.51
Compound (SMILES): CNC(=O)[C@H](CCCCNC(=N)N)NC(=O)c1csc([C@@H]2C[C@@H](N)CN2C(=O)c2cc3c(Cl)cccc3s2)n1
InChIKey: SNRDAMCHFNCWJG-SKDZVZGDSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma Kallikrein: The example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 10 min at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at room temperature. After initiation with substrate, reactions were incubated for 18 hr at room temperature and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 591.16, ALogP 2.42, HBA 8, HBD 6, PSA 179.32, QED 0.12
Compound (SMILES): Cc1cc2[nH]c(Nc3cccc(C(=O)NO)c3)nc2cc1Br
InChIKey: FLUSTWYPWBTCFQ-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: HDAC Inhibition Assays: This example describes in vitro inhibition properties of exemplary HDAC11 inhibitors for various HDACs. HDAC inhibition assays were performed using an electrophoretic mobility shift assay at Nanosyn, Inc. (Santa Clara, Calif.). Full length human recombinant HDAC proteins were expressed in the baculoviral system and purified by affinity chromatography. A human recombinant HDAC3 was co-expressed with nuclear receptor corepressor (Ncor2). The following peptide substrates were used: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2 for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4, HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound, enzyme, and substrate were performed in reaction buffer (comprised of 100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% Triton X-100) at 25° C. and quenched by the addition of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescence intensity of the electrophoretically separated de-acetylated product and substrate peptide were measured and analyzed using the LabChip® 3000 microfluidic electrophoresis instrument (Perkin Elmer/Caliper Life Sciences). The IC50 values of inhibitors were determined by fitting the %-inhibition curves with 4 parameter dose-response model using XLfit 4 software (IDBS).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 361.2, ALogP 3.5, HBA 4, HBD 4, PSA 90.04, QED 0.42
Compound (SMILES): O=C(NC1CCOCC1)c1cc(C2CC2NC2CCC2)cs1
InChIKey: FQYSZTCPJNEABK-UHFFFAOYSA-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 320.46, ALogP 2.65, HBA 4, HBD 2, PSA 50.36, QED 0.88
Compound (SMILES): CCCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3cnc(OC)nc3)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)O1
InChIKey: ULFYYEPIFHBMQE-RSXGOPAZSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 563.61, ALogP 5.2, HBA 8, HBD 2, PSA 106.43, QED 0.29
Compound (SMILES): Cn1cc(-c2nc3c(-c4ccc(CNC(=O)CCC(C)(C)C)c(F)c4)ccnc3[nH]2)cn1
InChIKey: ORKNLVNZQBGBDW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK Enzyme: Briefly, 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; developed in-house) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT. 5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27° C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel. Switzerland).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 434.52, ALogP 4.61, HBA 5, HBD 2, PSA 88.49, QED 0.46
Compound (SMILES): CCOc1c([C@@H](C)n2nc(C)c3c(N)ncnc32)cc(Cl)c(F)c1C1COC(=O)N1
InChIKey: UCYDWQYLQRHHCX-PKEIRNPWSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kdelta Scintillation Proximity A3 Assay: [γ-33P]ATP (10 mCi/mL) was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 434.86, ALogP 3.3, HBA 8, HBD 2, PSA 117.18, QED 0.63
Compound (SMILES): Cc1ncc(C)n2nc(CCc3cn4ccccc4n3)nc12
InChIKey: HBENBNYEZORWNQ-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Pharmacology Assay: In one embodiment, the compounds provided herein were assayed for their ability to inhibit human PDE-10A. In one embodiment, the activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 292.35, ALogP 2.17, HBA 6, HBD 0, PSA 60.38, QED 0.58
Compound (SMILES): Cn1c(C(=O)N[C@@H]2CCN(C(=O)OC(C)(C)C)C2)cc2ccc(Br)cc21
InChIKey: MLMXKCQSFFLFCR-CQSZACIVSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical Fluorescence Intensity Kinetic Assay/Biochemical Fluorescence Intensity Assay: USP30 Biochemical Fluorescence Intensity Kinetic Assay.Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. USP30 CD (Boston Biochem E-582 or 57-517, #64-0057-050 Ubiquigent) was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0, 0.0005, 0.001, 0.005, and 0.01 μl/well. Buffer was optimised for optimal temperature, pH, reducing agent, salts, time of incubation, and detergent. Reactions were initiated by the addition of Ubiquitin-Rhodamine 110 (U-555, Boston Biochem) at a final concentration of 100 nM. Reactions were incubated at room temperature and read every 2 min for 120 min. Readings were performed on a Pherastar Plus (BMG Labtech). Excitation 487 nm; λ Emission 535 nm.USP30 Biochemical Fluorescence Intensity IC50 AssayDilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.001 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of Ubiquitin-Rhodamine 110 (U-555; Boston Biochem) at a final concentration of 100 nM. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 487 nm; 2 Emission 535 nm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 422.32, ALogP 3.68, HBA 4, HBD 1, PSA 63.57, QED 0.8
Compound (SMILES): COC(=O)[C@@H](C)Cn1c(=O)[nH]/c(=N\c2ccc(OC(C)C)c(Cl)c2)n(Cc2ccc(OC)cc2)c1=O
InChIKey: RNNNFOLPKYQXBX-INIZCTEOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 516.98, ALogP 2.88, HBA 9, HBD 1, PSA 116.91, QED 0.44
Compound (SMILES): COC(=O)CCc1cc2nc(Nc3ccc(OC(C)C)cc3)n([C@@H]3C[C@H](C)CC(C)(C)C3)c2cc1C
InChIKey: ROSVXSYEBMTPHP-GBXCKJPGSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μL and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μL of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/mL FMN, 2 μL/ml decanal/ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 491.68, ALogP 7.37, HBA 6, HBD 1, PSA 65.38, QED 0.33
Compound (SMILES): Cn1nccc1-c1cccc(C(=O)N[C@H](CCN2CC[C@@H](CCc3ccc4c(n3)NCCC4)C2)C(=O)O)c1
InChIKey: DJOICTNSUFQOEV-CJFMBICVSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 516.65, ALogP 3.37, HBA 7, HBD 3, PSA 112.38, QED 0.38