CHEMBL504323 (+)-berbamine Berbamine BDBM50241654
O-(4-Ethoxybutyl)berbamine BDBM50293304 CHEMBL454806 O-(4-ethoxybutyl)-berbamine
BDBM50427811 CHEMBL2325764 BM-1074
BDBM50427812 CHEMBL2325763 BM-1075
BDBM670949 BDBM50188614 BM-513
US10106501, Example BM BDBM293154
[4-(2-Benzenesulfonylamino-ethyl)-phenoxy]-acetic acid(sulotroban) BDBM50002765 [4-(2-Benzenesulfonylamino-ethyl)-phenoxy]-acetic acid (BM 13177) Sulotroban BM 13177 BM-13177 CHEMBL8273 [4-(2-Benzenesulfonylamino-ethyl)-phenoxy]-acetic acid
BM 573 BDBM50188619 N-tert-butyl-N'-[2-(4-methylphenylamino)-5-nitrobenzenesulfonyl]urea 1-(2-(p-toluidino)-5-nitrophenylsulfonyl)-3-tert-butylurea CHEMBL210602 BM-573
Daltroban BM 13505 (daltroban){4-[2-(4-Chloro-benzenesulfonylamino)-ethyl]-phenyl}-acetic acid BM-13505 {4-[2-(4-Chloro-benzenesulfonylamino)-ethyl]-phenyl}-acetic acid CHEMBL71685 BDBM50002805
CHEMBL310852 BDBM50004734 N-Methyl-N-(1-methyl-4-pyrrolidin-1-yl-but-2-ynyl)-acetamide N-Methyl-N-(1-methyl-4-pyrrolidin-1-yl-but-2-ynyl)-acetamide (BM 5) N-Methyl-N-(1-methyl-4-pyrrolidin-1-yl-but-2-ynyl)-acetamide (R) N-Methyl-N-(1-methyl-4-pyrrolidin-1-yl-but-2-ynyl)-acetamide (S)
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- In Vitro Kinase Assay The IC50 for the different compounds was determined with Invitrogen Z'-LYTE kinase assay kit Ser/Thr 1 peptide PV3174 by following the manufacturer's instructions. To determine the IC50 of receptor tyrosine kinase, an assay based on the ELISA principle with kinases from Biomol (N-terminally fused to GST), POD-coupled antibody from Calbiochem (PY20)) and BM chem chemiluminescence ELISA substrate from Roche was performed.
- Phospho ELISA Assay Adult human dermal lymphatic microvascular endothelial cells (HMVEC-dLyAD) (Cat# CC-2810, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in Endogro MV complete (Cat#SCME004, Millipore) at 200,000 cells/well (volume 1 mL), and the plates incubated at 37 C. and 5% CO2 for 6 hours. The media was replaced with Endogro Basal (Cat # SCME-BM, Millipore)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37 C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 uL of Total VEGFR2 capture antibody (Part #841888, Human Total VEGFR3/Flt4 ELISA Kit, Cat # DYC3491, R&D Systems), or Phospho VEGF R3Capture antibody (Part #841885, Human Phospho VEGF R3/Flt4 ELISA Kit, Cat# DYC2724, R&D Systems). The plates were covered and incubated at room temperature overnight. The coating antibody was flicked out and the plates washed three times with Wash Buffer.
- Enzyme Activity In Vitro Assay I. Experimental Materials and Equipment1. Malachite green phosphate detection kit (R & D Systems, Cat #, DY996)2. Recombinant human 5′-nucleotidase (CD73) (R & D Systems, Cat #, 5795-EN)3. HEPES buffer (Gibco, Cat #, 15630-080)4. CMP (Sigma, Cat #, C1006)5. DMSO (Fisher Chemical, Cat #, D128-1)6. NaCl 5M (Boston Bioproducts, Cat #, BM-244)7.384-well plate (Fisher, Cat #, 5795-EN)8. TECAN plate reader (TECAN)II. Experimental ProcedureCompounds are first dissolved in DMSO to 10 mM as a stock solution. When determining the IC50 of the compound, prepare 3-fold serial dilutions with a highest concentration of 125 μM for a total of 12 concentration points and ensure each dilution containing equal amount of DMSO. In each well of 384-well plate, 0.34 nM of recombinant human 5′-nucleotidase (CD73) was pre-incubated at 37° C. for 2 hours with the compounds tested in assay buffer containing 20 mM HEPES buffer (pH 7.4), 137 mM NaCl, 0.001% Tween 20. The final reaction volume of the reaction in each well was 12 μL. The highest concentration of compound was 125 μM and the DMSO concentration was 1.25%. After pre-incubation, 3 μL of CMP dissolved in assay buffer was added to each reaction. The final CMP concentration was 45 μM. The reaction was incubated at 37° C. for 15 minutes. Then 3 μL of Malachite Green Reagent A was added to each reaction. Spin the plate briefly in centrifuge for 30 seconds. After incubation for additional 10 minutes at room temperature, 3 μL of malachite green Reagent B was added to each reaction. Spin the plate briefly in centrifuge for 30 seconds. After 20 minutes of incubation at room temperature, the signal was read on a TECAN reader at OD620.
- Sandwich ELISA Assay For measuring the autophosphorylation of the endogenous Tie2 receptor in the cell lysates prepared, a self-prepared anti-Tie2 antibody directed against the N-terminus of the Tie-2 receptor (1.09 mg/ml) and an HRP-coupled anti-phosphotyrosine antibody (Sigma, A4595, clone pY-20) were used. White 96-well ELISA plates (Lumitrac600, Greiner, #655074) were incubated with 100 μl per well of a 1:1000 dilution of the anti-Tie2 antibody in coating buffer [15 mM Na2CO3, 35 mM NaHCO3, pH 9.6] with shaking at 4° C. overnight. The coated ELISA plates were washed three times with 250 μl PBST buffer [0.1% Tween-20 in PBS], the wells were blocked with 250 μl 3% BSA in PBST buffer with shaking at RT for 1-6 h and washed three more times with 250 μl PBST buffer. From the cell lysate plates thawed in the fridge, in each case 100 μl of lysate were transferred into the coated wells of the ELISA plates, and the plates were incubated with shaking at 4° C. overnight. The plates were washed three times with in each case 250 μl of PBST buffer, and 100 μl of a 1:5000 dilution of the anti-phosphotyrosine HRP antibody in 3% Prionex (Calbiochem, #529600) in PBST were then pipetted into each well and the plates were incubated with shaking and protected from light at 4° C. overnight. The plates were washed three times with in each case 250 μl of PBST, and 100 μl of chemiluminescent substrate [BM Chemiluminescence ELISA Substrate (POD) Reagent A and B 1:100, Roche, #1582950] were then pipetted into each well, and after 3 min the plates were measured using a luminescence reader.