SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1196

1196

SLAS Discovery 22(10)

conditions of either excess ATP or CHKtide. The rates were
plotted against variable substrate concentration and subjected to standard Michaelis-Menten analysis to derive Km
and Vmax values. Values were normalized for volume and
protein concentration across the two technologies for comparative purposes.
MALDI TOF data processed by the FlexAnalysis 4.0
software were exported as a .csv file using FlexAnalysis
Batch Process (Compass 2.0) and further processed in
Microsoft Excel (Microsoft, Redmond, WA). For peak area
calculation, the isotopic distribution was taken into account
by determining a peak area from 2699.4 to 2702.6 m/z for
the unphosphorylated CHKtide and 2779.8 to 2782.2 m/z
for the phosphorylated CHKtide.
SIK2 activity was measured by the percent phosphorylation
(%P) of the CHKtide substrate by the following equation:


( Peak Area Phosphorylated Product )

 ×100.
 ( Peak Area Phos product ) + ( Peak Area Substrate ) 

Raw data from the ADP Hunter assay (relative fluorescence
units [RFU]) were also exported as a comma delimited (.csv)
file. All subsequent primary single-point screening data processing and analysis for both assays were conducted within
Activity Base version 8.1.2.12 using Activity Base XE
Runner version 8.1.1 (IDBS, Guilford, UK). Data were normalized and expressed as a percentage effect (PE) value for
each test compound as follows:
MALDI TOF data:
 %Ptest − %Phigh

PE = 
× 100 
 %Plow − %Phigh



ADP Hunter data:
 RFU test − RFU high

PE = 
× 100  ,
 RFU low − RFU high



where test is the signal associated with the test compound,
high is the median of the no-effect signal (reaction in presence of enzyme only), and low is the median of maximum
effect control signal (reaction in absence of enzyme [biochemical] or presence of staurosporine [MALDI TOF]).
The performance of the assay on each screening plate
was evaluated using internal controls to determine robust
signal-to-background (s:b) and robust Z′ values, which
were calculated as follows:
s:b =

high
,
low

 ( 3 × sMADhigh ) + ( 3 × sMADlow ) 
Z′ = 1 − 
 ,

( high − low )



where high refers to either RFUhigh or %Phigh and low
corresponds to either RFUlow or %Plow, as appropriate.
sMADhigh is 1.4826 × median absolute deviation of signal
associated with the no-effect control, and sMADlow is the
1.4826 × median absolute deviation of signal calculated for
the maximum effect control.
All database querying for global data analysis and report
creation was undertaken using the Dotmatics Browser (version 4.9.623.45008-s) and Vortex analysis software (version
2015.12.46651.25; Dotmatics, Bishop Stortford, UK).

SIK Cellular Assay
The human bone osteosarcoma cell line U2OS (ATCC
HTB-96) was subcultured and grown in 96-well plates
(µClear bottom, cat. 655090; Greiner, Stonehouse, UK).
After 24 h, cells were treated for 1 h with the small-molecule inhibitors staurosporine, HG-9-91-01, KIN112,
MRT67307, and MRT199665. DMSO at 0.1% and secondary antibody-only controls were included on each plate. All
compounds started at 10 µM and were serially diluted with
eight replicates per treatment group. Cells were fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS) and
permeabilized with 1% NP-40 in PBS for 10 min each,
respectively. Following a blocking step with 3% goat serum
(Sigma) for 45 min, cells were incubated overnight at 4 °C
with rabbit anti-CRTC3 (1:350; Abcam, Cambridge, UK)
followed by 1 h with goat anti-rabbit Alexa 488 (1:300;
Invitrogen, Carlsbad, CA) and 1 µg/mL 4′,6-diamidino2-phenylindole (DAPI; Invitrogen). Blocking and antibody
solutions were made in 1% bovine serum albumin (BSA)-
PBS. Plates were subsequently imaged on a PerkinElmer
(Coventry, UK) Operetta high-content wide-field fluorescence imaging system with a 20× objective and identical
parameters used for all well and plates: DAPI (em 445 nm),
Alexa 488 (em 525 nm), and digital phase contrast (ex 740
nm) with four fields of view imaged per well in an identical
pattern. Quantification was performed using the Columbus
database and analysis software. Nuclear intensity of CRTC3
was quantified via nuclear identification with DAPI, with
subsequent subtraction of the secondary-only control
(Alexa 488 signal) from all samples. Results are expressed
as mean fluorescence intensity ± standard deviation (SD).

Results and Discussion
To screen for inhibitors of the SIK2 kinase, we developed
and applied three distinct high-throughput assays (Fig. 1): a
well-established fluorescence-based, enzymatically coupled assay, ADP Hunter; a label-free MALDI TOF assay
screening for the phosphorylated peptide substrate; and a
cell-based assay using the nuclear translocation to the SIK
substrate CRTC3 in response to SIK inhibition. For the in
vitro kinase assays, human, recombinant full-length SIK2
Table of Contents for the Digital Edition of SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017
