1. Chemical probe candidate for the understudied kinase GAK
SGC-GAK-1
Chemical probe candidate for the understudied kinase GAK

2. The Numb Associated Kinase Family
Cyclin G Associated Kinase (GAK)
Understudied kinase: 54 citations
bibliometric analysis (2016)
NAK Family: AAK1, BIKE, GAK, and MPSK/STK16
Expression: broad - highest in testes
Function: GAK is involved in clathrin coating, trafficking, lysosomal sorting and receptor mediated endocytosis. In the nucleus,
GAK is required for proper centrosome maturation.

3. GAK Disease Associations

4. Known inhibitors of GAK
Clinically approved EGFR inhibitors with potent GAK activity
Selective GAK inhibitor - PMID:
Kinases with Kd ≤ 1 µM
GAK
KIT
PDGFRB
FLT3
MEK5
PDGFRA
CSF1R
CLK2
GAK

5. Binding Pose of 4-Anilinoquinoline
BACK CLEFT
DFG MOTIF
HINGE
BACK CLEFT
DFG MOTIF
HINGE

6. In vitro Potency Assay Value SGC-GAK-1 SGC-GAK-1N Construct DSF ∆Tm
GAKA-c028
ITC KD
4.5 nM
Not tested
GAKc028
FRET Ki
3.1 nM
2500 nM
DiscoverX
1.9 nM
7100 nM
NP_
Probe: SGC-GAK-1
Control: SGC-GAK-1N

7. Kinome Selectivity by KinomeSCAN
SGC-GAK-1
Kinase
Kd (nM)
Ratio vs GAK
GAK
1.9 -
RIPK2
110 58
ADCK3
190
100
NLK
520
274
AVCR1
980
516
GAK
Probe: SGC-GAK-1
SGC-GAK-1N
Kinase
Kd (nM)
GAK
7100
IRAK3
740
RIPK2
4900
NLK
6500
Control: SGC-GAK-1N

8. Cellular target engagement
GAK IC50 = 110 ± 35 nM
RIPK2 IC50 = 360 ± 280 nM
Probe: SGC-GAK-1
GAK IC50 > 50 µM
RIPK2 IC50 > 50 µM
Control: SGC-GAK-1N
DiscoverX GAK InCELL Pulse enzyme complementation assay
SGC-GAK-1 IC50 = 200 nM; SGC-GAK-1N IC50 > 50 µM

9. RIPK2 Control: HY-19764
Other known RIPK2 inhibitors surveyed have significant GAK activity
RIPK2 IC50 = 2.2 nM
GAK IC50 > 50 μM

10. Antiviral activity Cell viability DENV infection % Probe: SGC-GAK-1
Dengue viral entry and packing   
Cell viability
DENV infection %
Probe: SGC-GAK-1
Control: SGC-GAK-1N
Concentration (µM)
Concentration (µM)
SGC-GAK-1 EC50= 0.92 µM
SGC-GAK-1N EC50= >10 µM

11. Summary – SGC-GAK-1 Potency
Single digit nM GAK potency by 3 in vitro methods
FRET GAK Ki = 3.1 nM
DiscoverX GAK KD = 1.9 nM
ITC GAK KD = 4.5 nM
Cellular target engagement:
NanoBRET GAK IC50 = 110 nM
InCELL Pulse GAK IC50 = 200 nM
Kinase selectivity
No kinases within 30x in binding
RIPK2 potency is within 2-4x in cellular target engagement
To control for RIPK2 activity, HY can be employed (potent RIPK2 activity; GAK inactive)
Negative Control
SGC-GAK-1N is a close structural relative with GAK IC50 > 50 µM
Probe: SGC-GAK-1
Control: SGC-GAK-1N

12. Experimental—DSF assay
Differential scanning fluorimetry (DSF) assays: Starting from a 100 µM stock, GAK (14-351) was diluted to 1 µM in buffer 100 mM K2HPO4 pH7.5 containing 150 mM NaCl, 10% glycerol and 1X dye (Applied Biosystems catalog ). The protein/dye mixture was transferred to a 384-well PCR microplate with 20 µL per well. Compounds at 10 µM in DMSO were added next, in 20 nL volume, using a liquid handling device setup with a pin head to make a final 10 µM compound in the assay plate. The final DMSO concentration in all wells was 0.1 %, including the reference well with DMSO only. Thermal shift data was measured in a qPCR instrument (Applied Biosystems QuantStudio 6) programmed to equilibrate the plate at 25 °C for 5 min followed by ramping the temperature to 95 °C at a rate of 0.05 °C per s. Data was processed on Protein Thermal shift software (Applied Biosystems) fitting experimental curves to a Boltzmann function to calculate differential thermal shifts (ΔTm) referenced to protein/dye in 0.1 % DMSO only. The compounds were screened against a purified GAK (14-351) construct according to previously reported procedures. (Methods Mol Biol 2012, 795, )

13. Experimental—ligand binding displacement assay
Ligand binding displacement assays:Inhibitor binding was determined using a binding-displacement assay, which tests the ability of the inhibitors to displace a fluorescent tracer compound from the ATP binding site of the kinase domain. Inhibitors were dissolved in DMSO and dispensed as 16-point, 2x serial dilutions in duplicate into black multi-well plates (Greiner). Each well contained either 0.5 nM or 1 nM biotinylated kinase domain protein ligated to streptavidin-Tb-cryptate (Cisbio), 12.5 nM or 25 nM Kinase Tracer 236 (ThermoFisher Scientific), 10 mM Hepes pH 7.5, 150 mM NaCl, 2 mM DTT, 0.01 % BSA, 0.01 % Tween-20. Final assay volume for each data point was 5 µL, and final DMSO concentration was 1%. The plate was incubated at room temperature for 1.5 h and then read using a TR-FRET protocol on a PheraStarFS plate reader (BMG Labtech). The data was normalized to 0 % and 100 % inhibition control values and fitted to a four parameter dose-response binding curve in GraphPad Software (version 7, La Jolla, CA, USA). The determined IC50 values were converted to Ki values using the Cheng-Prusoff equation and the concentration and Kd values for the tracer (previously determined).

14. Experimental—Competition binding assays
Competition binding assays: Competition binding assays were performed at DiscoverX as described previously [24]. Kinases were produced either as fusions to T7 phage3, or were expressed as fusions to NF-κB in HEK-293 cells and subsequently tagged with DNA for PCR detection. In general, full-length constructs were used for small, single-domain kinases, and catalytic domain constructs including appropriate flanking sequences were used for multidomain kinases. Briefly, for the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands to generate affinity resins. The liganded beads were blocked to reduce non-specific binding and washed to remove unbound ligand. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds prepared as 100× stocks in DMSO. DMSO was added to control assays lacking a test compound. Assay plates were incubated at 25 °C with shaking for 1 h, and the affinity beads were washed extensively to remove unbound protein. Bound kinase was eluted in the presence of nonbiotinylated affinity ligands for 30 min at 25 °C with shaking. The kinase concentration in the eluates was measured by quantitative PCR. The KINOMEscan® panel of approximately 400 wild type human kinase assays was run as a single measurement at 1 µM. KD values were determined using 11 serial threefold dilutions of test compound and a DMSO control.

15. Experimental—NanoBRET
HEK-293 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (GE Healthcare), with incubation in a humidified, 37°C/5% CO2 incubator. N- or C-terminal NanoLuc/Kinase fusions were encoded in pFN31K or pFN32K expression vectors (Promega), including flexible Gly-Ser-Ser-Gly linkers between Nluc and each kinase. HEK-293 were transfected with NLuc/target fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, Nluc/target fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:10 (mass/mass), after which FuGENE HD was added at a ratio of 1:3 (g DNA: μL FuGENE HD). FuGENE HD complexes thus formed were combined with HEK-293 cells suspended at a density of 2 x 105 (1 mL complex: 20 mL cell suspension), followed by incubation in a humidified, 37°C/5% CO2 incubator for 20 hr. Following transfection, cells were washed and resuspended in Opti-MEM. BRET assays were performed in white, 96-well plates (Corning) at a density of 2 x 104 cells/well. All chemical inhibitors were prepared as concentrated stock solutions in DMSO (Sigma-Aldrich) and diluted in Opti-MEM (unless otherwise noted) to prepare working stocks. Cells were equilibrated for 2 h with energy transfer probes and test compound prior to BRET measurements. BRET reported probes were prepared at a working concentration of 20X in tracer dilution buffer (12.5 mM HEPES, 31.25% PEG-400, pH 7.5). The energy transfer probes were added to the cells at concentrations optimized for each target. To measure BRET, NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega) were added according to the manufacturer’s recommended protocol, and filtered luminescence was measured on a GloMax Discover luminometer equipped with 450 nm BP filter (donor) and 600 nm LP filter (acceptor), using 0.5 s integration time. Milli-BRET units (mBU) are calculated by multiplying the raw BRET values by Apparent tracer affinity values (EC50) were determined using the sigmoidal dose-response (variable slope) equation available in GraphPad Prism: Y = Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) Competitive displacement data were then plotted with GraphPad Prism software and data were fit to Equation 1 to determine the IC50 value.

16. Acknowledgements Funding Sources
Christopher Asquith, James Bennett, Benedict-Tilman Berger, Tuomo Laitinen, Szu-Yuan Pu, Mike East, Paulo Godoi, Jing Wan, Antti Poso, Apirat Chaikuad, Stefan Knapp, Lee Graves, H. Shelton Earp, Carrow Wells, David Drewry, Graham Tizzard, Daniel Treiber, Jonathan Elkins, Shirit Einav, Susanne Müller-Knapp, William Zuercher, Timothy Willson
Funding Sources