lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1ccnc(N2CC[C@H]3CCN(C(=O)c4ccccc4-n4nccn4)C[C@H]32)c1
InChIKey: BNDDJOWAWNWUOT-YLJYHZDGSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Assay: The utility of the compounds in accordance with the present invention as orexin receptor OX1R and/or OX2R antagonists may be readily determined without undue experimentation by methodology well known in the art, including the FLIPR Ca2+ Flux Assay. In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined.
Measurement: IC50 = 40.5 nM (pChEMBL 7.389999866485596)
Descriptors: MW 399.46, ALogP 2.27, HBA 7, HBD 0, PSA 90.94, QED 0.67
Compound (SMILES): COCCOc1nccc2cc(Nc3n[nH]c4nccnc34)ccc12
InChIKey: VFABHYARLBFYCZ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGuR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGuR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured. Approximately two and a half minutes later, a concentration of mGuR4 orthosteric agonist (e.g. glutamate or L-AP4) eliciting approximately 20% (EC20) of the maximal agonist response was added to the cells, and the response was measured. Two minutes later, a concentration of mGluR4 agonist (e.g. glutamate or L-AP4) eliciting 80% (EC80) of the maximal agonist response was added to the cells, and the response was measured. For rat mGuR4/GIRK experiments, a baseline was established for approximately five seconds, disclosed compounds were added, and either an EC20 or EC80 concentration of agonist was added approximately two and one half minutes later.
Measurement: EC50 = 35.2 nM (pChEMBL 7.449999809265137)
Descriptors: MW 336.36, ALogP 2.67, HBA 7, HBD 2, PSA 97.84, QED 0.52
Compound (SMILES): CC(C)c1ncccc1-c1ncc2c(n1)N(Cc1ccc(-c3nc(C(F)(F)F)cn3C3CN(C)C3)cc1)C(=O)C2
InChIKey: XPORHKULYYHEDY-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 547.59, ALogP 5.12, HBA 7, HBD 0, PSA 80.04, QED 0.33
Compound (SMILES): COCc1cc(-c2[nH]c3ccc(C4CCN(CC(=O)N5CC(C)(C)C5)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: YDWUXOGKHLOMDN-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 528.7, ALogP 5.2, HBA 6, HBD 1, PSA 78.76, QED 0.36
Compound (SMILES): Cn1ccc(-c2cc(C(=O)Nc3cnc(CN)nc3-c3ccccc3)ccc2C(F)(F)F)n1
InChIKey: BAMRETPYUFMNKC-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Functional Activity Assay: TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shcl is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shcl-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.All reagents were purchased from DiscoverX, except for the receptor agonists (NGF, BDNF, NT3) which were purchased from Peprotech. Cells were expanded and frozen into cryovials, and stored in the vapor phase of liquid nitrogen, and thawed immediately before use. Thawed cells were added to a 384-well plate at 7500 cells/well, and allowed to incubate overnight. Compound at various concentrations was added the following morning and allowed to incubate on cells for 1 h. Then, NGF was added at a concentration sufficient to elicit 80% of a maximal response and allowed to incubate for 3 h at ambient temperature.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 452.44, ALogP 4.27, HBA 6, HBD 2, PSA 98.72, QED 0.47
Compound (SMILES): CCCNC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3ccc(OC)cc3S(C)(=O)=O)n[nH]2)C1
InChIKey: RNYHEDWENUCLMZ-WMLDXEAASA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.67 nM (pChEMBL 9.170000076293945)
Descriptors: MW 478.57, ALogP 2.78, HBA 7, HBD 3, PSA 139.48, QED 0.5
Compound (SMILES): CC(C)(CNC(=O)C1CCN(c2ncc(C(=O)Oc3ccc(C(=N)N)cc3)s2)CC1)C(=O)O
InChIKey: BDJUDABBRAYMKT-UHFFFAOYSA-N
Target: Enteropeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P98073
Assay: Enteropeptidase Inhibition Assay: The inhibitory activity of the enteropeptidase inhibitor synthesized using the purified Recombinant Human Enteropeptidase and the substrate Acetyl-Asp-Asp-Asp-Asp-Lys-AFC (BioVision) was measured. 7.2 ng/mL of Enteropeptidase diluted with a buffer (20 mM Tris, 50 mM NaCl, pH 7.5) in a 96 well plate (Costar), 30 μM of Acetyl-Asp-Asp-Asp-Asp-Lys-AFC, several concentrations of Enteropeptidase inhibitors (1% DMSO concentration) were dispensed such that a final volume was 100 μL, and then the enzyme reaction was carried out at 30° C. for 1 hour. At this time, 1% DMSO, a substrate and Enteropeptidase instead of the compound were dispensed onto positive control wells, and 1% DMSO and a substrate were dispensed onto negative control wells. The enzyme reaction was started using an excitation wavelength of 380 nm and an emission wavelength of 500 nm in a fluorescence spectrometer, and then the rate of increase in fluorescence (milli-units per min) between 20 and 60 minutes was measured.
Measurement: Ki = 9.7 nM (pChEMBL 8.010000228881836)
Descriptors: MW 473.56, ALogP 2.09, HBA 8, HBD 4, PSA 158.7, QED 0.2
Compound (SMILES): CC(C)[C@H]1CN(c2cccc(S(C)(=O)=O)c2)CCN1C[C@@H]1CC[C@@H](C(F)(F)F)OC1
InChIKey: GEXZSCVAMWWQAV-DBVUQKKJSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki = 333 nM (pChEMBL 6.480000019073486)
Descriptors: MW 448.55, ALogP 3.59, HBA 5, HBD 0, PSA 49.85, QED 0.69
Compound (SMILES): COc1cccc(-c2nc3ccc(C(=O)NCCCn4ccnc4)cc3nc2-c2ccccc2)c1
InChIKey: TUCFZBOXCRNNOI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 463.54, ALogP 4.99, HBA 6, HBD 1, PSA 81.93, QED 0.33
Compound (SMILES): O=C(NS(=O)(=O)[C@@H]1CCC[C@H]1O)c1ncc(N2C(=O)[C@@H]3C[C@H]2C[C@H]3OCc2c(-c3c(Cl)cccc3Cl)noc2C2CC2)cc1F
InChIKey: GHOWCLGXEUYKFX-WRMOXONVSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Binding Assay: The affinity of FXR ligands for the ligand binding domain of FXR was determined using a commercially available human FXR ligand binding assay (LanthaScreen, Thermofisher Cat # PV4833). The purified ligand binding domain of human FXR tagged with GST (glutathiones-S-transferase) is incubated with a terbium labelled anti-GLT antibody and a fluorescein-labelled SRC2-2 peptide (LKEKHKILHRLLQDSSSPV (SEQ ID NO.: 1)). Binding of FXR ligands to the FXR ligand binding domain promotes binding of the fluorescein-labelled SRC2-2 peptide. This causes a FRET signal between the terbium-labelled anti-GST antibody and the fluorescein-labelled SRC peptide which are both bound to the FXR ligand binding domain.Test compounds are dissolved in DMSO and a 3-fold serial dilution series is generated, then further diluted into assay buffer. The compounds are mixed with 5 nM GST-tagged FXR ligand binding domain, 5 nM Tb-labelled anti-GST antibody and 500 nM fluorescein-labelled SRC2-2 peptide in a pH7.4 buffer. The reaction is incubated at room temperature for 1 hour, then the FRET signal is measured as the ratio of the 520 nm/495 nm emission following excitation at 340 nm. The change in FRET signal is plotted against the test article concentration and fit to a 3-parameter logistical equation.
Measurement: EC50 = 6.31 nM (pChEMBL 8.199999809265137)
Descriptors: MW 679.55, ALogP 4.74, HBA 9, HBD 2, PSA 151.93, QED 0.33
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3cc(F)c(C(C)(C)O)cc3n([C@H](c3ccc(F)cc3)C3CCOCC3)c2c1
InChIKey: BHEQYIQLHLSTDG-GDLZYMKVSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 531.61, ALogP 5.82, HBA 7, HBD 1, PSA 77.99, QED 0.31
Compound (SMILES): CCCCNc1ncc(-c2ccc(CCN(C)C)cn2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: MJKYJDTWPAMHCX-WGSAOQKQSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 412.58, ALogP 3.57, HBA 7, HBD 3, PSA 86.2, QED 0.51
Compound (SMILES): COc1nccc(Sc2ncc(N3CCC4(CCC[C@H]4N)CC3)nc2N)c1Cl
InChIKey: BVMGFHBKWCPQQH-CYBMUJFWSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat#3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat# D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then quenched by the addition of 5 μl of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 420.97, ALogP 3.36, HBA 8, HBD 2, PSA 103.18, QED 0.78
Compound (SMILES): CC(C)CC(=O)N1C[C@H](F)[C@H](NC(=O)c2cccc(-c3cc(C(F)(F)F)c4c(N)ncnn34)c2F)C1
InChIKey: MAONMVLPPNVFNT-JKSUJKDBSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 29000 nM (pChEMBL 4.539999961853027)
Descriptors: MW 510.47, ALogP 3.46, HBA 6, HBD 2, PSA 105.62, QED 0.51
Compound (SMILES): CS(=O)(=O)NCC1(c2ccc(N3CCC[C@@H](NC(=O)Nc4ncc(Cl)cc4Cl)C3=O)cc2)CC1
InChIKey: FKXYIPZSHLGKKA-GOSISDBHSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 1.7 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 flurorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 675 nM (pChEMBL 6.170000076293945)
Descriptors: MW 526.45, ALogP 3.29, HBA 5, HBD 3, PSA 120.5, QED 0.51
Compound (SMILES): C[C@@H]1C[C@H](NC(=O)c2cc(C3CC3)on2)CCN1S(=O)(=O)CCCN
InChIKey: MHOHMFSGWGOOOF-DGCLKSJQSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 55.9 nM (pChEMBL 7.25)
Descriptors: MW 370.48, ALogP 0.81, HBA 6, HBD 2, PSA 118.53, QED 0.73
Compound (SMILES): CCCC(=O)n1nc(-c2ccccn2)nc1NCc1ccc(F)cc1
InChIKey: YNHHXWUXGOCAAU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: KLKB1 Activity Assay: Human KLKB1 protein was obtained from Enzyme Research Labs. The chromogenic substrate S-2302 was obtained from DiaPharma. KLKB1 was assayed in buffer containing 0.05 M Tris (pH 7.4), 0.01 M NaCl and 0.2% w/v PEG-8000. The final concentration of enzyme used was 3 nM KLKB1. The final concentration of substrate used was 250 μM S-2302 for KLKB1. All assays were performed in 96-well microtiter plates at room temperature (RT). The enzyme and inhibitor were pre-incubated for 10 minutes then substrate was added and read at 405 nm in a SpectraMax Plus Spectrophotometer (Molecular Devices). Inhibitor IC50 values were determined by adding test compound as ten point, three-fold serial dilutions in buffer solution, as known in the art. The plate was read at 10 minutes after substrate addition.
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 339.37, ALogP 3.53, HBA 6, HBD 1, PSA 72.7, QED 0.74
Compound (SMILES): CCCCNc1ncc(C(=O)NCc2ccc3ccccc3n2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: BXUFOWVQUPXSQM-KESTWPANSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 448.57, ALogP 3.88, HBA 7, HBD 4, PSA 112.06, QED 0.37
Compound (SMILES): C[C@H]1[C@H](c2nccs2)OC(=O)N1c1cc(-c2cnc(N)nc2C(F)(F)F)nc(N2CCOCC2)n1
InChIKey: ONCWHTARVIIEDJ-IINYFYTJSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Biochemical Assays for PI3Kalpha, PI3Kbeta: The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. (L-alpha-phosphatidylinositol (PI), Liver, Bovine) were obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), Phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2(Avanti, Cat. No. 840046X) or L-α-phosphatidylinositol (PI) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274). L-α-Phosphatidylserine (PS) was from Avanti Polar Lipid (Cat. No. 840032C), n-Octylglucoside Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison/WI, USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676) as described in section 8.2. L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under nitrogen beam. It was then resuspended in 3% OctylGlucoside by vortexing and stored at 4° C. 5 μL of a mix of PI/OG with the PI3Ka and Pi3Kb subtypes were added. Kinase reactions were started by addition of 5 μl of ATP-mix containing in a final volume 10 μL 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP, and occurred at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (standard) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). NVP-BGT226 was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 508.49, ALogP 2.91, HBA 11, HBD 1, PSA 132.48, QED 0.56
Compound (SMILES): Cc1cc(C2(c3cccc(C(F)(F)F)c3)N=C(N)N(C)C2=O)ccc1OC(F)F
InChIKey: JNHPCUDMTGBLRN-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: in vitro BACE inhibition assay: Approximately 200 nM to 10 μM substrate, approximately 10 to 200 pM enzyme, and approximately 0.1 nM to 10 μM of the agent(s), in aqueous solution, at an approximate pH of 4-7, at approximately 37° C., for a time period of approximately 10 minutes to 3 hours. These incubation conditions are illustrative only, and can be varied as required for the particular assay components and/or desired measurement system. Optimization of the incubation conditions for the particular assay components should account for the specific alpha-secretase and/or beta-secretase enzyme used and its pH optimum, any additional enzymes and/or markers that might be used in the assay, and the like. Such optimization is routine and will not require undue experimentation.
Measurement: IC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 413.35, ALogP 3.65, HBA 4, HBD 1, PSA 67.92, QED 0.78
Compound (SMILES): CCOC(=O)NC(CCCCC(=O)Nc1ccccc1N)C(=O)Nc1cccc(OC2CC2)c1
InChIKey: SJFBOJTZIUBOCM-UHFFFAOYSA-N
Target: Histone acetyltransferase p300/Myocyte-specific enhancer factor 2D (PROTEIN COMPLEX), Homo sapiens
UniProt: Q14814
Assay: Inhibitory Effects of the Compounds on MEF2D and P300 Protein Interactions: Detection of MEF2D and P300 interactions. The same system that was used in Example 19 is used in here to detect MEF2D and P300 interactions. In this assay, two separate DNA constructs were made with MEF2D fused with the GAL4 DNA binding domain (GAL4-MEF2D) and P300 fused with the viral transactivator VP-16 (HDAC4-VP16). Human epithelial carcinoma cells (HeLa) were co-transfected with the two DNA constructs (GAL4-MEF2D and P300-VP16) and a reporter plasmid (Gal4Luc). The DNA constructs resulted in expression of the GAL4-MEF2D and P300-VP16 fusion proteins within the cell. MEF2D and P300 protein interactions were detected as a result of the GAL4-MEF2D and P300-VP16 protein complex binding a DNA promoter on the reporter plasmid (Gal4Luc) and driving cellular expression of a luciferase gene. The measured luciferase protein signal was proportional to the amount of MEF2D and P300 protein interactions occurring with the cell.
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 468.55, ALogP 4.06, HBA 6, HBD 4, PSA 131.78, QED 0.27
Compound (SMILES): C[C@H](Oc1cc(-n2nc(C3CCC3)n(C)c2=O)c(F)cc1C(=O)Nc1ccccc1F)C(F)(F)F
InChIKey: LBMCQUKZMYMYSZ-LBPRGKRZSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay 1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 496.44, ALogP 4.7, HBA 6, HBD 1, PSA 78.15, QED 0.5
Compound (SMILES): CNC(=O)c1cc(C)c(F)c(-c2cnn3cc(-c4cnn(C)c4)c(OC4CCOC4)nc23)c1
InChIKey: SIWLVOKRZCQNMV-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR Enzymatic Assay: The inhibitor potency of the exemplified compounds was determined in an enzyme discontinuous assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.2 μL was transferred to the wells of a 384-well plate. A 5 μL/well volume of enzyme isoforms of FGFR (-1, -2, -3 wild-type and mutant isoforms, -4) including phosphorylated and un-phosphorylated proteins diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated with inhibitor for 5 to 15 minutes at ambient temperature. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The reaction was initiated by the addition of a 5 μL/well volume containing both biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP in assay buffer. The 10 μL/well reaction concentration of the peptide substrate was 500 nM whereas the ATP concentration was maintained near or below the ATP Km. The ATP Km values were pre-determined in a separate series of experiments. The reaction plate was incubated at 25° C. for 1 hr and the reactions were ended with the addition of 5 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 45 mM EDTA, 600 nM staurosporin, with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 10 minutes at ambient temperature before scanning on a PheraStar plate reader (BMG Labtech) instrument.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 450.47, ALogP 2.77, HBA 8, HBD 1, PSA 95.57, QED 0.5
Compound (SMILES): COc1ccc(F)cc1-c1c(F)cnc2[nH]c(C3=CCN(CC(=O)N[C@H]4CC[C@H](O)CC4)CC3)cc12
InChIKey: IJNMSWQNUBKIMG-WGSAOQKQSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 496.56, ALogP 4.03, HBA 5, HBD 3, PSA 90.48, QED 0.48
Compound (SMILES): CC(C)[C@H]1CN(c2cccc(S(C)(=O)=O)c2)CCN1CC1CCC(C(F)(F)F)N(C)C1
InChIKey: NTWOSTRWTSTGLC-TWMPVWRBSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 461.59, ALogP 3.51, HBA 5, HBD 0, PSA 43.86, QED 0.67
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(N(C)CC(F)(F)F)nc1Cl)C3c1ncc(C2CC2)cc1F
InChIKey: PKYTWNNMNMIUMN-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 610.04, ALogP 5.11, HBA 9, HBD 0, PSA 88.75, QED 0.21
Compound (SMILES): CS(=O)(=O)Nc1nnc2cc(OC3C4CC5CC(C4)CC3C5)c(C3CC3)cn12
InChIKey: ODBLMALRJHFUOR-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): The following patch voltage clamp electrophysiology studies were performed on representative compounds of the invention using human embryonic kidney cells (HEK), permanently transfected with an expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit, grown in culture media containing 10% FBS, 1% PSG, and 0.5 mg/mL G418 at 37° C. with 5% CO2. HEK cells used for the electrophysiology (EP) recordings had a passage number of less than 40 for all studies and were used within three days from the time of plating. NaV1.7 and NaV1.5 cDNAs (NM_002977 and AC137587; SCN5A, respectively) were stably expressed in HEK-293 cells. The β1 subunit was coexpressed in both the NaV1.7 and NaV1.5 cell lines.Sodium currents were measured using the patch clamp technique in the whole-cell configuration using either a PatchXpress automated voltage clamp or manually using an Axopatch 200B (Axon Instruments) or Model 2400 (A-M systems) amplifier. The manual voltage clamp protocol was as follows: Borosilicate glass micropipettes were fire-polished to a tip diameter yielding a resistance of 2-4 Mohms in the working solutions. The pipette was filled with a solution comprised of: 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaC12, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA; and adjusted to pH 7.2 with CsOH. The external solution had the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaC12, 1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH. In some studies, the external sodium was reduced by equimolar replacement with choline. Osmolarity in the CsF internal and NaCl external solutions was adjusted to 300 mOsm/kg and 310 mOsm/kg with glucose, respectively. All recordings were performed at ambient temperature in a bath chamber with a volume of 150 μL. Control sodium currents were measured in 0.5% DMSO. Controls and representative compounds of the invention were applied to the recording chamber through a 4-pinch or 8-pinch valve bath perfusion system manufactured by ALA Scientific Instruments.
Measurement: IC50 = 344 nM (pChEMBL 6.460000038146973)
Descriptors: MW 402.52, ALogP 3.18, HBA 6, HBD 1, PSA 85.59, QED 0.83
Compound (SMILES): O=C(O)c1ccc(NC(=O)C2c3cccc(N4CCN(CC(F)(F)F)CC4)c3CCN2C(=O)c2cn(-c3cccc(Cl)c3F)nn2)cc1
InChIKey: HLCWXSMXMUHDBM-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 686.07, ALogP 4.82, HBA 8, HBD 2, PSA 123.9, QED 0.27