lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(C(C)(F)F)c2)cc1-c1cc(N2CCOC[C@@H]2C)n(C)c(=O)c1
InChIKey: KUWKOKQQSGVPBT-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 482.53, ALogP 4.34, HBA 6, HBD 1, PSA 76.46, QED 0.59
Compound (SMILES): CN(C)c1ccc2c(c1)CN(c1cccc(-c3nncn3C3CC3)n1)C2=O
InChIKey: GPKPSFNBUNOXBQ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ASK1 Kinase Assay: The ASK1 enzymatic assay was run following Promega ASK1 Kinase Enzyme System (Cat #V3881). The kit provides the protocol, enzymes and all reagents necessary to run an assay. Firstly, the compounds, enzyme, substrate and ATP were diluted in provided assay buffer. The final concentration of the enzyme was 50 nM, substrate (Myelin basic protein) 1 μg/ml and ATP 10 μM. The compound and the enzyme were pre-incubated in a 384 well white solid bottom plate (Greiner, Cat#784075) for 10 minutes. After incubation, the substrate and ATP were added and incubated for further 60 minutes. After 60 minutes, ADP-Glo™ was added and plate was incubated for another 40 minutes. After 40 minutes, Kinase Detection Reagent was added and the plate was incubated for 45 minutes. After 45 minutes, plate was read on Perkin Elmer EnVision using luminescence read (0.5 seconds/well).
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 360.42, ALogP 2.9, HBA 6, HBD 0, PSA 67.15, QED 0.72
Compound (SMILES): O=C1NCc2nc(-c3c(F)cccc3F)nc(Nc3ccc(C(=O)N4CC(O)C4)cc3)c21
InChIKey: WMCMZRUUGDNZTL-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 437.41, ALogP 2.23, HBA 6, HBD 3, PSA 107.45, QED 0.58
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)c2cccc3[nH]ncc23)c2ccccc2N(Cc2c(C)ccc3ccccc23)C1=O
InChIKey: SCHUKIUZPJPNGL-KMRXNPHXSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged 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-(c-biotin)-OH 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/mL 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. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 55.1 nM (pChEMBL 7.260000228881836)
Descriptors: MW 560.66, ALogP 4.31, HBA 5, HBD 3, PSA 110.43, QED 0.29
Compound (SMILES): Nc1ccc(-c2ccncn2)cc1NC(=O)N1Cc2ccncc2C1
InChIKey: FASOYXRDVWLWFU-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Reporter Displacement Assay: HDAC2 containing a C-terminal HIS-Tag (Proteros) and fluorescently-labeled anti-HIS-antibody are diluted in one vial in assay buffer 50 mM Tris, pH 8.0, 1 mM DTT, 150 mM NaCl, and 0.01% Tween20. Components are pre-incubated for 30 min, then a small volume of reporter probe (Proteros) is added from a highly concentrated stock and incubation is continued for another 30 min. Final concentrations after adding the reporter probe amount to 20 nM HDAC2, 4 nM antibody, and 180 nM probe.
Measurement: Kd = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 332.37, ALogP 2.67, HBA 5, HBD 2, PSA 97.03, QED 0.7
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(OC)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: PIXGPCPPSIRBCJ-SIKLNZKXSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 485.54, ALogP 3.7, HBA 6, HBD 2, PSA 80.65, QED 0.51
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OC[C@]5(F)CC[C@@](C)(O)CC5)c(Cl)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: PXMGHKWJLJTPIN-QWKHHPMFSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: TR-FRET (Time-Resolved Fluorescence-Resonance-Energy-Transfer) assay: TR-FRET (Time-Resolved Fluorescence-Resonance-Energy-Transfer) assay.
Measurement: Ki = 0.014 nM (pChEMBL 10.850000381469727)
Descriptors: MW 905.92, ALogP 9.37, HBA 10, HBD 3, PSA 149.98, QED 0.11
Compound (SMILES): c1csc(-c2cc(N3CCOCC3)nc3c(-c4ccn[nH]4)nccc23)c1
InChIKey: GRELDEQHIMPJNF-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 122 nM (pChEMBL 6.909999847412109)
Descriptors: MW 363.45, ALogP 3.59, HBA 6, HBD 1, PSA 66.93, QED 0.6
Compound (SMILES): COc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CCCCC4)c(C)sc23)cc1NS(C)(=O)=O
InChIKey: BHPXOXXXCYOLCU-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Activity Inhibition Assay: 1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.2. Compound was formulated in 100% DMSO in a concentration gradient, and added to a 384-well plate to a final DMSO concentration of 1%.3. PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ enzymes were diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, and transferred to a 384-well plate and incubated with the compound for a certain time.4. Substrate was diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, 25 μM ATP; then added to a 384-well plate to initiate the reaction, and for PI3Kα, PI3Kβ and PI3Kγ at room temperature reacted for 1 hr, for PI3Kδ at room temperature reacted for 2 hrs. For PI3Kβ and PI3Kγ, it was still necessary to further add 10 μL ADP-Glo Detection Reagent, and then equilibrated at room temperature for 30 minutes.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 532.69, ALogP 3.26, HBA 10, HBD 1, PSA 109.78, QED 0.49
Compound (SMILES): COc1cc(C2CCCN(C)C2)ccc1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: CNCRALKDGCSMDY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.14 nM (pChEMBL 9.850000381469727)
Descriptors: MW 389.46, ALogP 3.3, HBA 7, HBD 2, PSA 102.75, QED 0.69
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#N)c1cc2ncc(Cl)n2nc1-c1ccc2c(c1)OCCO2
InChIKey: PWFVRDACZCBVNO-NSHDSACASA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Transcreener KINASE Assay: Briefly, the Transcreener KINASE Assay was designed as a simple two-part, endpoint assay as follows:1) Preparation of 25 uL kinase reaction: the 25 uL kinase reaction was performed by preparing a reaction mixture containing 10 uL kinase buffer (50 mM HEPES, 100 mM NaCl, 1 mM EGTA, 0.03% CHAPS, 3 mM MgCl2, and freshly supplemented 1 mM DTT), and 10 uL 30 uM PIP2 and 10 uM ATP, 5 uL test compound solution (the compound was dissolved in DMSO, the final concentrations of the compound in the reaction mixture were at 1 uM, 0.3 uM, 0.1 uM, 0.037 uM, 0.012 uM, 0.0041 uM, 0.0014 uM and 0.0005 uM, and final concentration of DMSO in the reaction mixture was 2%) or 5 uL control (2% DMSO). The reaction mixture was added into desired wells of a 96-well plate. The plate was sealed and incubated for 80 min at room temperature.2) Next, 25 uL ADP detection mix was added into each well. The plate was sealed again and incubated for 60 min at room temperature. Then fluorescence polarization was measured by Tecan Infinite F500 Reader.Data was analyzed and IC50 values were generated using the add-in software for Microsoft Excel, Xlfit (version 5.3).
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 448.87, ALogP 3.24, HBA 10, HBD 2, PSA 136.27, QED 0.48
Compound (SMILES): CC1(C)CN(c2ccccc2NC(=O)Nc2ccc(OC(F)(F)F)cc2)c2c(O)ccc(-c3ccncc3Cl)c21
InChIKey: YKSVLCAZQWYDLJ-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 568.98, ALogP 8.08, HBA 5, HBD 3, PSA 86.72, QED 0.23
Compound (SMILES): COc1ccc(Nc2cc(-c3ccccc3OC)ncn2)cc1F
InChIKey: QZIULHJPRUECDI-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: Fluorescent Polarization Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations. Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kianse buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 325.34, ALogP 4.04, HBA 5, HBD 1, PSA 56.27, QED 0.77
Compound (SMILES): O=C(NO)c1cc(Nc2nc3cc(Cl)c(Cl)cc3o2)ncn1
InChIKey: IFKMIAWLUVIHFH-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: Time Resolved Fluorescence (TRF) Assay: The probe binding HDAC11 assay was performed using a time resolved fluorescence (TRF) assay format. Recombinant N-terminal GST tag full-length human HDAC11 was expressed and purified from baculovirus in Sf9 insect cells (SignalChem, # H93-30G-1000). Each assay was performed in 1536 black well microplates (Corning, #3936) in a final volume of 8 Î¼L in assay buffer containing 50 mM HEPES (pH 7.5), 50 mM KCl, 50 mM NaCl, 0.5 mM GSH (L-Glutathione reduced, Sigma #G4251), 0.03% BGG (0.22 Î¼M filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). 100 nL of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into respective wells of 1536 assay plates for a final test concentration range of 25 Î¼M to 1.3 nM respectively. The final concentration in the assay of HDAC11 and probe (a fluorescein labeled HDAC 11 inhibitor) was 2.5 nM and 20 nM respectively. 4 Î¼L of 2Ã probe and 2Ã anti-GST Terbium (Cisbio, #61GSTXLB) was added to assay plates followed by 4 Î¼L of 2ÃHDAC11. Plates were incubated for 16 hours at room temperature before time resolved fluorescence was read on the Envision (Excitation at 340 nm, and Emission at 485 nm and 535 nm, Perkin Elmer).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 340.13, ALogP 2.79, HBA 7, HBD 3, PSA 113.17, QED 0.5
Compound (SMILES): Cc1c(C(=O)NC2(C(F)F)CC2)cc(Nc2nc(-c3cc4c(nc3F)C(C)(C)C(=O)N4C3CC(C)(N4CCCCC4)C3)cc3ncn(C(C)C)c23)c(F)c1F
InChIKey: RPARKMSQZWXCNI-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBHPK1 IC50 Assay: Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 764.84, ALogP 8.1, HBA 8, HBD 2, PSA 108.28, QED 0.13
Compound (SMILES): Cc1cc(OC(F)F)c(C)cc1Nc1nc(=O)c(Cl)cn1Cc1cc(F)cc(F)c1
InChIKey: YBSVGYKVFLAVHN-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 441.81, ALogP 5.19, HBA 5, HBD 1, PSA 56.15, QED 0.54
Compound (SMILES): CN(C)C(=O)/C=C/CC[C@H](OC(=O)N(C)C)C(=O)Nc1cccn(Cc2cc3cc(F)cc(CCC(F)(F)F)c3[nH]2)c1=O
InChIKey: NNHZDHYDDBJELP-LNLBBOMNSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 607.61, ALogP 4.44, HBA 6, HBD 2, PSA 116.74, QED 0.25
Compound (SMILES): O=c1[nH]ncc(N2Cc3c(F)ccc(OCCO)c3C2)c1C(F)(F)F
InChIKey: IDDKBPYIVFJFDB-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 359.28, ALogP 1.82, HBA 5, HBD 2, PSA 78.45, QED 0.81
Compound (SMILES): O=c1[nH]ncc(N2Cc3cccc(OC4CCN(C(c5ccccc5)c5ccccc5)CC4)c3C2)c1C(F)(F)F
InChIKey: HXSUCSVCTSWKJF-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 546.59, ALogP 5.94, HBA 5, HBD 1, PSA 61.46, QED 0.32
Compound (SMILES): CC(=O)N1CCCCC1COC(=O)Nc1cc(-c2cccs2)ccc1N
InChIKey: SBFYCTBUKVTFCR-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC1: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. Trypsin Coupled Assay: HDAC1-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tweem-20 (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.18 ug/ml HDAC1 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 18uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 17550 nM (pChEMBL 4.760000228881836)
Descriptors: MW 373.48, ALogP 3.95, HBA 5, HBD 2, PSA 84.66, QED 0.79
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@@H](NC(=O)COCCCOCCCCOCCCOc2ccc(Nc3ncc(I)c(NCCN(C)C(=O)C4CCC4)n3)cc2)C(C)(C)C)cc1
InChIKey: YBFBUZSEMDFXJF-PPZGWQTASA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: In vitro degradation assay: In vitro degradation assay protocol for compounds 3-33: Panc02.13 cells were purchased from ATCC and cultured in RPMI-1640 (Gibco), supplemented with 15% FBS (ATCC) and 10 Units/mL human recombinant insulin (Gibco). PROTAC treatments were carried out in 12-well plates for 16 h. TLR3 agonist Poly I:C (Invivogen; tlr1-pic) was added for the final 3 h. Cells were harvested, and lysed in RIPA buffer (50 mM Tris pH8, 150 mM NaCl, 1% Tx-100, 0.1% SDS, 0.5% Sodium Deoxycholate) supplemented with protease and phosphatase inhibitors. Lysates were clarified at 16,000 g for 10 minutes, and supernatants were separated by SDS-PAGE. Immunoblotting was performed using standard protocols. The antibodies used were TBK1 (Cell Signaling#3504), pIRF3 (abcam#ab76493), and GAPDH (Cell Signaling#5174).
Measurement: Kd = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 1126.17
Compound (SMILES): COc1cnc2c(Nc3ccc(F)c([C@]4(C)CS(=O)(=O)[C@@](C)(F)C(N)=N4)c3)ncnc2c1
InChIKey: DYKMPQOLZUUJCU-VQTJNVASSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vivo Inhibition of Beta-Secretase: Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
Measurement: IC50 = 2.68 nM (pChEMBL 8.569999694824219)
Descriptors: MW 462.48, ALogP 2.6, HBA 9, HBD 2, PSA 132.45, QED 0.6
Compound (SMILES): Fc1cccc(-c2ccc(-n3ccc4ccncc43)nc2)n1
InChIKey: QFTJCGLTHBZSOD-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Tau binding assay: For displacement tau binding assay, unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000× final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg/mL from original 30 mg/mL volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight/assay tube. [3H]-6 was prepared at 100× final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 0.25 nM. The assay plate was incubated at room temperature (25° C.) for 90 minutes. Unbound and bound ligand were separated by filtration of bound onto GF/C filter plates (pre-treated for 30 min with 0.2% polyethyleninamine) using a Packard UniFilter Harvester and washing away unbound with 2.5 ml per well ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hour in a vacuum oven and 50 μl/well MicroScint-20 were added. Plates were counted 1 min per well by Packard Topcount. Total amount of radioligand used in the assay was determined by counting 25 μl of 10× ligand stock. Data was analyzed using Activity Base software to generate 4 p fit of dose responses; and, Ki values were calculated.
Measurement: Ki = 1.41 nM (pChEMBL 8.850000381469727)
Descriptors: MW 290.3, ALogP 3.62, HBA 4, HBD 0, PSA 43.6, QED 0.53
Compound (SMILES): COc1ccc(-c2sc(NC(=O)CC3CCCC3)nc2C)cc1S(=O)(=O)Nc1ccc(F)c(Cl)c1
InChIKey: LNXQTIGMFLESCX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-kinase beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBF8
Assay: Kinase Activity Assay: In some embodiments, the subject compounds inhibit a P14-kinase, as determined by a kinase activity assay, e.g., by an assay that determines the level of incorporation of radiolabeled phosphate from [γ-32P]-ATP into a substrate molecule after treatment with a subject compound, relative to a control, by measuring the beta-particle emission rate using a scintillation counter or phosphorimaging. In certain embodiments, the subject compounds have an IC50 value for PI4K-IIIβ of less than about 1 μM, less than about 0.2 μM, less than about 0.1 μM, less than about 10 nM, less than about 1 nM, or even less, such as described in Tables 2-3. In certain embodiments, the subject compounds have an IC50 value for PI4K-IIIα of less than about 50 μM, less than about 10 μM, less than about 1 μM, less than about 0.1 μM, less than about 10 nM, less than about 1 nM, or even less, such as described in Tables 2-3. In certain further embodiments, the subject compounds have an IC50 value for PI4K-IIIβ of 50 μM or less, [etc., etc.], 10 nM or less, 6 nM or less, or even less, such as described in Tables 2-3. In certain further embodiments, the subject compounds have an IC50 value for the PI3-kinase p110α-p85 complex of between about 8 and about 10 nM, between about 8 μM and about 10 μM, or even more.
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 538.07, ALogP 6.24, HBA 6, HBD 2, PSA 97.39, QED 0.35
Compound (SMILES): NCc1cccc(-c2nc(C(=O)O)c3ccc(CN4CCCC4)n3n2)c1
InChIKey: QBGOAHVHBBRRIM-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 351.41, ALogP 2.15, HBA 6, HBD 2, PSA 96.75, QED 0.73
Compound (SMILES): c1cc(-c2cc3ccccc3[nH]2)cc(N2CCC(NCCN3CCOCC3)CC2)c1
InChIKey: SKMSDGOUUSSDHT-UHFFFAOYSA-N
Target: Transitional endoplasmic reticulum ATPase (SINGLE PROTEIN), Homo sapiens
UniProt: P55072
Assay: Biological Assay: To optimize p97 inhibitors, the C-5 trifluoromethylated trifluoromethylated indole 12 was generated as a promising lead structure. In the ADP-Glo assay, it was determined that this compound exhibits a 5.96±1.66 μM IC50 value (Table 1), after which the effect of replacing the CF3 with an SF5-group at the C-5 position as shown for compound 13 was explored. The ADP-Glo assay can be conducted as described in Chou, T.-F.; Bulfer, S. L.; Weihl, C. C.; Li, K.; Lis, L. G.; Walters, M. A.; Schoenen, F. J.; Lin, H. J.; Deshaies, R. J.; Arkin, M. R., Specific inhibition of p97/VCP atpase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains. J. Mol. Biol. 2014, 426, 2886-2899.
Measurement: IC50 = 910 nM (pChEMBL 6.039999961853027)
Descriptors: MW 404.56, ALogP 3.73, HBA 4, HBD 2, PSA 43.53, QED 0.66
Compound (SMILES): CCOc1ccnc(Nc2n[nH]c3c2CN(C(=O)N2C[C@@H](C)N(C)C[C@@H]2C)C3(C)C)n1
InChIKey: VFPDTTYPIIITKQ-KGLIPLIRSA-N
Target: Protein kinase C beta type (SINGLE PROTEIN), Homo sapiens
UniProt: P05771
Assay: Protein Kinase C beta 2 Assay: A typical assay was carried out on a 96-well, clear microtiter plate in a Molecular Devices spectrophotometer for 20 minutes at 30° C. in 0.1 mL of assay buffer containing 50 mM HEPES, pH 7.4, 5 nM PKC, 23 units of pyruvate kinase, 33 units of lactate dehydrogenase, 0.15 mM peptide, 0.1 mM ATP, 1 mM DTT, 4 mM PEP, 8 mM MgCl2, 0.3 mM NADH, 60 mM CaCl2, 10 mg/mL PS, 50 ng/mL PMA, 7.5% DMSO and from about 10,000 nM to 0.169 nM compound inhibitor. Stock solutions of 3-sn-phosphatidyl-L-serine (PS) and phorbol-12-myristate-13-acetate (PMA) were sonicated for 30 seconds just prior to addition to assay buffer and assays were initiated by the addition of 100 μM ATP.
Measurement: Ki = 4.24 nM (pChEMBL 8.369999885559082)
Descriptors: MW 428.54, ALogP 2.54, HBA 7, HBD 2, PSA 102.51, QED 0.77
Compound (SMILES): CN/C=C1/C=C(c2cccc3c2N[C@H](C)CC(=O)N3)C=CC1=N
InChIKey: WYESCIONGOQOKS-HOGZDCNDSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 831 nM (pChEMBL 6.079999923706055)
Descriptors: MW 308.38, ALogP 2.91, HBA 4, HBD 4, PSA 77.01, QED 0.68
Compound (SMILES): COc1ccc(CC(=O)NC(=N)N[C@H](Cc2ccccc2)C(=O)NCc2ccc(F)c(OC)c2)cc1F
InChIKey: HMXZVWXMUIFZPC-JOCHJYFZSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCA-GKPILFFRLK(Dnp)d-RNH2 (Enzo Life Sciences, Lörrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 μl of each substance solution with serially diluted substance concentration were added to in each case 4 μl of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 μl of substrate solution (final concentration 5 μM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 510.54, ALogP 3.09, HBA 5, HBD 4, PSA 112.54, QED 0.25
Compound (SMILES): CCOc1c(F)c(SC)c2[nH]ncc2c1-c1ccn2nc(NC(=O)[C@@H]3C[C@@H]3F)cc2c1
InChIKey: AKTPBYQEULBXMB-OCCSQVGLSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 443.48, ALogP 4.43, HBA 6, HBD 2, PSA 84.31, QED 0.43
Compound (SMILES): CN(C)CCc1ccc(-c2cnc(NCc3cccc4c3CCO4)n3cnnc23)cc1
InChIKey: RNUJULGJVKPSBQ-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 8.3 nM (pChEMBL 8.079999923706055)
Descriptors: MW 414.51, ALogP 3.44, HBA 7, HBD 1, PSA 67.58, QED 0.5