lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)C(C)(C)CC(=O)N1CCC(c2cn(C)c3ncc(NC(=O)c4cccc(C#N)c4)c(C(F)(F)F)c23)CC1
InChIKey: GQVYFUTZXPRWGL-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Assay of Co-Activator Recruitment by TR-FRET: Specifically, in one embodiment the aforementioned assay was performed as outlined below. The assay was carried out in black polystyrene, 384-well plates in a total assay volume of 50.5 μL. The assay buffer contained 50 mM TRIS¿HCL pH 7.5, 1 mM NaCl, 2 mM MgCl2, 0.5 mg/mL bovine serum albumin, and 5 mM dithiothreitol. The final concentration of reagents was 6.3 nM RORC2 LBD, 200 nM SRC1-2, 50 nM streptavidin APC, 1 nM Europium-labeled anti-His antibody, and varying concentrations of compounds such that final concentration of DMSO is 1% (v/v). The assay steps were: (1) dispensing 500 μL compound at 100× final concentration in DMSO (test wells) or DMSO only (control wells for no inhibition); and (2) dispensing 50 μL mixture of the other assay components including receptor (test wells) or excluding receptor (control wells for maximal inhibition). Assay mixtures were incubated are room temperature for 3 hr and read in EnVision 2100 Multilabel Reader (PerkinElmer Life Sciences) at Excitation Filter 320, Emission Europium Filter 615, Emission APC Filter 665, Dichroic Mirror D400/D630. TR-FRET signal was determined by calculating the ratio of 665 nm by 615 nm and IC50 values of compounds of the invention (Table 1) were determined by the non-linear regression analysis of dose response curves.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 553.63, ALogP 6.49, HBA 5, HBD 1, PSA 91.02, QED 0.38
Compound (SMILES): Cc1nc2cc(C(F)(F)F)c(N3CCN(C)CC3)c(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2cnsc12
InChIKey: MEYAISJZHOLGMY-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 4.77 nM (pChEMBL 8.319999694824219)
Descriptors: MW 640.09, ALogP 5.75, HBA 10, HBD 0, PSA 100.17, QED 0.23
Compound (SMILES): CC(C)N1CCN(CCNC2CCN(c3cccc(-c4cc5ccccc5[nH]4)c3)CC2)C(=O)C1
InChIKey: FUZHCAZRFGKXGF-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 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 459.64, ALogP 3.95, HBA 4, HBD 2, PSA 54.61, QED 0.56
Compound (SMILES): CCNc1nc(Nc2cc(C3(C)CCOC3=O)nn2C2CC2)ncc1Br
InChIKey: QEAXZAQDYNKCMP-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:    LRRK2 G2019S enzyme    Substrate (LRRKtide)    ATP    TR-FRET dilution buffer    pLRRKtide antibody    384-well assay plate    DMSOEnzyme Reaction Conditions    50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT    5 nM LRRK2    134 μM ATP    60 minute reaction time    23° C. reaction temperature    10 μL total reaction volumeDetection Reaction Conditions    1× TR-FRET dilution buffer    10 mM EDTA    2 nM antibody    23° C. reaction temperature    10 μL total reaction volumeCompounds were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2× ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using a 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 421.3, ALogP 3.15, HBA 8, HBD 2, PSA 93.96, QED 0.69
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Cl)n2)c2ccc(Nc3cncnc3)cc12
InChIKey: HYNKZTKMOWBTPY-ACJLOTCBSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz1 and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 537.94, ALogP 2.29, HBA 9, HBD 3, PSA 161.02, QED 0.3
Compound (SMILES): CC(C)[C@@H]1CN2C(=CC3C=NC(Nc4ccc(N5CCNCC5)cn4)=NC32)C(=O)N1
InChIKey: YGRZRIXNWACOMU-RCDNUKIMSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biological Activity Assay: Kinase enzymatic reactions were performed in 384-well microplates using a 12-channel Caliper LabChip instrument as a detection device. The enzymatic phosphorylation of a peptide results in a change in net charge, enabling electrophoretic separation of product from substrate. As substrate and product are separated, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured, reflecting enzyme activity. In the presence of an inhibitor, the ratio between product and substrate is altered. The signal of the product decreases, while the signal of the substrate increases.For the measurement of CDK2/cyclinE activity, enzyme (0.22 nM) was incubated with 100 mM ATP and the phosphoacceptor substrate peptide (1 mM) for one hour. For the measurement of CDK4/CyclinD activity, enzyme (0.85 nM) was incubated with 200 mM ATP and the phosphoacceptor substrate peptide (1 mM) for three hours. Potential inhibitor compounds (as HCl salts) were tested using 12-point dose response curves in single point at the Km for ATP.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 408.51, ALogP 0.64, HBA 8, HBD 3, PSA 97.25, QED 0.68
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3c(N4CCN(CC(N)=O)CC4)ccc(S(C)(=O)=O)c3n([C@H](c3ccccc3)C3CCOCC3)c2c1
InChIKey: VXYPWPFNZRVAMN-MGBGTMOVSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The binding of compounds to bromodomain BRD4 (44-168), BRD4 (333-460), and BRD4 (1-477 or 44-460) was assessed using a time resolved fluorescent resonance energy transfer binding assay (1), that measures the binding of a fluorescently labeled probe molecule to the bromodomain protein. The bromodomain protein, fluorescent probe molecule (either a biotinylated histone peptide or a fluorescently labeled small molecule), and dose-responsed test compound are incubated together to reach thermodynamic equilibrium. In the absence of a test compound, the bromodomain and small molecule are bound, resulting in a high fluorescent signal. In the presence of a sufficient concentration of inhibitor, this interaction is disrupted resulting in a lost of fluorescent resonance energy transfer.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. All data was normalized to 16 high vehicle wells and 8 low reference control wells, and then a four parameter curve fit was applied:Y=a+((b−a)/(1+(10x/10c)d)Where 'a' is the minimum, 'b' is the Hill slope, 'c' is the IC50, and 'd' is the maximum.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 656.81, ALogP 3.32, HBA 11, HBD 1, PSA 141.47, QED 0.27
Compound (SMILES): COc1ccc(Cn2c(CC3CCCC3)cn3c(C4CCOCC4)ncc3c2=O)cc1
InChIKey: TUNXEGCQHJFZRL-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 421.54, ALogP 4.18, HBA 6, HBD 0, PSA 57.76, QED 0.6
Compound (SMILES): CCOC(=O)c1csc(CN(Cc2ccc(OCc3cccnc3)cc2)Cc2cccnc2)n1
InChIKey: KGAQRVZWPHHDGP-UHFFFAOYSA-N
Target: Collagenase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P45452
Assay: Activity Assay: Stock solution of substrate (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was prepared in DMSO at a concentration of 6 mM. Assays were performed in an assay buffer (50 mM Tris pH 7.5, 300 mM NaCl, 10 uM ZnSO4, 5 mM CaCl2, 0.005% Brij-35). The final DMSO concentration in all assays was 1.0%. The used substrate concentration was 3.33 uM. The assays were carried out at 37° C. The fluorescence changes, resulting from the substrate cleavage, were detected using excitation at 340 nm and emission wavelength of 405 nm using a Wallac 1420 Microplate Reader. The reaction mixtures were preincubated with the inhibitors for 30 minutes. The reactions were started by the addition of MMP substrate, and the fluorescence intensity changes were measured after 60 minutes. The obtained fluorescence values were normalized and % inhibition values calculated. Dose response curves were determined from 6 concentration points using three fold dilution steps, each concentration point was determined in dupllicate. IC50 values were calculated from the dose response curves. FIGS. 1-4 show the dose response curves of Compound 1 and ilomastat on on MMP1, -2, -9, -13 activities respectively.
Measurement: IC50 = 19000 nM (pChEMBL 4.71999979019165)
Descriptors: MW 474.59, ALogP 4.89, HBA 8, HBD 0, PSA 77.44, QED 0.29
Compound (SMILES): Cc1cc(-c2cncc([C@@]34CC[C@@H](c5cc(-c6c(F)cccc6F)nnc53)C4(C)C)n2)nn1C[C@@H](O)CO
InChIKey: KSHHKZYKCVOLOF-AUPSIVQCSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 518.57, ALogP 3.94, HBA 8, HBD 2, PSA 109.84, QED 0.4
Compound (SMILES): O=C1N[C@]2(CO1)C[C@H](C(=O)N1CC3(CC(c4ccc5c(c4)CCC5)C3)C1)C2
InChIKey: IQSNBZSTQQYWMH-CXGRPWHSSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Biological Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition.
Measurement: IC50 = 0.62 nM (pChEMBL 9.210000038146973)
Descriptors: MW 366.46, ALogP 2.77, HBA 3, HBD 1, PSA 58.64, QED 0.88
Compound (SMILES): Cc1nc2cnc(N3CCN(CC(C)(C)F)CC3)c(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)NS(C)(=O)=O)csc12
InChIKey: UBMCKLCISZORNS-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 753.28, ALogP 4.54, HBA 13, HBD 1, PSA 163.41, QED 0.21
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc6c(c5)CCO6)nc4n3CC3CC3)n(C)c12
InChIKey: RGTFQUSSGOKNRJ-XBPDSQQVSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 588.71, ALogP 5.17, HBA 8, HBD 1, PSA 100.43, QED 0.29
Compound (SMILES): C[C@H](Nc1nccc(N(CC(C)(C)C)S(C)(=O)=O)n1)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: PDFBGBSIUQDAET-ZDUSSCGKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 M Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 463.99, ALogP 3.96, HBA 6, HBD 2, PSA 108.05, QED 0.57
Compound (SMILES): Cn1ccc2c(Cl)c(C(=O)N3CCN4C[C@@H](c5ccc(Cl)cc5)CC[C@@H]4C3)cnc21
InChIKey: KFLWPEHWYYBSLA-FUHWJXTLSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition Assay: In one embodiment, the present invention provides compounds of formula (Ie) and their pharmaceutically acceptable salts as described herein, wherein said compounds of formula (Ie) and their pharmaceutically acceptable salts have an IC50 for MAGL below 25 μM, preferably below 10 μM, more preferably below 5 μM as measured in an assay comprising the steps of: list-style a) providing a solution of a compound formula (e), or a pharmaceutically acceptable salt thereof, in DMSO; b) providing a solution of MAGL (recombinant wild-type) in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid); c) adding 1 μL of compound solution from step a) to 19 μL of MAGL solution from step b); d) shaking the mixture for 1 min at 2000 rpm; e) incubating for 15 min at RT; f) adding 20 μL of a solution of 4-nitrophenlyacetate in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid, 6% EtOH); g) shaking the mixture for 1 min at 2000 rpm;h) incubating for 5 min at RT; i) measuring the absorbance of the mixture at 405 nm a first time; j) incubating a further 80 min at RT;k) measuring the absorbance of the mixture at 405 nm a second time; l) substracting the absorbance measured under i) from the absorbance measured under k) and calculating the slope of absorbance; wherein: i) the concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof in the assay after step f) is in the range of 25 μM to 1.7 nM; ii) the concentration of MAGL in the assay after step f) is 1 nM; >iii) the concentration of 4-nitrophenylacetate in the assay after step f) is 300 μM; and iv) steps a) to 1) are repeated for at least 3 times, each time with a different concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 443.38, ALogP 4.58, HBA 4, HBD 0, PSA 41.37, QED 0.58
Compound (SMILES): CC(C)n1nnnc1-c1nc(NC(=O)c2cc(-c3cnn(C4CC4)c3)c(N3CCCC3)cn2)cs1
InChIKey: LNZFJTYMAYAYIU-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 490.6, ALogP 3.82, HBA 11, HBD 1, PSA 119.54, QED 0.42
Compound (SMILES): Cc1ccc(-n2ncnc2[C@@H]2O[C@H](CO)[C@H](O)[C@H](n3cc(-c4cc(F)c(F)c(F)c4)nn3)[C@H]2O)cc1
InChIKey: JIASLAPPHPKIBS-WHCFWRGISA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Biological Assay: The Gal-3 assays were performed in 384 white Opti plates in three replicates at room temperature with gentle shaking at 250-300 rpmFrom the original stocks, 2.525× working stock concentrations of His-tagged recombinant human Gal-3 (hGal-3) and that of B-ASF were prepared. From the working stock, 20 μl of hGal-3 (15 nM) and 20 μl B-ASF (15 nM) were added to the plates. In Negative Control, only hGal-3 was added. A concentration range of 50× working stocks were prepared for the compounds in 100% DMSO. Aliquots of 1 uL of the compounds were added to the wells and pre-incubated with 20 μl hGal-3 per well for 30 minutes Then 20 μl B-ASF wereadded and incubated for another 1 hour. To detect the signal, 5 μL (final conc. of 1.0 nM) terbium labelled Anti-His antibody was added and incubated for 30 min followed by adding 5 μL (final conc. of 20 nM) Streptavidin d2 and incubation for another 1 hour. The assay signal was detected using HTRF screen protocol (Excitation wavelength=340 nm, emission wavelength=615 nm/665 nm) on Envision 2104 Multilabel Reader. Data analysed using Toolset and Curve Master. Results are reported in the experimental section (IC50 in μM).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 502.45, ALogP 1.65, HBA 10, HBD 3, PSA 131.34, QED 0.35
Compound (SMILES): O=C(NC1CC1)c1nc2cccnn2c1-c1cc(F)c(Cl)cc1F
InChIKey: IJGCWHSMOTWWLO-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays: The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays use the Scintillation Proximity Assay (SPA) technology to measure the inhibition of human recombinant PDE4A1, PDE4B3, PDE4C1, and PDE4D3 enzyme activity by compounds in vitro. The PDE4A1, PDE4B3, PDE4C1, and PDE4D3 assays are run in parallel using identical parameters, except for the concentration of enzyme (80 pM PDE4A3, 40 pM PDE4B3, 40 pM PDE4C1 and 10 pM PDE4D). The assays are performed in a 384-well format with 50 uL assay buffer (50 mM TRIS pH7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4A3, PDE4B1, PDE4C1, and PDE4D to convert ¿20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 uM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 uL of 8 mg/ml yitrium silicate SPA beads (Perkin Elmer) stops the reaction. The plates are sealed (TopSeal, Perkin Elmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 348.74, ALogP 3.22, HBA 4, HBD 1, PSA 59.29, QED 0.74
Compound (SMILES): O=C(O)c1cccc(S(=O)(=O)N2CCC(c3n[nH]c4ccc(C(c5ccc(Cl)cc5)c5ccc(Cl)cc5)cc34)CC2)c1
InChIKey: ANXXADRQGPNLCU-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Biological Assay: CB1 and CB2 receptors are Gi-coupled GPCR. Activation of CB1 and CB2 receptors results in a decrease in cAMP production. An inverse agonist of the CB1 or CB2 receptor results in the opposite effect, an increase of cAMP production. The principle of this assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the fluorophore d2. The tracer binding is quantified by a Mab anti-cAMP labeled with Eu3+TBP-NHS Cryptate (supplied as part of the assay kit). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
Measurement: EC50 = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 620.56, ALogP 7.32, HBA 4, HBD 2, PSA 103.36, QED 0.19
Compound (SMILES): O=C(NCC(O)CNC1Cc2ccccc2C1)c1ccc(N2CCCCC2=O)cc1
InChIKey: LMIXENLBDITFMW-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: iochemical Assay: Compounds of the invention may be tested for in vitro activity in the following assay: A histone H4 derived peptide is used as substrate (amino acid sequence: Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-NH2). Full-length PRMT5 enzyme (NCBI Reference sequence NP-006100.2) was co-expressed with Hiss-MEP50 in insect cells and purified via Nickel immobilized metal affinity and gel filtration chromatograph.The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37° C. Reaction progress was measured using the Transcreener™ EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 min before being read on a PerkinElmer EnVision™ plate reader in fluorescence polarisation mode. IC50 values were obtained from the raw readings by calculating percent inhibition (% I) for each reaction relative to controls on the same plate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/positive reactions, respectively), then fitting the % I data vs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I])^D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC50 value, and D is the slope.
Measurement: IC50 = 7295 nM (pChEMBL 5.139999866485596)
Descriptors: MW 407.51, ALogP 2.05, HBA 4, HBD 3, PSA 81.67, QED 0.66
Compound (SMILES): O=C(Nc1ccc(-c2cccc3nc(NC(=O)C4CC4)nn23)cc1)C1CCC1
InChIKey: BWKNUDQJFOBENL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 375.43, ALogP 3.48, HBA 5, HBD 2, PSA 88.39, QED 0.71
Compound (SMILES): CCCc1c(-c2cccc3c2N[C@H](C)CC(=O)N3)n(C)c2ccccc12
InChIKey: ZVVXYZBNCGUJGW-CQSZACIVSA-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 = 851 nM (pChEMBL 6.070000171661377)
Descriptors: MW 347.46, ALogP 4.94, HBA 3, HBD 2, PSA 46.06, QED 0.7
Compound (SMILES): C[C@@H]1Oc2cc(-c3ccnc(Nc4ccnn4C)n3)cc3nnc(n23)[C@H]1Cc1ccccc1
InChIKey: SGEUXVVXMIAVCA-YJBOKZPZSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 < 0.02 nM (pChEMBL 10.699000358581543)
Descriptors: MW 438.5, ALogP 3.77, HBA 9, HBD 1, PSA 95.05, QED 0.45
Compound (SMILES): CNC(=O)C1CCc2c(-c3ccc(C(=O)O)cc3F)nn(C(=O)c3c(Cl)cccc3C3CC3)c2C1
InChIKey: FTDNRINDDKYCMJ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 mg/ml bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per ml in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO:1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 495.94, ALogP 4.46, HBA 5, HBD 2, PSA 101.29, QED 0.54
Compound (SMILES): COc1cc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4sc(N5CC(F)(F)C5)nc4C3)CC2)on1
InChIKey: OJUNMSDCPOWMOQ-WKILWMFISA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Assay: 225 nL of the solutions of the non-specific ligand or the compounds of the present invention at each concentration (in case of vehicle, final concentration 0.3% DMSO) were added in each well of a 384-well white/clear bottom microplate (3706, Corning). Jump-In HEK Cell membranes (final reaction amount: 2 μg protein/well), SPA beads (final reaction amount: 0.2 mg/well) and the buffer were mixed and the mixed solution was left still for more than or equal to 1 h at 4° C. Then, 50 μL of the mixture was added to each well of the plate. In addition, 25 μL of 3.6 nM [3H]-Metylspiperone (final concentration: 1.2 nM) was added to each well. The plate was sealed by putting TopSeal-A 96/384 well (6050185, PerkinElmer) on the top of the plate, mixed using stirring deaerator (Welltornado, FK-62, Sakaki) and incubated for 120 min at 25° C. After incubation, the radioactivity of [3H]-Metylspiperone which was binded to D2 receptor was determined by liquid scintillation counter (1450 Microbeta, PerkinElmer) in each well. Non-specific binding was calculated based on the radioactivity of [3H]-Metylspiperone in the presence of 10 μM non-labeled Butaclamol. The total binding was calculated using the radioactivity of [3H]-Metylspiperone in the absence of the compounds of the present invention (Vehicle). The Ki values were calculated from dose-response curves.
Measurement: Ki > 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 495.6, ALogP 3.26, HBA 8, HBD 1, PSA 83.73, QED 0.6
Compound (SMILES): N#Cc1ccc(CN2CCC(NC(=O)c3ccc(C(=O)N4CCC(Oc5ccc(SC(F)(F)F)cc5)CC4)cn3)CC2)cc1
InChIKey: CUGRGOQWDRJFLR-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 623.7, ALogP 5.64, HBA 7, HBD 1, PSA 98.56, QED 0.32
Compound (SMILES): CCC(=O)c1c[nH]c(C(=O)NC(c2ccccc2)C2CCNCC2)c1
InChIKey: DPIRAFYDRLKCMA-UHFFFAOYSA-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).
Measurement: IC50 = 10040 nM (pChEMBL 5)
Descriptors: MW 339.44, ALogP 3.08, HBA 3, HBD 3, PSA 73.99, QED 0.71
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1nc(NC2CCCN(C(=O)C(C)(C)C)C2)ncc1Cl
InChIKey: VTHSPYLYWLACHY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 456.98, ALogP 4.45, HBA 6, HBD 3, PSA 99.25, QED 0.56
Compound (SMILES): CN(C)C1CCC(CNC(=O)c2cccc3cccnc23)N(C(=O)c2ccccc2-c2ccccc2)C1
InChIKey: PQHANPYFHWQTGR-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 492.62, ALogP 4.87, HBA 4, HBD 1, PSA 65.54, QED 0.42
Compound (SMILES): Cn1c(CCc2nc3ccccc3o2)nc2c3cccnc3ccc21
InChIKey: CATDHNSGUMFFIY-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Pharmacology Assay: In one embodiment, the compounds provided herein were assayed for their ability to inhibit human PDE-10A. In one embodiment, the activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 328.38, ALogP 4.05, HBA 5, HBD 0, PSA 56.74, QED 0.5
Compound (SMILES): CN1C(=N)N[C@](C)(c2cc(NC(=O)c3cnc(OCC(F)(F)F)cn3)ccc2F)C2(CC2)S1(=O)=O
InChIKey: NGKDQMRAUSDSAI-GOSISDBHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 516.48, ALogP 2.36, HBA 7, HBD 3, PSA 137.37, QED 0.52