lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN1CCN(c2ccc(Nc3nc(-c4ccsc4)nc4cn[nH]c(=O)c34)cc2)CC1
InChIKey: OLCKMCZRMYBMCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition of Enzymatic Syk Kinase Activity: 2.5 μL of 10% DMSO or compound in 10% DMSO was added to the wells in a 96 well V-bottom plate. Optimized concentration of in-house Syk enzyme (different batches of Syk (356-635) kinase domain) were used at optimized concentrations) ranging from 0.035 ng to 7.5 ng/reaction diluted in assay buffer was added to a total volume of 12.5 μL). Compound and protein were incubated for 30 minutes at room temperature on a plate shaker. Ten μL of a substrate mix containing 100 μM ATP (0.25 μL), γ-P32-ATP (0.1 μL; 10 μCi/μL), pG4T (0.25 μL; 10 mg/mL) and 1× assay buffer (9.4 μL) was added to all the wells. Samples were incubated at 30° C. for 10 minutes after mixing. The reaction was stopped by the addition of 8N HCl (13 μL) containing 100 mM ATP. Thirty μL of sample was transferred to the center of a 2×2 cm2 Whatman P81 chromatography paper. After allowing the sample to dry for one minute, the assay squares were washed 3 times for 5 minutes each in ortho-phosphoric acid (0.5%) and once in acetone. Assay squares were dried for 15 minutes in a 30° C. oven and transferred to 96 well optiplate. Microscint-O reagent (100 μL, Perkin Elmer) was added to each well, the plate was sealed with Topseal-A microplates and incubated for 10 minutes at room temperature at very low speed on rocker and the plate was read in the Topcount NXL instrument.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 433.54, ALogP 3.33, HBA 8, HBD 2, PSA 90.04, QED 0.5
Compound (SMILES): Nc1cccc(C[S+]([O-])c2ccc(Cl)cc2NS(=O)(=O)c2ccc(Cl)cc2F)c1
InChIKey: FFCRIZFJMNJSST-UHFFFAOYSA-N
Target: C-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41597
Assay: Biological Assay: HEK-Gqi5 cells stably expressing CCR2 were cultured in (DMEM high glucose, 10% FBS, 1% PSA, 400 μg/ml geneticin and 50 μg/ml hygromycin. Appropriate positive control chemokines (MCP-1, MIP1A or RANTES) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MuItiPROBE robotic liquid handling systems.
Measurement: IC50 = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 473.38, ALogP 4.82, HBA 4, HBD 2, PSA 95.25, QED 0.4
Compound (SMILES): O=C(Cc1ccc(O)c(OC(F)(F)F)c1)Nc1ccc(CCCCc2nnc(NC(=O)Cc3ccccn3)s2)nn1
InChIKey: DHPBNOLWZCELSM-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Recombinant Enzyme Assa: Compounds were assessed for their ability to inhibit the enzymatic activity of a recombinant form of Glutaminase 1 (GAC) using a biochemical assay that couples the production of glutamate (liberated by GAC) to glutamate dehydrogenase (GDH) and measuring the change in absorbance for the reduction of NAD+ to NADH. Substrate solution was prepared (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K2HPO4, 0.1 mg/ml BSA, 1 mM DTT, 20 mM L-glutamine, 2 mM NAD+, and 10 ppm antifoam) and 50 μL added to a 96-well half area clear plate (Corning #3695). Compound (2 μL) was added to give a final DMSO concentration of 2% at 2× the desired concentration of compound. Enzymatic reaction was started with the addition of 50 μL of enzyme solution (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K2HPO4, 0.1 mg/ml BSA, 1 mM DTT, 10 ppm antifoam, 4 units/ml GDH, 4 mM adenosine diphosphate, and 4 nM GAC) and read in a Molecular Devices M5 plate reader at 20° C. The plate reader was configured to read absorbance (λ=340 nm) in kinetic mode for 15 minutes. Data was recorded as milli-absorbance units per minute and slopes were compared to a control compound and a DMSO-only control on the same plate. Compounds with slopes less than the DMSO control were considered inhibitors and plate variability was assessed using the control compound.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 587.58, ALogP 4.26, HBA 10, HBD 3, PSA 152.11, QED 0.21
Compound (SMILES): COc1ccc(OC[C@@H](C)NCCCF)cc1[C@@H]1c2[nH]c3ccccc3c2C[C@@H](C)N1CC(F)(F)CO
InChIKey: DLSUONXFKGICCB-DRGJWEBESA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: ERalpha Binding Assay: The ability of compounds to bind to isolated Estrogen Receptor Alpha Ligand binding domain (ER alphaLBD (GST)) was assessed in competition assays using a LanthaScreen Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) detection end-point. For the LanthaScreen TR-FRET endpoint, a suitable fluorophore (Fluormone ES2, ThermoFisher, Product code P2645) and recombinant human Estrogen Receptor alpha ligand binding domain, residues 307-554 (expressed and purified in-house) were used to measure compound binding. The assay principle is that ER alpha-LBD (GST) is added to a fluorescent ligand to form a receptor/fluorophore complex. A terbium-labelled anti-GST antibody (Product code PV3551) is used to indirectly label the receptor by binding to its GST tag, and competitive binding is detected by a test compound's ability to displace the fluorescent ligand, resulting in a loss of TR-FRET signal between the Tb-anti-GST antibody and the tracer. The assay was performed as follows with all reagent additions carried out using the Beckman Coulter BioRAPTR FRD microfluidic workstation:1. Acoustic dispense 120 nL of the test compound into a black low volume 384 well assay plates.2. Prepare 1× ER alpha-LBD/Tb-antiGST Ab in ES2 screening buffer and incubate for 15 minutes.3. Dispense 6 μL of the 1× AR-LBD/Tb-anti-GST Ab reagent into each well of the assay plate followed by 6 μL of Fluorophore reagent into each well of the assay plate4. Cover the assay plate to protect the reagents from light and evaporation, and incubate at room temperature for 4 hours.5. Excite at 337 nm and measure the fluorescent emission signal of each well at 490 nm and 520 nm using the BMG PheraSTAR.Compounds were dosed directly from a compound source microplate containing serially diluted compound (4 wells containing 10 mM, 0.1 mM, 1 μM and 10 nM final compound respectively) to an assay microplate using the Labcyte Echo 550. The Echo 550 is a liquid handler that uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions and the system can be programmed to transfer multiple small nL volumes of compound from the different source plate wells to give the desired serial dilution of compound in the assay which is then back-filled to normalise the DMSO concentration across the dilution range.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 519.61, ALogP 4.86, HBA 5, HBD 3, PSA 69.75, QED 0.3
Compound (SMILES): CCN(CC)C1CCN(S(=O)(=O)N2[C@@H]3CC[C@H]2C[C@@H](NC(=O)c2cc(C4CC4)on2)C3)CC1
InChIKey: QUHPORBEVXETCI-FNLKRUPLSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient 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 = 12.7 nM (pChEMBL 7.900000095367432)
Descriptors: MW 479.65, ALogP 2.33, HBA 6, HBD 1, PSA 98.99, QED 0.61
Compound (SMILES): CC(C)(O)[C@]1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(O[C@H]3CC[C@H](O)CC3)cc2O1
InChIKey: KRFTVOULWMYDOE-TZPNTHKFSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 466.54, ALogP 3.13, HBA 8, HBD 3, PSA 118.21, QED 0.53
Compound (SMILES): Cc1[nH]c(-c2cc(N)ccc2Oc2ccccc2)c2c1C(=O)CC(C)C2
InChIKey: KYAWXAUITZHJRN-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM EDTA, 0.01% Triton X-100, 1 mM DTT) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM−0.85 nM compound, 8 nM bromodomain, 1 nM antibody and 100 or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).TR-FRET data were normalized to the means of 24 no-compound controls (high) and 8 controls containing 1 μM un-labeled probe (low). Percent inhibition was plotted as a function of compound concentration and the data were fit with the 4 parameter logistic equation to obtain IC50s Inhibition constants (Ki) were calculated from the IC50s, probe Kd and probe concentration. Typical Z′ values were between 0.65 and 0.75. The minimum significant ratio was determined to evaluate assay reproducibility (Eastwood et al., (2006) J Biomol Screen, 11: 253-261).
Measurement: Ki = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 346.43, ALogP 5.13, HBA 3, HBD 2, PSA 68.11, QED 0.65
Compound (SMILES): C[C@@H]1CC[C@@H](c2noc(C(C)(C)O)n2)CN1C(=O)c1ccccc1-n1ncnn1
InChIKey: FNYZXCNSTQAKRI-CHWSQXEVSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR assay: The utility of the compounds in accordance with the present invention as orexin receptor OX1R and/or OX2R antagonists may be readily determined without undue experimentation by methodology well known in the art, including the FLIPR Ca2+ Flux Assay (Okumura et al., Biochem. Biophys. Res. Comm. 280:976-981, 2001). In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 μM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 184 nM (pChEMBL 6.739999771118164)
Descriptors: MW 397.44, ALogP 1.68, HBA 9, HBD 1, PSA 123.06, QED 0.71
Compound (SMILES): N#Cc1ccc(CN2CCC(NC(=O)c3ccc(C(=O)N4CC(Oc5ccc(C#N)cc5)C4)cn3)CC2)cc1
InChIKey: WKWOWUPNJQOYOV-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 520.59, ALogP 3.12, HBA 7, HBD 1, PSA 122.35, QED 0.51
Compound (SMILES): C=C1C[C@@H](c2nc3c(OC)ccc(OC)c3[nH]2)N(C(=O)c2cc(C)ccc2-n2nccn2)C1
InChIKey: ZPMYPIMEGJXJAF-SFHVURJKSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 767 nM (pChEMBL 6.119999885559082)
Descriptors: MW 444.5, ALogP 3.61, HBA 7, HBD 1, PSA 98.16, QED 0.47
Compound (SMILES): O=C(Nc1cnn(Cc2cccc(F)c2)c1)c1cc(C2=CCCCC2)on1
InChIKey: GQRXCEDYPSPQRL-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 366.4, ALogP 4.27, HBA 5, HBD 1, PSA 72.95, QED 0.73
Compound (SMILES): Cc1nn(C2(C#N)CC2)cc1Nc1ncc(C(F)(F)F)c(C2CC2)n1
InChIKey: KRNSNIACPFSXHK-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 > 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 348.33, ALogP 3.63, HBA 6, HBD 1, PSA 79.42, QED 0.91
Compound (SMILES): CCCn1ncc2c(=O)n(Cc3ccc(OC)cc3)c(C)cc21
InChIKey: GMPCYHZOARRAOF-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 311.38, ALogP 2.97, HBA 5, HBD 0, PSA 49.05, QED 0.73
Compound (SMILES): CN/C=C(\C=N)c1ccc2c(c1)[C@H](C)CCN2c1cccc2c1N[C@H](C)CC(=O)N2
InChIKey: AWKZKSSSAAVVMA-HYSYFVHKSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 403.53, ALogP 4.68, HBA 5, HBD 4, PSA 80.25, QED 0.56
Compound (SMILES): CCCOc1c(OC)ccc(-c2cncc(C3COB(O)C3)c2)c1F
InChIKey: MANABQSIOLMRPG-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Cytokine Assays: Cytokine inhibitory activity is determined by measuring the effect of test agent on the release of the cytokines IL-4, IL-13 and IFNγ from human peripheral blood mononuclear cells (PBMCs) stimulated with the T-cell mitogen phytohemagglutinin-L (PHA-L). At the time of assay, human PBMCs (Astarte Biologics) are removed from cryopreservation, thawed quickly at 37° C., diluted in assay medium (RPMI-Gibco 1640 medium, 10% heat inactivated fetal bovine serum (HIFBS), 2 mM glutamine and 10 mM HEPES pH 7.4) and then centrifuged at 250×g for 5 minutes. The resulting cell pellet is re-suspended in assay medium to a concentration of approximately 5×106 cells/mL and 50 μL of this cell suspension (approximately 250,000 cells) is added to each well of a 384-well cell culture microtiter plate (Perkin Elmer). Varying concentrations of test compound are then diluted in assay medium and added to the assay plate wells in a volume of 25 μL. After −10 minutes at room temperature (21° C.) the cells are stimulated by the addition of 25 μL of PHA-L (14 μg/mL; Millipore) and the assay plate is placed in an incubator at 37° C. in a humidified environment in 5% carbon dioxide. The final assay conditions are approximately 250,000 human PBMCs per well in assay medium containing 3.5 μg/mL PHA-L and the indicated final concentration of test compound (approximately 10 μM to 9.5 μM by 4-fold dilution). The final concentration of DMSO in the assay is approximately 0.25%. After 48 hours, the assay plate is removed from the incubator and centrifuged at 250×g for 5 minutes. A portion of the resulting cell supernatant is then used to determine the amount of IL-4, IL-13 and IFNγ in each well. Cytokine measurements are made using human IL-4, IL-13 or IFNγ HTRF assay kits (CisBio) following the manufacturer's assay protocol. The concentrations and resulting effect values for tested compounds are plotted and the concentration of compound required for 50% effect (IC50) is determined with a four-parameter logistic dose response equation (E-WorkBook, ID Business Solutions Ltd.).
Measurement: IC50 = 2.36 nM (pChEMBL 8.630000114440918)
Descriptors: MW 345.18
Compound (SMILES): Oc1ccc2c(c1)[C@@]13CCCC[C@H]1[C@@H](C2)N(CC1COC1)CC3
InChIKey: RTGBFYXGEAQCNA-DFQSSKMNSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: Affinities of various compounds were measured in vitro using competitive radioligand binding assays. Serial dilutions of test compounds were incubated with membranes prepared from CHO-K1 cells expressing the mu opioid receptor (MOR; for opioid receptor binding) or rat forebrain membranes (for NMDA receptor binding). 2 nM [3H] Naloxone (MOR) or 0.2 nM [3H] MK801 (NMDA) were used as the specific, competitive radioligands. 10 uM Naloxone (MOR), or 10 uM MK801 (NMDA) was used to determine non-specific binding. Bound radioactivity was measured using a scintillation counter & IC50 values for test compounds were determined by non-linear regression analysis using a one-site competition model (Graph Pad Prizm). Due to each test compound being solubilized in a buffered system for the assay, the results presented herein reflect the free base activity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 313.44, ALogP 3.1, HBA 3, HBD 1, PSA 32.7, QED 0.91
Compound (SMILES): COc1ncc(F)c(-c2cnn(C)c2C(=O)Nc2cc3nc(-c4ccccc4)nn3cc2F)n1
InChIKey: VZTASVSZGOSENP-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 462.42, ALogP 3.13, HBA 9, HBD 1, PSA 112.12, QED 0.43
Compound (SMILES): OCC1O[C@@H](S[C@@H]2O[C@H](CO)C(O)C(n3cc(-c4cccc(F)c4)nn3)[C@H]2O)C(O)[C@@H](n2cc(-c3cccc(F)c3)nn2)[C@H]1O
InChIKey: YGIDGBAHDZEYMT-YFJDYNSLSA-N
Target: Galectin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09382
Assay: Evaluation of Kd Values: The affinity of compounds S8a-c, S11, S16, S20a-b and S21 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe as described Sörme, P., Kahl-Knutsson, B., Huflejt, M., Nilsson, U. J., and Leffler H. (2004) Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions. Anal. Biochem. 334: 36-47, (Sörme et al., 2004) and Monovalent interactions of Galectin-1 By Salomonsson, Emma; Larumbe, Amaia; Tejler, Johan; Tullberg, Erik; Rydberg, Hanna; Sundin, Anders; Khabut, Areej; Frejd, Torbjorn; Lobsanov, Yuri D.; Rini, James M.; et al, From Biochemistry (2010), 49(44), 9518-9532, (Salomonsson et al., 2010). The assay was adapted to be able to measure the high affinity of the present compound for galectin-3 by using a probe (SY) constructed to have high affinity for galectin-3 based on the structure of 3,3′-Dideoxy-3,3′-di-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]-1,1′-sulfanediyl-di-β-D-galactopyranoside, which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 648.65, ALogP -0.12, HBA 15, HBD 6, PSA 201.26, QED 0.15
Compound (SMILES): CC(C)Sc1ncccc1-c1cc(F)c(N2CCC(CC(=O)O)CC2)c(F)c1
InChIKey: XLZZKXADYBQGQZ-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 406.5, ALogP 5.22, HBA 4, HBD 1, PSA 53.43, QED 0.67
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4cnn(S(=O)(=O)C5CC5)c4)c4c(N)ncnn34)c2)C(=O)C1(C)C
InChIKey: IQOIZYHLEKJRIR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 548.63, ALogP 2.16, HBA 10, HBD 1, PSA 148.79, QED 0.4
Compound (SMILES): Cc1c(C(=O)Nc2ccc3[nH]nc(-c4ccoc4)c3c2)nn(C)c1C#N
InChIKey: CCKOQCMZDHPZJW-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 346.35, ALogP 2.99, HBA 6, HBD 2, PSA 112.53, QED 0.59
Compound (SMILES): O=C(NCc1nc2cnccc2[nH]1)c1ccc2nc(-c3ccc(F)cc3)c(-c3ccc(F)cc3)nc2c1
InChIKey: BSHKDAXSTWMKNH-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 492.49, ALogP 5.44, HBA 5, HBD 2, PSA 96.45, QED 0.33
Compound (SMILES): CO[C@H]1c2c(S(C)(=O)=O)ccc(Oc3cc(F)cc(C#N)c3)c2CC1(F)F
InChIKey: PVCCPQLOECPRHD-KRWDZBQOSA-N
Target: Endothelial PAS domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99814
Assay: Scintillation Proximity Assay (SPA): HIF-2a: The total assay volume was about 100 uL in the following configuration: 2 uL compound in 100% DMSO, 88 uL buffer with protein and probe and 10 uL of SPA beads. The compound was diluted in a master plate consisting of a 10-point dose response with a 3-fold compound dilution from 100 nM to 5 nM. Assays were run on a 96-well plate in which one column, designated as the high signal control, contained DMSO with no compound and another column, designated as the low signal control, contained no protein. Prior to plating out of compound, a buffer solution, consisting of 25 mM TRIS pH 7.5 (Sigma), 150 mM NaCl (Sigma), 15% Glycerol (Sigma), 0.15% BSA (Sigma), 0.001% Tween-20 (Sigma), 150 nM Compound 183 and 100 nM HIF-2Î± HIS TAG-PASB Domain, was made and allowed to equilibrate for 30 minutes. Compounds that were to be tested were then plated in to a 96-well white clear bottom Isoplate-96 SPA plate (Perkin Elmer). To the compounds, 88 uL of the buffer solution was then added, the plate was covered with a plastic cover and then aluminum foil, placed onto a shaker and equilibrated for 1 hour. After equilibration, 10 uL of a 2 mg/mL solution of YSi Cu His tagged SPA beads (Perkin Elmer) were then added to each well of the plate, covered and equilibrated for another 2 hours. The plates were then removed from the shaker, placed into a 1450 LSC and luminescence counter MicroBeta Trilux (Perkin Elmer) to measure the extent of probe displacement.
Measurement: IC50 = 17000 nM (pChEMBL 4.769999980926514)
Descriptors: MW 397.37, ALogP 3.77, HBA 5, HBD 0, PSA 76.39, QED 0.79
Compound (SMILES): COc1cc(-c2[nH]c3cc(C)c(C4CCC(N(C)CCC#N)CC4)nc3c2C(C)C)cn2ncnc12
InChIKey: OCQIAZUKOBKAIB-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 5888 nM (pChEMBL 5.230000019073486)
Descriptors: MW 485.64, ALogP 5.58, HBA 7, HBD 1, PSA 95.13, QED 0.37
Compound (SMILES): COc1cc(OCc2cccc(NC(=O)c3c(F)cccc3C(F)(F)F)c2)c2cc(-c3cn4nc(OC)sc4n3)oc2c1
InChIKey: YCJMSULZIUHTCU-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: FLIPR Assay in PAR4-Expressing HEK293 Cells: FLIPR-based calcium mobilization assay in HEK293 cells was used to measure PAR4 antagonism, agonism, and selectivity against PAR1. The activity of the PAR4 antagonists of the present invention were tested in PAR4 expressing cells by monitoring H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2-induced intracellular calcium mobilization. Counter screens for agonist activity and PAR1 antagonist activity were also performed. Briefly, PAR1/PAR4-expressing HEK293 cells were grown in DMEM (Life Technology, Grand Island, N.Y.) containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin, 10 μg/mL blasticidin, and 100 μg/mL Zeocin at 37° C. with 5% CO2. Cells were plated overnight prior to the experiment in a black 384-well Purecoat Amine clear bottom plate (Becton Dickinson Biosciences, San Jose, Calif.) at 10,000 cells/well in 30 μL growth medium and incubated in a humidified chamber at 37° C. with 5% CO2 overnight. Prior to compound addition, the cell medium was replaced with 40 L of 1× calcium and magnesium-containing Hank's Balanced Saline Solution (HBSS) (with 20 mM HEPES) and 1:1000 diluted fluorescent calcium indicator (Codex Biosolutions, Gaithersburg, Md.). After a 30 minute incubation period at 37° C. and a further 30 minute incubation and equilibration period at room temperature, 20 μL test compound (diluted in 1× HBSS buffer) was added at various concentrations at 0.17% dimethyl sulfoxide (DMSO) final concentration. Changes in fluorescence intensity were measured using a Functional Drug Screening System (FDSS, Hamamatsu, Japan) to determine agonist activities. The cells were then incubated for 30 minutes at room temperature followed by addition of 20 μL of agonist peptide for antagonist activity measurement. The PAR4 agonist peptide (H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2) and the PAR1 agonist peptide (SFFLRR) were routinely tested to ensure a proper response at the EC50 value in the assay (5 μM for PAR4 agonist peptide and 2 μM for PAR1 agonist peptide). Compound potency was derived from 11-point concentration-response curves.
Measurement: EC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 612.56, ALogP 7.21, HBA 9, HBD 1, PSA 100.12, QED 0.18
Compound (SMILES): CCN(CC)CCNC(=O)CCC1CN(C(=O)O[C@@H]2CC[C@]3(CO3)[C@@H]([C@@]3(C)O[C@@H]3CC=C(C)C)[C@@H]2OC)C1
InChIKey: MUIGSLXUSDEOIM-UFRBKFNPSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Biological Enzymatic Assay: Flag tagged Human recombinant MetAP2 expressed and isolated for use as the enzyme source. 10 mM stock solutions of compounds were prepared in 100% DMSO and further diluted in 100% DMSO required concentration to 1 mM stocks. The stock compound solutions and DMSO vehicle controls were diluted to target final compound concentrations using assay buffer to a final concentration of 50 mM HEPES containing 100 mM NaCl, pH adjusted to 7.5. The MAS peptide was formulated to a 7.5 mM stock in distilled water and prior to use further diluted 1:4. Amino acid oxidase was prepared as a stock solution (6.2 mg/ml) and prior to use further diluted 1:49.6 in distilled water. A 250 μM solution of MnCl2 was prepared in advance of thawing an aliquot of MetAP2 enzyme. 40 μl of enzyme was mixed with 100 μl of MnCl2 then further diluted in assay buffer to a final concentration of 16 μg/ml. To test for compound effect on MetAP2 enzyme activity, 5 μl of test compound, 10 μl of MAS substrate/amino acid oxidase mixture, 10 μl of MetAP2 was added to test wells in a 384 well black plate with blank wells containing no enzyme, replaced with 10 μl of assay buffer. All compounds were tested in duplicate on two occasions on the same day. The final in well concentrations of the assay were: 1% DMSO, 0.272 μg/ml MetAP2, 10 μM MnCl2, 50.0 μg/ml (0.225 U/ml) amino acid oxidase, and 0.75 mM MAS. The plate was sealed with a TopSeal A cover and mixed briefly on an orbital mixer at 900 rpm. The plate was incubated for a further 25 minutes at 25° C. A 5× stock of Amplex buffer was prepared (0.25M sodium phosphate, pH 7.4) and stored at 4° C. When preparing for use the stock was diluted with distilled water Amplex Ultraread stock solution was prepared at 2.57 mg/ml in 100% DMSO and stored in 50 μl aliquots at −20° C. 20 μl of 505 U/ml. Horse radish peroxidase was diluted in 990 ml of Amplex buffer, 100 μl of this was combined with 50 μl of Amplex Ultrared in 4850 ml of 1× Amplex buffer to generate sufficient detection reagent for a 384 well plate. 25 μl detection reagent was added to each well of the test plate, which was re-sealed and mixed briefly on an orbital shaker. The plate was transferred to an Envision Multi-label reader and RFU measured corresponding to excitation 531 nm and emission 595 nm At the end of the MetAP2 incubation 25 μl Amplex/HRP mixture per well was added and the plate read plate on a plate reader.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 535.73, ALogP 3.37, HBA 7, HBD 1, PSA 96.17, QED 0.29
Compound (SMILES): CC(C)[C@H](NC(=O)[C@@H](NC(=O)CCc1cc(F)cc(F)c1)c1ccccc1)C(=O)C(F)(F)F
InChIKey: PRGQFSDVTIKPGX-PMACEKPBSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore, whose emission is quenched in the intact peptide.Assay buffer: 500 mM Tris pH 8.0, 200 mM NaCl, 0.025% CHAPS, 0.005% BSGEnzyme: human HtrA1 Cat-PDZ, final concentration 1 nMSubstrate: Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys, final concentration 500 nM (from Innovagen Cat: SP-5076-1, Lot: 89584.02)Mca=(7-Methoxycoumarin-4-yl)acetylDnp=2,4-DinitrophenylFinal volume: 51 μlExcitation 320 nm, emission 390 nmAfter a pre-incubation of the HtrA1 protease for 30 min with compounds, substrate is added to the wells and initial RFU is measured. Upon incubation for 2 hours at RT, the enzymatic activity cleaved the substrate releasing fluorescent Mca-peptide conjugate and the final RFU value is measured. The presence of inhibitors leads to a decreased final RFU.
Measurement: IC50 = 218 nM (pChEMBL 6.659999847412109)
Descriptors: MW 470.44, ALogP 4.03, HBA 3, HBD 2, PSA 75.27, QED 0.54