lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)C(=O)c1ccc(-c2cnc(N)c(-c3nnc(-c4ccccn4)o3)n2)cc1
InChIKey: NWVSZAIYUZBPNA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 387.4, ALogP 2.54, HBA 8, HBD 1, PSA 123.92, QED 0.57
Compound (SMILES): Cc1ccc(-c2noc(C)c2COc2ccc(C(=O)N[C@H]3CCOC3)nn2)cn1
InChIKey: AKFWDSCDLSTTJA-HNNXBMFYSA-N
Target: Gamma-aminobutyric acid receptor subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14867
Assay: Electrophysiology: Electrophysiological experiments were performed on days 3 to 5 after the micro-injection of mRNA. During the experiment the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCl2 1, CaCl2 1 (pH 7.4). Oocytes were impaled by two glass microelectrodes (resistance: 0.4 MΩ) which were filled with a solution containing KCl 1M+K-acetate 1.5 M and voltage-clamped to −80 mV. The recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA was added for 1.5 min at a concentration evoking approximately 20% of a maximal current response (EC20). After another rest interval of 2.5 min GABA was again added evoking a response of similar amplitude and shape. 0.5 min after the onset of this second GABA application the test compound, at a concentration corresponding to approximatively 30 fold its Ki, was added while GABA was still present. Current traces were recorded at a digitization rate of 10 Hz during and shortly before and after the GABA application.
Measurement: Ki = 8.1 nM (pChEMBL 8.09000015258789)
Descriptors: MW 395.42, ALogP 2.24, HBA 8, HBD 1, PSA 112.26, QED 0.68
Compound (SMILES): CN(CCc1ccccn1)C(=O)[C@@H]1CN(C2CCCCC2)CC[C@H]1NC(=O)c1cc(-c2ccc(F)cc2F)on1
InChIKey: FHDGXNIIGJYPHC-AOYPEHQESA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 551.64, ALogP 4.47, HBA 6, HBD 1, PSA 91.57, QED 0.44
Compound (SMILES): CN1CCN(C(=O)Cn2cc3cc(NC(=O)c4cccc(-c5cnn(C)c5)n4)ccc3n2)CC1
InChIKey: YUSQNTXFDIAXDP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 458.53, ALogP 1.86, HBA 8, HBD 1, PSA 101.18, QED 0.49
Compound (SMILES): COc1ccc(C(=O)N2CC3CN(c4ncc5ccccc5n4)CC3C2)c(OC)c1
InChIKey: SDVAQBZVRFSRSR-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Human Orexin 2 Receptor Radioligand Binding Studies: HEK293 stably expressing human orexin-2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12 (Gibco, Cat #11039), in DMEM, 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 1×HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 10 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and vortexed for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moravek Corporation, specific activity=27 Ci/mmol), diluted to a 20 nM concentration in PBS (5 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM (N-[2-(3,4-dimethoxyphenyl)ethyl]-N-methylnaphthalene-1-carboxamide, CAS Registry #1089563-88-1). The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 2658 nM (pChEMBL 5.579999923706055)
Descriptors: MW 404.47, ALogP 2.86, HBA 6, HBD 0, PSA 67.79, QED 0.67
Compound (SMILES): CC1(N)CCN(c2nc3[nH]nc(-c4cccc(Cl)c4Cl)c3nc2C=O)CC1
InChIKey: XBAFDNBZXMKIPD-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Enzymatic Assay: Phosphatase activity of full length wild-type PTPN11(PTPN11-WT) or PTPN11-E76K mutant enzyme was measured using the fluorogenic 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Molecular Probes) as the substrate. Enzyme (250 pM) was incubated with or without increasing concentrations of compounds in assay buffer (62.5 mM HEPES, 125 mM NaCl, 1 mM EDTA, 1.25 mM TECP, 0.1% BSA) for 30 min at room temperature. Reaction was initiated by addition of DiFMUP (50 μM) at room temperature in 384-well black plate with a final reaction volume of 20 uL in assay buffer. After 1 hour, DiFMUP fluorescence signal was measured (Ex:340/Em:460) using Envision plate reader. Dose-response curves were analyzed using IC50 regression curve fitting (GeneData Screener). Curves were normalized to a high controls without inhibitor, and low controls without enzyme. Results are given below in Table 1. Other compounds disclosed herein are expected to have activity similar to the results below, showing activity as PTPN11 inhibitors.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 405.29, ALogP 3.46, HBA 6, HBD 2, PSA 100.79, QED 0.65
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CC2(CC(Oc3cccc(C(F)(F)F)c3)C2)C1
InChIKey: ZXJFMWMVAIHRNO-UHFFFAOYSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BV23
Assay: Fluorescent Assay: ABHD6: Certain compounds were tested for their ABHD6 and dual ABHD6/MGL inhibitory activity, which is expressed as % of inhibition or IC50 values. The percentage of inhibition describes the percentage by which the inhibitor reduces the velocity/rate of 2-AG hydrolysis by ABHD6, and MGL or AEA hydrolysis by FAAH.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 451.29, ALogP 5.18, HBA 3, HBD 0, PSA 38.77, QED 0.6
Compound (SMILES): CNC(=O)c1ccc2c(n1)OC[C@@H]1CN(Cc3cnc4cc(C)c(=O)[nH]c4c3)CCN21
InChIKey: LUEKTFALTSGELH-HNNXBMFYSA-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 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 420.47, ALogP 1.07, HBA 7, HBD 2, PSA 103.45, QED 0.65
Compound (SMILES): N[C@H](Cc1ccccc1)[C@H](O)C(=O)NC[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1
InChIKey: QCWAVKSEDLCGHN-PWWKTKHKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 663.61, ALogP 3.7, HBA 6, HBD 4, PSA 132.96, QED 0.26
Compound (SMILES): C[C@@H]1Cc2c([nH]c3ccccc23)[C@@H](c2c(F)cnc(OCCNCCCF)c2Cl)N1CC(F)(F)CO
InChIKey: NXEDZKAQYWQSQZ-IQMFZBJNSA-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 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 528.98, ALogP 4.65, HBA 5, HBD 3, PSA 73.41, QED 0.25
Compound (SMILES): N#CC(Cc1ccc(O)c(O)c1)C(=O)Nc1nc(-c2ccccc2)cs1
InChIKey: QGZIIRIDEQPTAS-UHFFFAOYSA-N
Target: Double-strand break repair protein MRE11 (SINGLE PROTEIN), Homo sapiens
UniProt: P49959
Assay: Inhibition of human MRE11 expressed in insect High Five cells using fluorescent-labeled 5'-overhang DNA as substrate preincubated for 20 mins followed by substrate addition and measured after 20 mins by gel based assay
Measurement: IC50 = 3400 nM (pChEMBL 5.46999979019165)
Descriptors: MW 365.41, ALogP 3.54, HBA 6, HBD 3, PSA 106.24, QED 0.6
Compound (SMILES): Cn1c(C(=O)N[C@H]2CC[C@H](CCN3CCc4ccc(CCC(F)(F)F)nc4CC3)CC2)cccc1=O
InChIKey: JPNVWOLGTXJVFQ-XUTJKUGGSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.22 nM (pChEMBL 9.65999984741211)
Descriptors: MW 504.6, ALogP 4.05, HBA 5, HBD 1, PSA 67.23, QED 0.62
Compound (SMILES): C[C@@](O)(CNC(=O)c1nc(C(F)(F)F)c(C(F)(F)F)cc1N)C(F)(F)F
InChIKey: DOVVSHMLCFFGGB-SECBINFHSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: Potentiator Assay: Fischer Rat Thyroid (FRT) epithelial cells, stably expressing human ΔF508-CFTR were used as monolayer cultures on permeable supports. Cl− current was measured using the short circuit current technique, under an imposed basolateral to apical Cl− gradient in Ussing chambers. To measure stable Cl− currents, FRT cells were cultured for 48 h at 27° C. to facilitate the insertion of ΔF508 CFTR into the plasma membrane. Ussing chamber studies were likewise conducted at 27° C. Under these conditions, the effects of cumulative additions of test compounds on ΔF508 CFTR currents could be quantitated with both potency and efficacy endpoints. Compounds were added to both the apical and basloalteral sides subsequent to addition of 10 μM forskolin. Efficacy of compounds was compared to a known potentiator such as gensitein.
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 399.21, ALogP 2.74, HBA 4, HBD 3, PSA 88.24, QED 0.68
Compound (SMILES): Cc1ccc(-c2cc(-c3ccc(O)nc3)cc(S(N)(=O)=O)c2-c2nn[nH]n2)cc1
InChIKey: NWTSLAUWCFNYET-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 3.65 nM (pChEMBL 8.4399995803833)
Descriptors: MW 408.44, ALogP 2.26, HBA 7, HBD 3, PSA 147.74, QED 0.47
Compound (SMILES): C[C@H](NC(=O)[C@H]1CC(Cc2ccccc2)CN1)C(=O)NCc1cnc2c(c1)c(Cl)cn2C
InChIKey: ROPIKSRSEMFYDW-JIJYOHDDSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 453.97, ALogP 2.57, HBA 5, HBD 3, PSA 88.05, QED 0.51
Compound (SMILES): O=c1c2cccnc2ncn1Cc1nc([C@H]2[C@@H]3CN(c4ccc(Cl)cn4)C[C@@H]32)no1
InChIKey: HQVRXJTUSZIFBP-HALDLXJZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (15 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 421.85, ALogP 2.12, HBA 9, HBD 0, PSA 102.83, QED 0.49
Compound (SMILES): C[C@H](Nc1ncnc2ccc(Cl)nc12)c1nc2c(Cl)ccc(Cl)c2c(=O)n1N1CCNCC1
InChIKey: HDDXMIAMXZUMDY-NSHDSACASA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PIK3 Assay: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIPS for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site.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-PIPS, 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 is delay and 500 is read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, 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 = 8000 nM (pChEMBL 5.099999904632568)
Descriptors: MW 505.8, ALogP 3.41, HBA 9, HBD 2, PSA 100.86, QED 0.41
Compound (SMILES): O=S(=O)(Nc1cscn1)c1cc(Cl)c(OC2CCN(Cc3ccccc3)CC2)cc1F
InChIKey: YDTGVUJVMUOJJX-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
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 1-1.5 h 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 ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 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 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 482, ALogP 4.78, HBA 6, HBD 1, PSA 71.53, QED 0.53
Compound (SMILES): C#Cc1cnc2ccc(-c3c(-c4ccc(F)cc4)ncn3C(COC)C3CC3)nn12
InChIKey: JCPXJXLZCFLITK-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). 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 reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 401.45, ALogP 3.98, HBA 6, HBD 0, PSA 57.24, QED 0.46
Compound (SMILES): Cc1ncc(-c2nc3c(OC4CCN(C(=O)OC(C)(C)C)C4)ncnc3n2C)cn1
InChIKey: QAGUHFHZCUKXTL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 99 nM (pChEMBL 7)
Descriptors: MW 411.47, ALogP 2.52, HBA 9, HBD 0, PSA 108.15, QED 0.65
Compound (SMILES): CN1Cc2c(-c3c(F)ccc4cc(C5CCOCC5)ncc34)nc(C3CCOCC3)n2CC1=O
InChIKey: HAQSYYOVDCMOLK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4: 2.5 nM of BRD4(49-170) and 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
Measurement: IC50 = 219 nM (pChEMBL 6.659999847412109)
Descriptors: MW 464.54, ALogP 4, HBA 6, HBD 0, PSA 69.48, QED 0.59
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1-c1cn(C[C@H](C)O)cn1
InChIKey: HWUDIBHSVZRJER-AWEZNQCLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 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% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture 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 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 402.46, ALogP 2.75, HBA 9, HBD 2, PSA 117.47, QED 0.51
Compound (SMILES): C=CC(=O)Nc1cc(OC)ccc1Nc1nc(Nc2ccc(N3CCC[C@H]3CO)cc2C(F)(F)F)ncc1Cl
InChIKey: SEHPRSWSXUZSHE-INIZCTEOSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 562.98, ALogP 5.73, HBA 8, HBD 4, PSA 111.64, QED 0.24
Compound (SMILES): CCNC(=O)c1cc2c(-c3cc(C(C)(C)O)ccc3Oc3c(C)cc(SC)cc3C)cn(C)c(=O)c2[nH]1
InChIKey: IFWDZFWJUNJRBG-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 an approximately 3-fold serial dilution. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus#6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe.The final concentration of 1× assay mixture contained 2% DMSO, 12 nM His tagged BRD4 (BDI_K57-E168) and 100 nM probe or 4 nM His tagged BRD4 (BDII_E352-M457) and 30 nM probe, and 1 nM Europium-conjugated anti-His-tag antibody, and compound concentrations in the range of: 49.02 μM-0.61 nM or 0.98 μM-0.15 nM.After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 773 nM (pChEMBL 6.110000133514404)
Descriptors: MW 519.67, ALogP 5.64, HBA 6, HBD 3, PSA 96.35, QED 0.27
Compound (SMILES): CCn1ncc(-c2nc3c(O[C@H]4CCN(S(=O)(=O)c5cn(C)cn5)C4)ncnc3n2C)c1C
InChIKey: DHRIMGCJNKZAKO-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 471.55, ALogP 1.13, HBA 11, HBD 0, PSA 125.85, QED 0.41
Compound (SMILES): O=C(C1CCc2ccccc21)N1Cc2cccnc2Nc2ccccc21
InChIKey: RHRLXOILKKMRIP-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: R132H IDH1 Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation. The IC50 values of the exemplified compounds are included in the tables above in Examples section.Using the above biological assay, all compounds in the examples have IC50 of about 1 nM to about 40,000 nM, or more specifically, about 1 nM to about 20,000 nM, or even more specifically, about 5 nM to about 15,000 nM, or even more specifically, about 5 nM to about 10,000 nM, or even more specifically, about 5 nM to about 5,000 nM, or still more specifically, about 5 nM to about 1,000 nM. Such a result is indicative of the intrinsic activity of the compounds in use as an inhibitor of a mutant IDH1 enzyme. Specific IC50 activity data for the exemplified compounds disclosed herein is provided in the following table.
Measurement: IC50 = 1413 nM (pChEMBL 5.849999904632568)
Descriptors: MW 341.41, ALogP 4.4, HBA 3, HBD 1, PSA 45.23, QED 0.71
Compound (SMILES): CN1CCN(c2ccc(-c3cc(CN4CCN(CC5CC5)CC4)ccc3F)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)CC1
InChIKey: UCIMUGNNMDZMDT-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin label the protein in vivo. Purification was via Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40p1/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds, respectively, when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 626.7, ALogP 4.73, HBA 6, HBD 2, PSA 74.92, QED 0.35
Compound (SMILES): OCc1cc2c(Sc3ccc(Oc4ccccc4)cc3)cc(Cl)cc2[nH]1
InChIKey: DKQJJFJKSNBSHA-UHFFFAOYSA-N
Target: GTP-binding protein Rheb (SINGLE PROTEIN), Homo sapiens
UniProt: Q15382
Assay: In Vitro Kinase Assay: For the Rheb-dependent in vitro kinase assay, HA-GST-Rheb was first loaded with GTP-γ-S (Millipore #20-176) by incubating 600 ng/μL Rheb with 10 mM EDTA and 0.4 mM GTP-γ-S at 30° C. for 10 minutes. MgCl2 was added to a final concentration of 20 mM to stop the reaction. This mixture was used at 20× in the kinase assay.Loaded HA-GST-Rheb was then incubated with compound (final DMSO concentration 1%) for 30 minutes at room temperature. mTORC1, GFP-4E-BP1 substrate (Life Technologies #PV4759), and kinase assay buffer (HEPES pH 7.4, KCl, MgCl2) were added to the Rheb-compound mixture and incubated at room temperature for 20 minutes. ATP was then added, and the kinase reaction proceeded at 30° C. for 45 min before being stopped by the addition of SDS-PAGE loading buffer for Westerns or 10 mM EDTA for the LanthaScreen readout. During the kinase reaction, the final concentration of reagents is as follows: Rheb30 ng/μL, compoundvariable, GTPγS0.02 mM, EDTA0.5 mM, MgCl21 mM, mTORC1fixed volume of prep (molarity unknown), GFP-4E-BP1 substrate15 ng/μL, ATP0.5 mM. For LanthaScreen analysis (Thermo Fisher #PV4757), the manufacturer's instructions were followed and conditions were optimized for the amount of Rheb and mTORC1. The final concentration of reagents was as follows: 100 nM loaded HA-GST-Rheb, compound (variable), 0.5 mM EDTA, 10 mM MgCl2, 25 mM HEPES pH 7.4, 50 mM KCl, mTORC1 (fixed volume0.1 μL in 10 μL total volume), 0.4 μM GFP-4E-BP1 substrate, 0.5 mM ATP. Optimization is summarized in FIG. 1. Results were read on an EnVision reader (PerkinElmer) using 495ex/520em filters (Life Technologies, #PV00315).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 381.88, ALogP 6.26, HBA 3, HBD 2, PSA 45.25, QED 0.43
Compound (SMILES): COc1ccccc1-c1cc(C(=O)/N=c2\[nH]c3ccccc3n2[C@@H]2CC[C@H](O)[C@@H](O)C2)ccn1
InChIKey: ZMTRLEKKTLEDTH-VSDWSMLSSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (5 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at low ATP (final assay concentration 5 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 7.7 nM (pChEMBL 8.109999656677246)
Descriptors: MW 458.52, ALogP 3.23, HBA 6, HBD 3, PSA 112.73, QED 0.43