lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc2ncnc(Oc3cccc(NC(=O)Nc4cc(C(C)(C)F)on4)c3)c2cc1OCCCS(C)(=O)=O
InChIKey: LAOHEKBFEIOMCN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 573.6, ALogP 5.08, HBA 10, HBD 2, PSA 154.77, QED 0.23
Compound (SMILES): C=CC(=O)N1CCC(Nc2cnc3[nH]cc(C(=O)N[C@@H](C)CC)c3n2)CC1
InChIKey: OXIFJRNRMHPWEL-LBPRGKRZSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 370.46, ALogP 2.08, HBA 5, HBD 3, PSA 103.01, QED 0.68
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@@H](C)[C@@H](n3nc(C(C)(O)C(=O)O)c4ccccc43)C2)cc1
InChIKey: IDMAPSDMTPQWSO-FSHIIYSXSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki = 2505 nM (pChEMBL 5.599999904632568)
Descriptors: MW 473.55, ALogP 2.61, HBA 7, HBD 2, PSA 121.96, QED 0.54
Compound (SMILES): COc1cc2c(cc1-c1c(C)noc1C)[nH]c1ccnc(-c3cn(-c4ccccc4)nc3C)c12
InChIKey: KLKMODXQLGKDKQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25440
Assay: FP Competitive Binding Assay (No.350): Fluorescence Polarization (FP) competitive binding studies (see above) were carried out using the FAM labeled fluorescent probe Cpd. No. 350 to determine binding affinities of representative compounds to both BD1 and BD2 of BRD2, BRD3, and BRD4 proteins. Equilibrium dissociation constants (Id) values of Cpd. No. 350 to these six proteins were determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and proteins with increasing concentrations up to full saturation. Serial dilutions of testing protein were mixed with Cpd. No. 350 to a final volume of 200 μl in the assay buffer. In order to achieve large dynamic rages, particularly for BD1 bromodomains, 100 mM phosphate buffer (pH=6.5, 0.01% Triton X-100 (Sigma, 282103) being added right before assays) was used as the assay buffer. Final Cpd. No. 350 concentration was 1.5 nM for all proteins. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, round-bottom plates (Thermo Scientific, Waltham, Mass.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd values of Cpd. No. 350, which were calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), are 2.0, 2.2, 6.5, 0.6, 5.5, and 3.0 nM to BRD2 BD1 and 2, BRD3 BD1 and 2, and BDR4 BD1 and 2, respectively.
Measurement: Ki = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 449.51, ALogP 6.16, HBA 6, HBD 1, PSA 81.76, QED 0.35
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)COCCOCC3CCN(c4cn[nH]c(=O)c4C(F)(F)F)C3)CC2)nc1
InChIKey: DFPZIPIOHLFKOB-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6xHis and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and x is the compound concentration.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 535.53, ALogP 1.26, HBA 9, HBD 1, PSA 127.68, QED 0.47
Compound (SMILES): CC(C)OC(=O)N1CC2(CCN(c3ccc(-c4cc(-c5cnn(C)c5)cn5ncc(C#N)c45)cn3)CC2)C1
InChIKey: BJFCJIPBTHSSTB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type 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; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. 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 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 510.6, ALogP 4.12, HBA 9, HBD 0, PSA 104.58, QED 0.41
Compound (SMILES): COCCN1CC2CCCC(C1)C2(OC)c1ccnc(C(N)=O)c1
InChIKey: MBSYMYZEQJWFKU-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 333.43, ALogP 1.4, HBA 5, HBD 1, PSA 77.68, QED 0.85
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc4[nH]c(CN5CCOCC5)cc4c3N(c3cnn(C)c3)C2=O)c1F
InChIKey: VUCMYUCUMDTCFT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 539.54, ALogP 3.7, HBA 8, HBD 1, PSA 100.98, QED 0.4
Compound (SMILES): O=c1cc(C2CCNCC2)n2ncc(-c3nc(-c4ccc(F)s4)no3)c2[nH]1
InChIKey: VKJVSEXKIZLUER-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 386.41, ALogP 2.41, HBA 8, HBD 2, PSA 101.11, QED 0.56
Compound (SMILES): Cc1ccc2nc(N3CCC(N[C@H](C)C(C)C)CC3)c(-c3noc(C)n3)cc2c1
InChIKey: OFYZMMJWWIVRTC-MRXNPFEDSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 393.54, ALogP 4.5, HBA 6, HBD 1, PSA 67.08, QED 0.69
Compound (SMILES): CC(C)c1c(-c2ccccc2Cl)[nH]c2ncnn2c1=O
InChIKey: RHRALKFWGWQMPQ-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate-anti-H3K4Me1-2-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 3365 nM (pChEMBL 5.46999979019165)
Descriptors: MW 288.74, ALogP 2.86, HBA 4, HBD 1, PSA 63.05, QED 0.79
Compound (SMILES): CCOc1cc(Nc2nc(Nc3ccc(N4CCN(C)CC4)cc3)ncc2C)ccc1Cl
InChIKey: QTCDZETZWGLNQO-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alpha Screen Assay: AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay. The details are not described in this patent.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 452.99, ALogP 5.08, HBA 7, HBD 2, PSA 65.55, QED 0.52
Compound (SMILES): COc1cc(Nc2cc(CO)[nH]n2)nc(N(C)[C@@H]2C[C@H]3CCC[C@@H](C2)N3C(=O)C2CCC2)n1
InChIKey: NZKARKUMQHBGLV-BCDXTJNWSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK 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 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 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 455.56, ALogP 2.59, HBA 8, HBD 3, PSA 119.5, QED 0.58
Compound (SMILES): Cc1csc(C2CCN(C(C)c3ccc4c(c3)OCO4)CC2)n1
InChIKey: OHVXLGPSAHMXLV-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 330.45, ALogP 4.12, HBA 5, HBD 0, PSA 34.59, QED 0.85
Compound (SMILES): CC(C)(C)c1nc(N2CCC(O)C2)c2ncn(CC(=O)c3ccccc3Cl)c2n1
InChIKey: RKXUGSLDVUVSFJ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 101.5 nM (pChEMBL 6.989999771118164)
Descriptors: MW 413.91, ALogP 3.23, HBA 7, HBD 1, PSA 84.14, QED 0.66
Compound (SMILES): COCC(C)(C)C(=O)n1nc(-c2cccn(Cc3ccco3)c2=O)cc1NCc1ccc(Cl)s1
InChIKey: GQVVQCIUGAQKNX-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: KLKB1 Activity Assay: Human KLKB1 protein was obtained from Enzyme Research Labs. The chromogenic substrate S-2302 was obtained from DiaPharma. KLKB1 was assayed in buffer containing 0.05 M Tris (pH 7.4), 0.01 M NaCl and 0.2% w/v PEG-8000. The final concentration of enzyme used was 3 nM KLKB1. The final concentration of substrate used was 250 μM S-2302 for KLKB1. All assays were performed in 96-well microtiter plates at room temperature (RT). The enzyme and inhibitor were pre-incubated for 10 minutes then substrate was added and read at 405 nm in a SpectraMax Plus Spectrophotometer (Molecular Devices). Inhibitor IC50 values were determined by adding test compound as ten point, three-fold serial dilutions in buffer solution, as known in the art. The plate was read at 10 minutes after substrate addition.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 501.01, ALogP 4.99, HBA 9, HBD 1, PSA 91.29, QED 0.35
Compound (SMILES): CC(C)c1ccc2c(c1)Oc1cc(Cl)ccc1N=C2N1CCN(CC(C)(C)C(=O)O)CC1
InChIKey: HBMJSEBYZIMZIK-UHFFFAOYSA-N
Target: Histamine H1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35367
Assay: H1 and 5-HT2A Binding Assay: 1 μl of a compounds of the present disclosure and either reference compound (Table C) were transferred to assay plates. 1 μl of 0.2 mM Ketanserin or Pyrilamine was transferred to an assay plate according to the plate map for nonspecific binding (Low control: LC). 1 μl of DMSO was transferred to an assay plate according to plate map for total binding (High control: HC).100 μlof membrane stocks were dispensed into the plates, following the plate map. 100 of radio ligand was added. The plates were sealed and shaken at 300 rpm at room temperature for 1 hour. The Unifilter-96 GF/C filter plates were soaked with 50 of 0.3% PEI per well for at least 0.5 hour at room temperature.When the binding assays were complete, the reaction mixture was filtered through GF/C plates using Perkin Elmer Filtermate Harvester, and then each plate was washed 4 times with cold wash buffer. The filter plates were dried for 1 hour at 50 degrees. After drying, the bottom of the filter plate wells were sealed using Perkin Elmer Unifilter-96 backing seal tape. 50 μl of Perkin Elmer Microscint 20 cocktail was added. The top of the filter plates were sealed with Perkin Elmer TopSeal-A sealing film.
Measurement: Ki = 37.5 nM (pChEMBL 7.429999828338623)
Descriptors: MW 455.99, ALogP 5.38, HBA 5, HBD 1, PSA 65.37, QED 0.67
Compound (SMILES): C[C@@H]1CN(c2ccc(Nc3nccc(OCC4CCC(CO)CC4)n3)cc2)C[C@H](C)O1
InChIKey: GBPMNJYFAQXLCO-VCWRXUFXSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Enzymatic Assay: LRRKtide substrate (peptide sequence RLGRDKYKTLRQIRQ, derived from human ezrin [amino acids 561-573], moesin [amino acids 539-553] and radixin [amino acids 558-570], obtained from SignalChem, catalogue #L10-58, reconstituted in 20 mM Tris-HCl at pH 7.5 to a final concentration of 1 mg/mL, assay concentration 20 μM) and recombinant human LRRK2 (catalytic domain only [amino acids 970-2527], GST-tagged, expressed in insect cells, obtained from ThermoFisher Scientific, catalogue #PV4874, 0.35 mg/mL, assay concentration 30 nM) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM NaVO, 2 mM DTT, 1% DMSO). Compounds of interest (in DMSO, serial 3-fold dilution from 10 μM to 0.5 nM) or control (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [P]-ATP (Specific activity 10 μCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 426.56, ALogP 4.01, HBA 7, HBD 2, PSA 79.74, QED 0.69
Compound (SMILES): CNC(=O)c1nnc(N2CCN(CCOC)C2=O)cc1Nc1cccc(-c2ncn(C)n2)c1OC
InChIKey: KGMODVGXNCQUKB-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: DiscoverX's KINOMEscan™ is a popular platform for kinase profiling, frequently used in academic and industry witnessed by publications, therefore we selected this platform as a primary cell-free screening assay to determine the relative binding potency and guide chemistry SAR.Developed by DiscoverX, KINOMEscan™ employs proprietary active-site dependent competition binding assays to determine how compounds bind to kinases. KINOMEscan™ is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining TYK2 (JH2domain-pseudokinase), liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 111× stocks in 100% DMSO. All test compounds were shipped to DiscoverX in DMSO with concentration of 10 mM. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. Each compound was tested in duplicate. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions performed in polypropylene 384-well plate. Each was a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.The amount of kinase measured by qPCR (Signal; y-axis) is plotted against the corresponding compound concentration in nM in log 10 scale (x-axis). Binding constants (Kds) were calculated with a standard dose-response curve using the Hill equation:Response = Background + Signal - Background 1 + ( Kd Hill ⁢ ⁢ Slope / Dose Hill ⁢ ⁢ Slope ) The Hill Slope was set to −1.
Measurement: Kd < 1 nM (pChEMBL 9)
Descriptors: MW 481.52, ALogP 1.27, HBA 10, HBD 2, PSA 139.63, QED 0.46
Compound (SMILES): O=c1c(Cl)c(N2CCC(c3ccccc3)CC2)ccn1Cc1ccc(OC(F)(F)F)cc1
InChIKey: QMXOEWYJDSCILA-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]GTPγS Binding Assay: Measurement of mGluR2 positive allosteric modulatory activity of test compounds in membranes containing human mGluR2 was performed using frozen membranes that were thawed and briefly homogenized prior to pre-incubation in 96-well microplates (15 μg/assay well, 30 minutes, 30° C.) in assay buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl2, 50 μM GDP, 10 μg/ml saponin,) with increasing concentrations of positive allosteric modulator (from 0.3 nM to 50 μM) and either a minimal pre-determined concentration of glutamate (PAM assay), or no added glutamate. For the PAM assay, membranes were pre-incubated with glutamate at EC25 concentration, i.e. a concentration that gives 25% of the maximal response glutamate, and is in accordance to published data (Pin et al. (1999) Eur. J. Pharmacol. 375:277-294). After addition of [35S]GTPγS (0.1 nM, f.c.) to achieve a total reaction volume of 200 μl, microplates were shaken briefly and further incubated to allow [35S]GTPγS incorporation on activation (30 minutes, 30° C.). The reaction was stopped by rapid vacuum filtration over glass-fibre filter plates (Unifilter 96-well GF/B filter plates, Perkin-Elmer, Downers Grove, USA) microplate using a 96-well plate cell harvester (Filtermate, Perkin-Elmer, USA), and then by washing three times with 300 μl of ice-cold wash buffer (Na2PO4.2H2O 10 mM, NaH2PO4.H2O 10 mM, pH=7.4).
Measurement: EC50 = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 462.9, ALogP 5.83, HBA 4, HBD 0, PSA 34.47, QED 0.48
Compound (SMILES): COC1CCN(C(=O)N2CC(CC#N)(N3CCC(N[C@@H]4CC4c4ccccc4)CC3)C2)CC1
InChIKey: JDOVUIRQPWAANN-XMMISQBUSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay: LANCE LSD1/KDM1A demethylase assay 10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1×LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 451.62, ALogP 2.8, HBA 5, HBD 1, PSA 71.84, QED 0.72
Compound (SMILES): COC(=O)c1cccc(Oc2ccc(/N=c3\[nH]cc(C(F)(F)F)c(=O)n3Cc3ccc(Cl)cc3)cc2)n1
InChIKey: KPJWRINKPBJKEA-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% 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, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% 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, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 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, 150 nM ATP solution (25 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 4 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.
Measurement: IC50 = 683 nM (pChEMBL 6.170000076293945)
Descriptors: MW 530.89, ALogP 5.1, HBA 7, HBD 1, PSA 98.57, QED 0.35
Compound (SMILES): CCCC(CNC(=O)OC(C)(C)C)Nc1nc2ccccc2n2c(C)nnc12
InChIKey: REUJLJIGVWMBSK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 384.48, ALogP 3.69, HBA 7, HBD 2, PSA 93.44, QED 0.67
Compound (SMILES): CS(=O)(=O)c1cc(C2CC2)cn2cc(Cn3cc(C(=O)NCc4ncn5ccc(Cl)cc45)cn3)nc12
InChIKey: JXYCVCMSYBYHLY-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: 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 RT. After initiation with substrate, reactions were incubated for 10 min at RT 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 RT. After initiation with substrate, reactions were incubated for 18 h at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 < 1 nM (pChEMBL 9)
Descriptors: MW 524.01, ALogP 3.09, HBA 9, HBD 1, PSA 115.66, QED 0.35
Compound (SMILES): Cc1ccc(Nc2ncc(F)c(N[C@@H]3C[C@@H]4CCCN4C(C)(C)C3)n2)cc1-n1nnn(C2COC2)c1=O
InChIKey: RJRYGUYWFUAVBS-MSOLQXFVSA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 509.59, ALogP 2.8, HBA 11, HBD 2, PSA 115.02, QED 0.52
Compound (SMILES): Cc1nc2c(-c3nnc[nH]3)cc(N3CCOCC3)cc2n1-c1c(F)cnc2ccccc12
InChIKey: YUTDTGPYIGNWNN-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 μM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 429.46, ALogP 3.64, HBA 7, HBD 1, PSA 84.75, QED 0.47
Compound (SMILES): Fc1cc2nc(-c3cnnc(C(F)F)c3)n(C3CC3)c2cc1F
InChIKey: CHUIXOIVXUJWJC-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Metalloenzyme Activity: V79-4 cells expressing recombinant andrenodoxin and andrenodoxin reductase with either recombinant human CYP11B2 or CYP11B1 were prepared according to methods previously described (LaSala et al 2009 Anal Bioch 394:56-61). An enzyme enriched microsomal fraction was prepared from cellular lysates and subsequently used as the enzyme source for determining inhibitor IC50s. The substrate Km values were experimentally determined for 11-deoxycorticosterone (CYP11B2 substrate) and 11-deoxycortisol (CYP11B1 substrate). Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity. The percent activity in the presence of each inhibitor was calculated according to the following equation: % activity=(P−Pb)/(Pt−Pb), where P=the product formed in the presence of the inhibitor. The IC50 value was defined as the inhibitor concentration causing a 50% decrease in activity relative to the no inhibitor control reaction.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 322.27, ALogP 4.04, HBA 4, HBD 0, PSA 43.6, QED 0.68