AID	Name	Type	Comment
2689	Summary of Broad Institute MLPCN Serine Threonine Kinase 33 Inhibitor Project	summary	Summary assay

Keywords: STK-33 Kinase

Assay Overview: Purified STK33 Kinase is preincubated with potential inhibitors and then allowed to phosphorylate MBP at [ATP] = Km, which can be compared to other [ATP] experiments to determine competitive or non-competitive mechanism of action. Kinase activity is measured using the ADP Glo Kit (Promega) which converts ADP reaction product into a luminescent signal.

Expected outcome: Inhibitors of the STK33 will reduce the production of ADP, resulting in a decreased signal from the ADP Glo Kit.

Protocol:

Buffer Stock - 10 mL 1 M MOPS-Na pH 7.0 + 0.6 mL 500 mM EDTA + 10 mL 50% glycerol + 100 uL 10% Brij-35 -> 1L 0.2 u filter

10X BSA/BME - 50 mL Buffer + 50 mg BSA + 50 uL BME, aliquot 4X 10 mL -20C

Assay Buffer (AB) = 10 mL 10X BSA/BME + 90 mL Buffer Stock

STK33/MBP = 19 uL 26.5 uM STK33 + 78 uL 64 mg/mL MBP -> 10 mL w/ AB
ATP/Mg = 150 10 mM ATP + 500 uL 1M MgAc2 -> 10 ml w/ AB to a final conc of 0.15 mM
384 well plate assay

Add 20 uL AB to last column of 384 well white Aurora assay plate using combi dispenser (Thermo). Add 20 uL STK33/MBP to all but last column. Using a pintool, transfer 25 nL compound in DMSO stock solution into assay plate. Incubate 15 minutes at room temperature.

Add 5 uL ATP/Mg / well to all wells of plate to initiate kinase reaction. Incubate 60 minutes.

Add 25 uL / well ADP Glo Reagent I (Promega). Incubate 40-60 minutes.

Add 50 uL / well ADP Glo Reagent II (Promega). Incubate 30 minutes.

Read luminescence 0.1 sec integration time on Envision plate reader (Perkin Elmer).

PRESENCE OF CONTROLS: were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
No normalization was applied to the raw data signals.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

Grant Number: 1 R03 MH089854-01