GSK3beta Surface Plasmon Resonance (SPR) Assay Measured in Biochemical System Using Microfluidics - 2133-08_Inhibitor_Dose_DryPowder_Activity
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of binders of GSK-3beta with sub-micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity. more ..
BioActive Compounds: 35
Surface Plasmon Resonance, Biacore T100, Recombinant GST-GSK3beta, baculovirus Sf21 insect cells
The glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for several cellular pathways and plays a critical role in metabolism, transcription, development, cell survival, and neuronal functions. The overall objective is to identify one or multiple series of binders of GSK-3beta with sub-micromolar potency. Such compounds will become probe(s) with demonstrated kinase-selectivity. Structural series that demonstrates non-ATP competitive mechanism of action will be strongly preferred. After initial characterization of potency and selectivity, dry powders of promising compounds and their analogues were ordered or synthesized. This assay measures affinity of these compounds for GSK3beta. Approximately 2500 Response Units of GSK3beta (as a GST fusion from BPS Bioscience) was captured on a CM5 SensorChip and treated with varied doses of compounds (specific assay conditions see Protocol). The affinity was measured with reference subtracted values from FlowCell treated with recombinat GST. Positive control (CHIR99021 at 10 uM) was included in each plate to confirm activity of each flow cell.
Biomolecular interaction analysis: GSK3beta interaction with small molecules. Will see a decrease in Kd values as compound affinity increases.
GSK3a Surface Plasmon Resonance (SPR) Assay
A standard protocol utilizing Surface Plasmon Resonance on a Biacore T100 instrument to quantify kinetic relationships of small molecule inhibitors of GSK3a. Protocol will need to be checked for optimized conditions when switching instrument types.
Active GST-GSK3a (Millipore) was expressed in baculovirus Sf21 insect cells and purified via GST fusion on Ni/NTA-agarose gel. Protein was shipped and stored as a 1.4uM solution in tris buffered saline at -80 degrees C. Reference protein, recombinant GST, was arrived at in the same fashion.
a. Immobilization buffer from GE Lifesciences
b. Immobilization buffer (in house) PBS pH 7.4
c. Running buffer (in house) TBS pH 7.4
2. Biacore Materials
a. CM5 Sensorchip
b. 700uL small vials
c. 1500uL large vials
d. Small vial caps
e. Large vial caps
a. Recombinant GST- GSK3a
b. Recombinant GST
c. Anti GST antibody
1. In house PBS
a. Mix 900mL filtered DI water with 100mL 10X PBS buffer solution (Invitrogen)
2. In house TBS
a. Add 1.21g Tris and 8.7g NaCl to 950mL filtered DI water
b. pH using 6M HCl to 7.35
c. Fill to 1000mL mark
d. Filter and collect 900mL in one bottle
e. Filter and collect 100mL in second bottle
f. Add 450uL P20 detergent (GE LifeSciences) to bottle 1
g. Add 50uL P20 detergent to bottle 2
h. Mix thoroughly
i. Transfer 18mL buffer from bottle 1 to bottle 2 to make TBS running buffer
j. Add 18mL DMSO (Aldrich) to bottle 1 to make a 2% DMSO running buffer
GSK3a CM5 Sensor Chip Generation
1. Conditioning New Chip
a. Load PBS immobilization buffer into buffer shelf and insert buffer line A.
b. Load unused CM5 sensor chip into instrument per user manual
c. Prime chip with 6 minutes of buffer injection at 30uL/min
d. Initiate manual run and inject over flow cell (FC) 1 and 2 alternating injections of Lysine buffer (GE LifeSciences) and Sodium Hydroxide buffer (GE LifeSciences) at a flow rate of 30uL/min for 30 seconds per injection.
2. Immobilization of anti GST antibody
a. Prime chip again per step 1.c.
b. Generate anti GST antibody solution by mixing 4.5uL anti GST antibody (GE LifeSciences, GST Capture Kit) with 95.5uL immobilization buffer (GE LifeSciences, GST Capture Kit).
c. Put solution into 700uL small vial and cap
d. Repeat 2.b-2.c.
e. Place both vials in reagent rack 1 of Biacore T100 instrument and place into machine per user manual
f. Initiate immobilization of anti GST antibody by utilizing the immobilization Wizard protocol of Biacore T100 software.
g. Immobilize approximately 20,000 Response Units (RU) of anti GST antibody on FC1 and FC2
3. Capture Reference and Active Proteins on FC1 and FC2 respectively.
a. Remove PBS immobilization buffer from buffer rack and replace with TBS 2% DMSO running buffer.
b. Insert buffer line A
c. Repeat prime procedure from 1.c.
d. Generate GST protein solution by mixing 2uL of recombinant GST (GE LifeSciences, GST Capture Kit) with 98uL TBS 2% DMSO buffer
e. Generate GST- GSK3a protein solution by mixing 5uL of recombinant GST- GSK3a with 95uL TBS 2% DMSO running buffer.
f. Place GST protein solution in position B1 on reagent rack 1
g. Place GST- GSK3a protein solution in position C1 on reagent rack 1
h. Initiate a manual injection run and inject for 30 seconds at 5uL/min GST protein solution on FC1 to generate reference flow cell
i. Initiate a manual injection run and inject for 30 seconds at 5uL/min GST- GSK3a protein solution on FC2 to generate active flow cell
j. Capture approximately 1000 RU GST protein on FC1
k. Capture approximately 2500 RU GST- GSK3a protein on FC2
Analysis of Small Molecule GSK3a Inhibitors
1. Generation of analyte plate for assay
a. Using a standard deep volume 384 well plate, place 100uL of a 10uM TBS 2% DMSO solution of the desired analyte in wells A1 and A2.
b. Place 100uL of a 5uM TBS 2% DMSO solution of desired analyte in wells A3 and A4
c. Place 100uL of a TBS 2% DMSO solution 2.5um solution of desired analyte in wells A5 and A6
d. Repeat steps 1.a - - 1.c. with serially diluted analytes to 10nM in wells A21 and A22
e. Place 100uL of TBS 2% DMSO running buffer in wells A23 and A24
f. Repeat steps 1.a. - - 1.e. for subsequent analytes
2. Place analyte plate in Biacore T100 per user manual
3. Generate method that consists of the following
a. Analyte injection over FC1 and FC2 with reference subtraction
b. 30uL/min flow rate of analyte
c. 60 second contact time of analyte
d. 60 second wash time of running buffer
e. DMSO standard curve generation using 1, 1.5, 1.75, 2, 2.5, 2.75 and 3% DMSO in TBS running buffer.
4. Run analysis of compounds
5. Compounds were ranked according to KD determination utilizing the affinity measurement option in Biacore T100 software.
Calculation of Activity Score-Our benchmark compound (BRD-K16189898-001-04-5) was assigned a score of 100 as, prior to this examination, it was the best compound in this assay. Each compound tested gave a KD value. If this value was below 1,000nM it was then subtracted from 1,000 and divided by 10, then rounded to the nearest integer to give the activity score. i.e. BRD-A81474003-001-05-2 produced a KD of 130nM which translates to an activity score of 87 via: 1,000-130 = 870/10 = 87.
Calculation of Active Concentration-This is not a cellular assay, so an active concentration is actually a measurement of the affinity of the analyte for the target protein, a KD measurement. The calculation of the KDs for these analytes was done using software included in the Biacore T100 instrument based on the 1:1 binding model.
Determination of Activity Outcome-Overall activity outcome was determined by examining the KD of the analyte. A KD of <1000nM was determined to be active, a KD >1000nM was determined to be inactive.
Corrected KD Values-The KD values outlined in this document come from an absolute value that was subtracted from a reference value to give the reference subtracted value. This reference value is the response the compound showed against the benign protein GST.
** Test Concentration.
Data Table (Concise)