lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCS(=O)(=O)c1ccc([C@H](CO)NC(=O)c2ccc3c(c2)cc(CN2CCC(C(F)(F)F)CC2)n3C2CC2)cc1
InChIKey: BCTFFEPLQXCYLW-SANMLTNESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgammat 5xRORE Assay in Jurkat Cells (Assay 2): Compounds described herein were tested for RORγ inverse agonist activity in a cell-based, transcriptional activity assay. Secreted Nanoluc® luciferase was used as a reporter for transcriptional activity of the full-length RORγt in Jurkat cells (ATCC, Cat. # TIB-152). A reporter plasmid was constructed by inserting 5 repeats of the ROR Response Element (RORE) AAAGTAGGTCA (SEQ ID NO: 1) into a commercially available promoterless plasmid pNL1.3[secNluc] (Promega, Cat. # N1021) using KpnI and HindIII restriction sites. The expression plasmid for RORγt was purchased (Geneocopoeia, Cat. # EX-T6988-M02). Jurkat cells (30 million cells) were transfected with 11 μg of EX-T6988-MO2 and 26 μg of the reporter plasmid in OptiMEM media using Lipofectamine LTX and Plus reagents (Life Technologies, Cat. #15338-100). After 5-6 hrs of incubation at 37° C./5% CO2, the cells were collected, resuspended in phenol-red free RPMI media containing 10% (v/v) delipidated FBS (Hyclone, Cat. # SH30855.03) and dispensed into 96-well clear bottom tissue culture plates (CoStar, Cat. #3603), at 80,000 cells per well. Tested compounds were added to the cells in the same media (final concentration of DMSO was 0.1% (v/v)), and the plates were incubated at 37° C./5% CO2 for 16-18 hrs. Luciferase activity in the conditioned supernatants was determined with NanoGlo assay reagents (Promega, Cat.# N1130). Percent inhibition values were calculated based on the fully inhibited and non-inhibited (DMSO) controls, and the values were regressed against concentrations of the tested compounds to derive IC50 values using a four-parameter non-linear fitting model.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 577.67, ALogP 5.01, HBA 6, HBD 2, PSA 91.64, QED 0.37
Compound (SMILES): CCOc1cc(-c2ccc(N3CCN(C(=O)C4(COC)CC4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: GLFGFUIBWFTYKT-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 1264 nM (pChEMBL 5.900000095367432)
Descriptors: MW 460.54, ALogP 2.74, HBA 8, HBD 0, PSA 95.99, QED 0.53
Compound (SMILES): CCc1ccccc1OCC(C)(C)C(=O)NC1CCN(C)CC1
InChIKey: PUFNINORMBTKHT-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: SSTR4 I-125 Somatostatin Competition Binding Assay: This membrane-based assay measures the ability of compounds to competitively inhibit binding of I-125 labeled somatostatin to SSTR4 in membranes from CHO-K1 that overexpress SSTR4. Membranes from CHO-K1 cells overexpressing SSTR4 are purchased from Perkin Elmer (catalog number ES-524-M400UA). Test compounds are suspended in DMSO and then diluted in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, 1 mM CaCl2), 0.5% BSA) plus 0.2 nM I-125 labeled somatostatin (Perkin Elmer catalog number NEX389). Fifty μL of compound/I-125 somatostatin in assay buffer are added per well to 96-well poly-propylene plate. Then 1 μg of SSTR4 membranes in 50 μL assay buffer are added per well. The Plate is incubated for 60 minutes at room temperature. FilterMat A filters (Perkin Elmer catalog number 1450-421) are pre-soaked in 0.5% PEI (Sigma catalog number P3143). The contents of the assay plate are transferred to filters with a TomTech harvester and washed 5 times with 20 mM HEPES, 100 mM NaCl. The filters are dried in a microwave oven then transferred to sample bag containing a scintillator sheet (Perkin Elmer catalog number 1450-441). The scintillator sheets are melted to filters using a heat block. The filters are then read in a MicroBeta scintillation counter. Binding Ki curves are generated using Activity Base for Screening Data Management and the results are reported as pIC50.
Measurement: IC50 = 79.4 nM (pChEMBL 7.099999904632568)
Descriptors: MW 318.46, ALogP 2.86, HBA 3, HBD 1, PSA 41.57, QED 0.88
Compound (SMILES): Cc1ncc2c(n1)C[C@@](C)(c1sc(-c3cc(F)cc(-c4nnco4)c3)cc1Cl)NC2=N
InChIKey: NWZNGWLSGIKSDF-NRFANRHFSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis aAssay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values.
Measurement: Ki = 24.4 nM (pChEMBL 7.610000133514404)
Descriptors: MW 454.92, ALogP 4.74, HBA 7, HBD 2, PSA 100.58, QED 0.46
Compound (SMILES): C[C@H](NC(=O)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2)[C@H](O)c1ccc(C#N)cc1
InChIKey: HMXBTXMYJLIBLR-BEVDRBHNSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 25.7 nM (pChEMBL 7.590000152587891)
Descriptors: MW 430.51, ALogP 2.67, HBA 6, HBD 3, PSA 117.93, QED 0.57
Compound (SMILES): CC(=O)N1CCC(c2nc(-c3ccc(Oc4cc(F)cc(OCc5cnc(C)nc5)c4)cc3)c3c(N)nccn23)CC1
InChIKey: ADNLTCJFYJOAKH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay: Method A: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore®. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinease detection reagents. Phosphorylation of the substrate peptide was detected by fluorescence polarization measured with a Tecan 500 instrument.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 567.63, ALogP 5.31, HBA 9, HBD 1, PSA 120.76, QED 0.28
Compound (SMILES): Nc1nc2c(ncn2[C@@H]2O[C@H]3COP(=O)(S)OCCn4c(nc5cncnc54)COP(=O)(S)O[C@@H]2C3)c(=O)[nH]1
InChIKey: PWKZWJMHQAGGSJ-RFZXERIHSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Binding Assay: A human STING [3H] 2′,3′-cGAMP scintillation proximity competition binding assay was utilized to detect binding of compounds to STING protein. The competition binding assay was conducted with either a wild-type (WT, SEQ ID NO: 1) or an AQ variant (SEQ ID NO: 2) of truncated human STING protein that lacks the N-terminal four transmembrane domains and contains amino acids 155-379. The protein encoding plasmid contained a HIS tag, followed by an Avi tag and then a SUMO tag, which were followed by the STING coding sequence within the pET-28a (+) vector. The plasmid was transfected into a BL21 (DE3)/BirA E. coli co-expression strain. Protein was purified from the bacterial lysate using a cobalt-affinity column. The purified protein was dialyzed against PBS and then 20% glycerol was added prior to storage at −80° C.The assay was conducted in a 384-well plate containing final concentration of 25 nM biotin-STING protein and 17.5 nM [3H] 2′,3′-cGAMP in assay buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1% Fatty Acid Free BSA). Test compounds (2.5 μL) were added to the plate first followed by a 25 μL mixture of STING protein, streptavidin PVT SPA beads (Perkin Elmer) and 10 μL [3H] 2′,3′-cGAMP. Plates were incubated at room temperature for 18 hrs and read on a Wallac MicroBeta TriLux (Perkin Elmer). 2′,2′-cGAMP was used as a reference compound and had an IC50 of 0.562 μM using the WT STING protein and 0.098 M using the AQ STING protein.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 617.5, ALogP 1.85, HBA 16, HBD 4, PSA 213.48, QED 0.18
Compound (SMILES): Cc1n[nH]c2c1C(c1cc(C#CCCCN)cc(C(F)(F)F)c1)(C(C)C)C(C#N)=C(N)O2
InChIKey: ABVQWOKKUNIONW-UHFFFAOYSA-N
Target: Serine hydroxymethyltransferase, cytosolic (SINGLE PROTEIN), Homo sapiens
UniProt: P34896
Assay: Enzymatic Assay: For the SHMT1 and SHMT2 in vitro enzymatic assays, the rate of 5,10-methylene tetrahydrofolate formation catalyzed by SHMT1/2 was indirectly evaluated by coupling with excess MTHFD2, which converts NAD+ to NADH allowing for reaction monitoring by absorption at 340 nm. The reaction was started by addition of serine (1 mM final) to either human SHMT1 or human SHMT2 (2 mcg/mL), and human MTHFD2 (25 mcg/mL) in a buffer of 50 mM potassium phosphate (pH 7.4), 0.3 mM tetrahydrofolate, 7.5 mM dithiothreitol, 1.25 mM NAD+, and 4% DMSO. Inhibition of initial reaction velocity was determined by adding various inhibitors at different concentrations and monitored as described. IC50 may be calculated based on this assay.For the MTHFD2 in vitro assay, the rate of NADH formation is directly monitored at 340 nm. The reaction is started by addition of 0.125 uM (final) of 5,10 methylene tetrahydrofolate to MTHFD2 (2.5 mcg/mL), 50 mM potassium phosphate (pH 7.4), 7.5 mM dithiothreitol, 1.25 mM NAD+, and 4% DMSO.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 443.47, ALogP 3.86, HBA 5, HBD 3, PSA 113.74, QED 0.49
Compound (SMILES): CCNc1cc2c(cn1)c(N1CC3COCC3C1)nn2C1CCC(Oc2nccc(C(F)(F)F)c2F)CC1
InChIKey: IZSRKHLAIQIARI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzyme Assay: MER: Compounds of Formula I were screened for their ability to inhibit MER kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human MER cytoplasmic domain (5 nM) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 534.56, ALogP 5.06, HBA 8, HBD 1, PSA 77.33, QED 0.44
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C(C)C)CC4)cc3c2C(C)C)cc(C)n1
InChIKey: WSBHXZZDFFPYSN-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 460.67, ALogP 6.02, HBA 3, HBD 1, PSA 52.23, QED 0.49
Compound (SMILES): O=C(O)c1ccc(-c2nn(C(=O)c3c(Cl)cccc3C(F)(F)F)c3cc(-c4ccncn4)ccc23)cc1
InChIKey: RSRHBRNRWCKXDN-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.
Measurement: IC50 = 1441 nM (pChEMBL 5.840000152587891)
Descriptors: MW 522.87, ALogP 6.22, HBA 6, HBD 1, PSA 97.97, QED 0.3
Compound (SMILES): CC(C)(C)c1nc(N2CCN(CCCCNC(=O)N3CC(c4ccncc4)C3)CC2)cc(C(F)(F)F)n1
InChIKey: MRMFTAYATXSTSO-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Assay: The affinities of the target compounds were determined using radioligand binding experiments. All radioligand binding experiments were performed according to previously described methods (Luedtke R. R. et al. Synapse 2000, 38, 438-449; Chu, W. et al. Bioorg. Med. Chem. 2005, 13, 77-87; Taylor, M. et al. Synapse 2010, 64, 251-266). Stably transfected HEK cells expressing the human D2-long and the D3 dopamine receptor were developed using the pIRESneo2 bicistronic expression vector (Clontech, Palo Alto, Calif.). Levels of expression of human D2 and D3 dopamine receptors in HEK cells were 57,941±14,686 fmol/mg protein and 4202±1516 fmol/mg protein, respectively. Competition curves were performed using 125I-IABN. Membrane homogenates (50 μL) were suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5 and incubated with 50 μL of 125I-IABN at 37° C. for 60 min. Nonspecific binding was defined using 4 μM (+)-butaclamol. The radioligand concentration used was approximately equal to the Kd value, and the concentration of the competitive inhibitor ranged over five orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.4) and filtration over a glass-fiber filter (Schleicher and Schuell No. 32). Filters were washed with 10 mL of cold buffer, and the radioactivity was quantitated. A Packard Cobra gamma counter was used for 125I-labeled radioligands (efficiency=75%). The protein concentration was determined using a BCA reagent (Pierce) with bovine serum albumin as the protein standard. Data from competitive inhibition experiments was modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC50 value). Competition curves were modeled for a single site. Data from competition dose-response curves were analyzed using Tablecurve program (Jandel). IC50 values were converted to equilibrium dissociation constants (Ki values) using the Cheng and Prusoff correction.
Measurement: Ki = 605 nM (pChEMBL 6.21999979019165)
Descriptors: MW 519.62, ALogP 3.9, HBA 6, HBD 1, PSA 77.49, QED 0.56
Compound (SMILES): Nc1nc(Cl)nc2c1ncn2[C@@H]1O[C@H](COC(Cc2ccc(OCC(F)(F)F)cc2)(C(=O)O)C(=O)O)[C@@H](O)[C@@H]1F
InChIKey: HMLQFVKOTUYCKD-DMVWFOPNSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: In Vitro Enzyme Assay: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.5 nM and 50 μM, respectively. Reactions were allowed to proceed for 30 minutes at room temperature before the addition of 100 μL Malachite Green. After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer. The concentration of inorganic phosphate was determined using a phosphate standard curve.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 593.87, ALogP 1.77, HBA 11, HBD 4, PSA 192.14, QED 0.15
Compound (SMILES): C=CC(=O)N1CCC(COc2cc(-c3ccc(O)cc3)cn3cncc23)CC1
InChIKey: UEFJDEPPPRWNIO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK and Tyk2 Kinase Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 377.44, ALogP 3.51, HBA 5, HBD 1, PSA 67.07, QED 0.69
Compound (SMILES): COc1cc(C(=O)N2CCC[C@H]2c2nc(-c3cccc(F)c3C)no2)c(-n2nccn2)cc1C
InChIKey: SLPBBNNNUASMRF-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists 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 in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 462.49, ALogP 4.06, HBA 8, HBD 0, PSA 99.17, QED 0.44
Compound (SMILES): O=C(NO)c1ccc(CCCn2cc(CNC3CC3c3ccccc3)nn2)cc1
InChIKey: HJSROIWRJOQVCL-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Histone Deacetylase Assay : Histone deacetylase assay was done as per manufacturer's instructions. Briefly, assay buffer, 200 uM HDAC substrate (fluorogenic HDAC acetylated peptide substrate for class I HDACs (HDACs 1, 2, and 3) and class 2b HDACs (HDACs 6 and 10) and 1% BSA are taken as a master mix and aliquoted as 40 ul per well. Compounds (10×) were diluted in assay buffer and were added to respective wells of a black 96 well plate. HDAC6 human recombinant enzyme was thawed on ice and 5 μl (7 ng/ul) enzyme was added per well. The plate was incubated at 37° C. for 1 hour. Developer solution was then added (50 μl per well) and incubated at room temperature for 10 minutes. Fluorescence was measured at an excitation wave length of 350-380 nm and emission wavelength of 440-480 nm.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 391.48, ALogP 2.68, HBA 6, HBD 3, PSA 92.07, QED 0.39
Compound (SMILES): CN1CC(F)(COc2cc(-c3ccc(N4CCC(C)(NC(=O)c5ccccn5)CC4)nc3)c3c(C#N)cnn3c2)C1
InChIKey: MBMSXQYXGVYKCY-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 554.63, ALogP 3.48, HBA 9, HBD 1, PSA 111.68, QED 0.37
Compound (SMILES): CN/C=C(\C=N)c1ccc2c(c1)[C@@H](C)CCN2c1cccc2c1N[C@H](C)CC(=O)N2
InChIKey: AWKZKSSSAAVVMA-BVDQGFEBSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 2980 nM (pChEMBL 5.53000020980835)
Descriptors: MW 403.53, ALogP 4.68, HBA 5, HBD 4, PSA 80.25, QED 0.56
Compound (SMILES): Cn1cc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4ccc(OCC(F)F)nc4CC3)CC2)cn1
InChIKey: VXTINWNGKOZZIK-XGAFWQRZSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Inhibition for Dopamine D2 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.06HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 1.2 nM [H]-Methylspiperone ([HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer) Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.Kd: 0.272 nM(Preparation of Solutions of Non-Specific Ligand or the Compounds of the Present Invention)Butaclamol or the compounds of the present invention were weighed and DMSO was added to make a 10 mM solution. This solution was diluted to each concentration.(Preparation of Radioligand Solution),[H]-Methylspiperone was weighed and the buffer solution was added to make a 3.6 nM solution.(Preparation of SPA Beads Solution) SPA beads were weighed and stirred in water to make a 50 mg/mL solution. Using this solution, a mixture with the cell membranes was prepared.
Measurement: Ki = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 475.58, ALogP 3.17, HBA 6, HBD 1, PSA 72.28, QED 0.6
Compound (SMILES): CC(C)(C)c1nc(C(=O)Nc2cccc(-c3ccnc4[nH]c(-c5ccc(C(=O)N6CCOCC6)cc5)nc34)c2CO)no1
InChIKey: BBJONBDESPRKIZ-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 C481S Enzyme: The protocol below describes a microfluidic, off-chip mobility shift kinase assay to measure inherent potency of compounds against BTK C481S enzyme. The mechanics of the assay platform are described by the vendor (PerkinElmer, Hopkinton, Mass.) on their website at the following URL: http://caliperls.com.Briefly, 2.5× stocks of His-TEV-hsBTK(328-659)(C481S) from the Merck Serono Protein Purification Laboratory in Darmstadt, Germany (PCS, Q27/234), 1.6×ATP and appropriate KinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; Tufts University Core Facility custom synthesis) 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 (#4315) 384-well, sterile, flat-bottom 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 25° C. Following the incubation period, reactions were terminated by adding 65 uL quench buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (PerkinElmer, Mountain View, Calif.), 5% DMSO. Terminated reactions were monitored at −2 PSI, −3000 V/−700 Volts in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was quantified by laser-induced fluorescence measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 581.63, ALogP 4.19, HBA 9, HBD 3, PSA 159.36, QED 0.27
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3ccc(C(=O)NCC)s3)nc3ccccc31)C2
InChIKey: DPIFGUKPQIBTRZ-ODAJSGAPSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 0.93 nM (pChEMBL 9.029999732971191)
Descriptors: MW 477.59, ALogP 3.84, HBA 6, HBD 2, PSA 96.33, QED 0.53
Compound (SMILES): COc1cccc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(CNC(C)=O)c4OC)c4ccccc34)ccn2)c1
InChIKey: JSUAVXOHYRYOSZ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 633.75, ALogP 8.37, HBA 7, HBD 4, PSA 122.84, QED 0.12
Compound (SMILES): Cc1cnn(Cc2ccc(Cn3cnc(C(=O)NCc4ccc(CN)cc4C)c3)cc2)c1
InChIKey: HNTAKOBIRREIDW-UHFFFAOYSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: KLK1 assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 428.54, ALogP 3.18, HBA 6, HBD 2, PSA 90.76, QED 0.45
Compound (SMILES): CCOc1ccccc1-c1nc([C@@H]2CCCN2C(=O)c2cc(C)ccc2-n2nccn2)no1
InChIKey: MAOUWQSDIAWQEC-FQEVSTJZSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists 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 in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 444.5, ALogP 4, HBA 8, HBD 0, PSA 99.17, QED 0.44
Compound (SMILES): O=C(O)[C@H](CCN(CCCCc1ccc2c(n1)NCCC2)CC(F)F)Nc1ccnc(C(F)(F)F)n1
InChIKey: DKRIZDMOEGJABV-KRWDZBQOSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-2): A third series of exemplary compounds was selected for testing in the solid phase integrin αvβ6 binding assay. The compounds tested were compound samples prepared according to procedures described in the Synthetic Examples section, with the stereochemical purity as indicated in the Examples. As in Example B1, microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/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 μg/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 > 1000 nM (pChEMBL 6)
Descriptors: MW 516.52, ALogP 4.09, HBA 7, HBD 3, PSA 103.27, QED 0.27
Compound (SMILES): CC(C)c1cnc(OCc2cn(-c3ccc(F)c(C(F)(F)F)c3)nn2)nc1
InChIKey: WBKOUVTZNBJQEP-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 381.33, ALogP 3.92, HBA 6, HBD 0, PSA 65.72, QED 0.63
Compound (SMILES): NC1CCC(NC2CC23CCc2ccccc23)CC1
InChIKey: WEVDFRZQHZZSBI-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (50 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 50 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 93.8 nM (pChEMBL 7.03000020980835)
Descriptors: MW 256.39, ALogP 2.5, HBA 2, HBD 2, PSA 38.05, QED 0.85
Compound (SMILES): Cc1cccc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CN(C)CCN3)c2)c1
InChIKey: SQTIPABERDHCSI-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 454.96, ALogP 1.92, HBA 5, HBD 3, PSA 90.54, QED 0.58
Compound (SMILES): CC[C@@H](N)c1nc(N(C)C(C)C)cc2c1CN(c1cccc(-c3nnc4n3[C@@H](C)CC4)n1)C2=O
InChIKey: WDXXELAFEZBSNE-MAUKXSAKSA-N
Target: Serine/threonine-protein kinase D2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BZL6
Assay: Biochemical Selectivity Kinase Assay: PRKD2 (PKD2) and RPS6KA2 (RSK3) kinase assays were performed with Km-levels of ATP at the Thermo Fisher Scientific, Inc. (Madison, Wis.), using their Invitrogen SelectScreen fluorescence resonance energy transfer (FRET) Z′-LYTE technology, based on the differential sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage. Human recombinant full-length GST-tagged PRKD2 (PKD2) and RPS6KA2 (RSK3) were produced by Thermo Fisher Scientific, Inc. (Madison, Wis.). The kinase assays were conducted in 10-μL reactions. PRKD2 (PKD2) reactions contained 0.64-5.84 ng enzyme, 25 μM ATP (Km), 2 μM of Ser/Thr 17 (Z′-LYTE peptide substrate), 10 mM MgCl2, 0.01% BRIJ-35, 1 mM EGTA in 50 mM HEPES, pH 7.5. RPS6KA2 (RSK3) reactions were conducted similarly, and contained 0.5-9 ng enzyme, or 10 μM ATP (Km) and 2 μM Ser/Thr 06 peptide. After the 1-hour kinase reaction incubation, 5 μL of a 1:256 and 1:4096 dilution of Z′-LYTE Development Reagent A was added to the PRKD2 (PKD2) and RPS6KA2 (RSK3) reactions, respectively. The extent of kinase reactions, resulting in a change of FRET signal of the peptide substrate, was measured, and inhibition for each kinase was measured with respect to DMSO control and reported as an average of duplicate measurements.
Measurement: IC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 460.59, ALogP 3.66, HBA 8, HBD 1, PSA 106.06, QED 0.6