lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)COc1ccc2c(c1)N(C(=O)Cc1ccc(-c3n[nH]c(=O)c4ccccc34)cc1)CC2
InChIKey: GDCGZXVOEQJVGM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 455.47, ALogP 3.19, HBA 5, HBD 2, PSA 112.59, QED 0.46
Compound (SMILES): Cc1cc(Nc2cc(C(F)F)cc(N3CC(F)(C(=O)NCc4ccc(-n5cc(F)cn5)nc4)C3)n2)n[nH]1
InChIKey: CXSOZKDRXHKNEG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET V804L Gatekeeper Mutant Assay at KM: In each well of a 384-well plate, 7.5 nM-10 nM of mutant RET (ProQinase 1096-0000-1) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKDDIYFFFG-NH2) (SEQ ID NO: 5) and 10 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 515.48, ALogP 3.36, HBA 8, HBD 3, PSA 116.65, QED 0.31
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)-c1cc(O)cc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)C#CCN3[C@@H](C)COC[C@@H]3C)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: UCAHTEITWCOOLI-CSZAFGDPSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 1029.29
Compound (SMILES): CCOC(=O)C1CCCC(n2nc(-c3ccc(CNC(=O)c4ccccc4OC)cc3)c3c(N)ncnc32)C1
InChIKey: FPLDSFVCJJWFOY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTKC481S Binding Affinity: BTKC481S binding affinity of each compound tested was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. 5 nM Recombinant BTKWT kinase, varying concentrations of inhibitor, 2 nM LanthaScreen Eu anti-His Antibody and 30 nM Kinase Tracer 236 was incubated in 1× LanthaScreen Kinase Buffer A for 5 h. Recombinant BTKC481S kinase was purchased from SignalChem and all LanthaScreen components were purchased from Invitrogen. Measurements were performed in a reaction volume of 30 μL using half-area 96-well assay plates. The TR-FRET signal was read on a plate reader with an excitation wavelength of 340 nm and detection wavelengths of 615 and 665 nm. Binding affinity was determined for each compound by measuring TR-FRET signal at various concentrations of compound and plotting the relative fluorescence units against the inhibitor concentration to estimate the IC50 from log [Inhibitor] vs response using the Variable Slope model in Graphpad prism from Graphpad software (SanDiego, Calif.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 528.61, ALogP 4.31, HBA 9, HBD 2, PSA 134.25, QED 0.32
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(C4NC(=O)NC(C)=C4C(C)=O)cc3)nn(C3CCC(O)CC3)c2n1
InChIKey: BEOZHLFUBUPLOH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Inhibition Assay: The ectopic expression of Mer receptor tyrosine kinase (Mer) has been identified as a tumor cell survival gene product in Acute Lymphoblastic Leukemia (ALL) cells and a potential cause of ALL chemoresistance. Hence, we investigated whether the development of small molecule Mer inhibitors was possible.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 517.63, ALogP 4.4, HBA 8, HBD 4, PSA 134.06, QED 0.33
Compound (SMILES): CC(C)CCNc1nc(Nc2cnn(CC(C)(C)C)c2)ncc1C(N)=O
InChIKey: WWRBFLAECBKOFK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Kinase Activity Assay: JAK2 (the final concentration was 0.25 nM); ATP (the final concentration was 5 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 359.48, ALogP 3.02, HBA 7, HBD 3, PSA 110.75, QED 0.67
Compound (SMILES): O=C(CCc1nc2ccccc2c(=O)[nH]1)N1CCC(c2nnc(-c3ccccc3)o2)CC1
InChIKey: XZTBVJZEZAGWMB-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: PARP1 biochemical assay was purchased as a kit from Trevigen (cat.#4676-096-K) and used per manufacturers recommendations.PARP2 biochemical assay was purchased as a kit from BPS (cat.#80552) and used per manufacturers recommendations.
Measurement: IC50 = 3460 nM (pChEMBL 5.460000038146973)
Descriptors: MW 429.48, ALogP 3.31, HBA 6, HBD 1, PSA 104.98, QED 0.52
Compound (SMILES): COc1cccc(OC)c1-c1cc(C(=O)NC2(C(=O)O)C3CC4CC(C3)CC2C4)nn1-c1ccc(C(=O)N(CCCN)CCCN(C)C)cc1C(C)C
InChIKey: QIISNDUJUSNRAJ-UHFFFAOYSA-N
Target: Neurotensin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30989
Assay: Radioligand Binding Assay NTR1: In order to determine the binding affinity of compounds comprising a radiolabel for NTR1, a radioligand binding assay was carried out. A radioligand is a radioactive biochemical substance that is used for diagnosis or for research-oriented study of cellular receptor systems of the body. In in vivo systems it is often used to quantify the binding of a test molecule to the binding site of radioligand. The higher the affinity of the molecule, the more radioligand is displaced from the binding site. The amount of bound radioligand can be measured by scintillation counting and thereby quantified. This assay is commonly used to calculate binding constants of molecules to receptors. This example shows that conjugates of the present invention bind to NTR1 with high affinity.The NTR1 radioligand binding assay was performed by Cerep (Celle l'Evescault, France; Catalog reference 0109) according to Vita et al., FEBS Lett., 1993, 317, 139-142. NTR1 was prepared from CHO cells recombinantly expressing the human receptor and incubated with 0.05 nM 125I-(Tyr3-neurotensin) and serial dilutions of the test compounds. After 60 min incubation at 4° C. and washing to remove unbound neurotensin, bound radioactivity was measured by scintillation counting. The result for each test compound is expressed as IC50 concentration and provides a measure for the affinity of the test compound for NTR1.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 728.94, ALogP 5.43, HBA 9, HBD 3, PSA 152.25, QED 0.18
Compound (SMILES): CCc1cc2ncc(CN3CCN(c4ccc(C(=O)NC)nc4C#N)[C@H](C)C3)cc2[nH]c1=O
InChIKey: UQXREIKQEMWFLV-OAHLLOKOSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical (FP) Assay: Assays based on fluorescent polarization (FP) have been widely utilized in drug discovery due to the homogenous format, robust performance and lack of interference seen in other assays. To characterize our compounds, we utilized an assay measuring the displacement of a commercially available fluorescently labeled PARP 1/2 inhibitor (PARPi-FL, Tocris Biosciences, #6461) as exemplified in assays performed in WO2014/064149 and WO2021/013735A1. The assay was performed utilizing the following method: Compounds were dissolved in DMSO an Echo550 liquid handler was utilized to make serial dilations in the desired concentration range in Optiplate-384F plates. 100% DMSO was used for the high (with protein) and low (without protein) control samples. 20 nL of compound or DMSO alone was added to individual assay plate wells. PARP1 and PARP2 protein were expressed, purified and diluted in assay buffer containing 50 mM Tris pH 8.0, 0.001% Triton X-100, 10 mM MgCI2, 150 mM NaCl to a final concentration of 20 nM. The PARPi-FL was then added at a final concentration of 3 nM. The assay plate is centrifuged at 1000 rpm for 1 min and incubated for 4 h at room temperature.
Measurement: IC50 = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 445.53, ALogP 1.82, HBA 7, HBD 2, PSA 118.01, QED 0.61
Compound (SMILES): NCC1(c2ccc(F)cc2)CCCCC1
InChIKey: VPDDMTHLPLVCMM-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Functional Uptake Assay (hDAT): Inhibition of human dopamine reuptake transporter was assayed using the recombinant human dopamine transporter expressed in either CHO-K1 or HEK293 cells using a published method (Pristupa, Z. B. et al., Mol. Pharmacol. 45: 125-135, 1994). Either CHO-K1 or HEK293 cells expressing human recombinant dopamine transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 50 nM [3H]dopamine was then added for an additional timed incubation period (10 to 30 minutes). After washing the cells to remove [3H]dopamine not internalized, the cells were lysed, and the amount of tritium in the lysate was measured using a liquid scintillation counter to determine [3H]dopamine uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM nomifensine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]dopamine uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was nomifensine, for which the IC50 value of 11 nM was obtained in a typical experiment.
Measurement: IC50 = 1830 nM (pChEMBL 5.739999771118164)
Descriptors: MW 207.29, ALogP 2.99, HBA 1, HBD 1, PSA 26.02, QED 0.79
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2cccc3c(C(=O)O)cccc23)C1=O
InChIKey: MWGZMXNLKAOZAZ-AOMKIAJQSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 445.52, ALogP 3.11, HBA 4, HBD 3, PSA 98.74, QED 0.54
Compound (SMILES): C=CC(=O)N1CCN(C(=O)c2cc(Sc3cnc(Nc4ncc(C)nc4C)s3)c(C)cc2C)CC1
InChIKey: HWNNPBBHQOJHMO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 508.67, ALogP 4.53, HBA 8, HBD 1, PSA 91.32, QED 0.49
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3ccc(-c4cnco4)s3)nc3cc(Cl)ccc31)C2
InChIKey: IZIBDTDSPVBIEA-ODAJSGAPSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 508, ALogP 5.4, HBA 7, HBD 1, PSA 93.26, QED 0.37
Compound (SMILES): COc1cc(Cl)c(C)cc1C1CCN(C(=O)CC[C@@]2(C)NC(=O)NC2=O)CC1
InChIKey: WIXQHARDFJRBSX-HXUWFJFHSA-N
Target: Disintegrin and metalloproteinase domain-containing protein 17 (SINGLE PROTEIN), Homo sapiens
UniProt: P78536
Assay: hTACE assay: The basis for the assay is the cleavage of the substrate 5FAM-LAQAVRSSSRK-5TAMRA (SEQ ID No 3) (Anaspec, custom 34891) by human TACE (R&D SYSTEMS INC., Cat#930-ADB).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat#781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (25 mM Tris pH8.0, 2.5 μM ZnCl2, 0.01% CHAPS) containing TACE (0.05 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 5FAM-LAQAVRSSSRK-5TAMRA (5 μL, 5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 75 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 407.9, ALogP 2.74, HBA 4, HBD 2, PSA 87.74, QED 0.73
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(-c3cnn(Cc4cocn4)c3)nc3cnc4[nH]ccc4c32)CC1
InChIKey: YWBQPTNGCFVMGJ-RZDIXWSQSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 426.48, ALogP 4.46, HBA 8, HBD 1, PSA 114.14, QED 0.44
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(-c4cc5c(s4)CN(C)CC5)cc3c2C(C)C)cc(C)n1
InChIKey: KBAISJCJUJBCDO-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-κc Band 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 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-Bue 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 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 415.61, ALogP 6.69, HBA 3, HBD 1, PSA 31.92, QED 0.4
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H]2CN(C(=O)Nc3nc(CO)ns3)C[C@@H]2C1
InChIKey: XLPLHVMZOYBIBG-GDNZZTSVSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In vitro kinase assay: JAK3: In vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK3 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required by the experiment. JAK3 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 μM ATP and 1.2 μM substrate solution. The appropriate amount of JAK3 kinase (Invitrogen, Catalog Number: pv3855) was mixed with 4× buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/μL. To each well of a microplate [DELFIA® Streptavidin-coated clear plate (Perkin Elmer, Item: AAAND-0005)] 17.5 μL of ATP/substrate mixture, 5 μL of aqueous solution of a test compound (μL of pure water only were added to the control and blank), and 7.5 μL of the kinase solution prepared above (4× buffer only was added to the control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell signaling Technology, Catalog Number: 5465)] was added, and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction. Absorbance was measured on a Novostar microplate reader at a wavelength of 450 nm. IC50 values of test compounds were calculated from the data of the test compounds for inhibiting the activity of JAK3 kinase at different concentrations.
Measurement: IC50 = 201 nM (pChEMBL 6.699999809265137)
Descriptors: MW 414.5, ALogP 1.68, HBA 8, HBD 3, PSA 123.16, QED 0.59
Compound (SMILES): NC/C(=C\F)COc1ccc(S(=O)(=O)CN2CCCCC2=O)cc1
InChIKey: NXKARHTUNZOKGO-UKTHLTGXSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 356.42, ALogP 1.62, HBA 5, HBD 1, PSA 89.7, QED 0.8
Compound (SMILES): CC1CC2CNCCN2c2ncccc21
InChIKey: URUOICGPQOEMQJ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Radioligand Binding Assay: Radioligand binding assays were performed using the commercially available 5-HT2 receptor agonist [125I]DOI as the radioligand and nonspecific binding was determined in the presence of unlabeled DOI at a saturating concentration of 10 μM. Competition experiments utilized 5-HT2 receptor expressing HEK293 cell membranes obtained as described in Example 3 (15-25 μg membrane protein/well) and radioligand at final assay concentrations of 0.4 to 0.6 nM. Experiments comprised addition of 95 μL of assay buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2), 50 μL of membranes, 50 μL of radioligand stock, and 5 μL of test compound diluted in assay buffer to 96-well microtiter plates, which were then incubated for 1 h at room temperature. Assay incubations were terminated by rapid filtration through PerkinElmer F/C filtration plates under reduced pressure using a 96-well Packard filtration apparatus, followed by washing three times with ice cold assay buffer. Plates were then dried at 45° C. for a minimum of 2 h. Finally, 25 μL of BetaScint scintillation cocktail was added to each well and the plates were counted in a Packard TopCount scintillation counter. In each competition study, test compounds were dosed at ten concentrations with triplicate determinations at each test concentration.
Measurement: Ki = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 203.29, ALogP 1.37, HBA 3, HBD 1, PSA 28.16, QED 0.69
Compound (SMILES): Cc1nc(NCc2c(F)cccc2CN(C)C(C)(C)C)ccc1S(=O)(=O)Nc1cscn1
InChIKey: LAFKPVRCPUZXCV-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaC2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 23605 nM (pChEMBL 4.630000114440918)
Descriptors: MW 477.63, ALogP 4.63, HBA 7, HBD 2, PSA 87.22, QED 0.49
Compound (SMILES): O=C(Nc1ccc2cnccc2c1F)[C@@H]1CC1c1ccc(S(=O)(=O)Nc2ccccn2)cc1
InChIKey: NVHVDZKSZLWUNH-GFOWMXPYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 127 nM (pChEMBL 6.900000095367432)
Descriptors: MW 462.51, ALogP 4.31, HBA 5, HBD 2, PSA 101.05, QED 0.44
Compound (SMILES): Cc1cnc(-c2cc(OC3C4COCC3CN(C)C4)cc(C(=O)N[C@@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: UOQSAFNLRPUQCS-LONTXVJRSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9UBL9
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity : A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%.The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control)).
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 547.6, ALogP 4.37, HBA 8, HBD 1, PSA 89.47, QED 0.49
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(c2nc(=O)n(-c3c(C(C)C)cc(C4CCC4)nc3C(C)C)c3nc(-c4ccccc4F)c(Cl)cc23)C[C@H]1C
InChIKey: WNVWKTNBROXKMK-PKTZIBPZSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 8794 nM (pChEMBL 5.059999942779541)
Descriptors: MW 657.23, ALogP 7.76, HBA 7, HBD 0, PSA 84.22, QED 0.19
Compound (SMILES): Cc1cc2nc(N[C@@H](C)c3cccnc3)nc(C(=O)NC[C@H](C)O)c2s1
InChIKey: WMEKOMFCQQIPJY-JQWIXIFHSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 371.47, ALogP 2.68, HBA 7, HBD 3, PSA 100.03, QED 0.62
Compound (SMILES): N#CCCNC(=O)Cc1nc2cnc3[nH]ccc3c2n1C1CCC(CC#N)CC1
InChIKey: DZYJQAKCPQYEKD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzymatic Inhibition Assay: Substrate (NH2-KGGEEEEYFELVKK-CO2), internal standard peptide (NH2-SWGAIETDKEYYTVKD-CO2) and product peptide (for standard curve only) (NH2-KGGEEEEY-Pi-FELVKK-CO2), were purchased from AnaSpec (Fremont, Calif., USA). JAK1-JH1JH2 (574-1154 with a His-GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), JAK3-JH1JH2 (512-1124 with a GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), and Tyk2-JH1JH2 (8H_tev_580-1182-C936A-C1142A with a C-terminal tev (ENLYFQ-G) cleavage site) were purified internally. JAK2-JH1JH2 (532-1132 with a GST tag and C-terminal tev (ENLYFQ-G) cleavage site), was purchased from Invitrogen. LC/MS grade water and acetonitrile (ACN), were purchased from HoneyWell, Burdick & Jackson (Muskegon, Mich., USA). Dimethylsulfoxide 99.8% (DMSO) and trifluoroacetic acid 99.5% (TFA) were purchased from EMD Chemical (Gibbstown, N.J., USA). Adenosine triphosphate (ATP), 4-morpholinepropanesulfonic acid (MOPS), magnesium chloride (MgCl2), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), formic acid >95% (FA) and Tween-20 were purchased from Sigma (St Louis, Mo., USA). 384-well polypropylene plates, Cat #781280 were purchased from Greiner (Monroe, N.C.), RapidFire™ cartridge A C4 Column (Agilent Technologies, Santa Clara, Calif.).
Measurement: IC50 = 96.2 nM (pChEMBL 7.019999980926514)
Descriptors: MW 389.46, ALogP 3.13, HBA 6, HBD 2, PSA 123.18, QED 0.63
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2cccc(Cl)c2c(=O)n1[C@H]1C[C@@H](O)C1
InChIKey: PQANOGPKBULQEK-LPEHRKFASA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: a time-resolved fluorescence resonance energy transfer (TR-FRET) 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 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X, ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) 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 pM 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.
Measurement: IC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 426.87, ALogP 1.74, HBA 10, HBD 4, PSA 168.76, QED 0.48
Compound (SMILES): COc1cccc(OC)c1S(=O)(=O)Nc1noc2cc(Cn3ccnn3)cc(OC)c12
InChIKey: DLUHKUSWWRNIAE-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92993
Assay: Kat5 Assay: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT5 25 nM, 1 uM AcCoA, 2 uM H4 1-21 peptide, 30-minute reaction     2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 11700 nM (pChEMBL 4.929999828338623)
Descriptors: MW 445.46, ALogP 2.29, HBA 10, HBD 1, PSA 130.6, QED 0.43
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2ccn3nccc3n2)cc1NC1CCC(F)(F)C1
InChIKey: AODOHQYJWVDZCC-FQNRMIAFSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 477.49, ALogP 3.31, HBA 8, HBD 4, PSA 116.47, QED 0.39
Compound (SMILES): N#Cc1cccc(S(=O)(=O)Nc2cccc(-c3ccc([C@H]4C[C@@H]4N)cn3)c2)c1
InChIKey: NUABRMQYIQQHPM-UXHICEINSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Assay: Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of various concentrations of inhibitor (e.g., from 0 to 75 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in triplicate. After leaving the enzyme interacting with the inhibitor, 12.5 μM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 1 hour at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex® Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 30 extra minutes at room temperature in the dark. A 1 μM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex® Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan).
Measurement: Ki = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 390.47, ALogP 3.24, HBA 5, HBD 2, PSA 108.87, QED 0.7
Compound (SMILES): CCn1ccc(-c2cc3c(ccc4nnc(C)n43)n2Cc2ccccc2)n1
InChIKey: XULUCKUBDPIXHG-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 356.43, ALogP 3.92, HBA 6, HBD 0, PSA 52.94, QED 0.49
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCC(N(C)C5CCC5)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: LQODGZWGUQPSNB-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 = 2376 nM (pChEMBL 5.619999885559082)
Descriptors: MW 471.65, ALogP 5.47, HBA 6, HBD 1, PSA 65.35, QED 0.42