AAK1 (AP2 associated kinase 1) (IC50 = 270 nM; Ki = 9 nM)

In HT1080 cells, SGC-AAK1-1 (1.25 μM) dose-dependently activates WNT-driven BAR activity and significantly reduces AP2M1 (T156) phosphorylation [1]. SGC-AAK1-1 increases β-catenin-dependent transcription and β-catenin protein stability by inhibiting AAK1 kinase activity [1].

Binding-displacement assays [1]
The TR-FRET ligand binding-displacement assays for AAK1, BMP2K, GAK and STK16 were performed as previously described (Asquith et al., 2018). Inhibitor binding was determined using a binding-displacement assay, which measures the ability of 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. Each well contained either 0.5 nM or 1 nM biotinylated kinase domain protein ligated to streptavidin-Tb-cryptate, 12.5 nM or 25 nM Kinase Tracer 236, 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 kinase domain proteins were expressed in E. coli as a fusion with a C-terminal AVI tag (vector pNIC-Bio3, NCBI reference JN792439) which was biotinylated by co-expressed BirA, and purified using the same methods as used previously (Asquith et al., 2018). After setting up the assay plate it was incubated at room temperature for 1.5 hours and then read using a TR-FRET proto Residue ranges were AAK1: 31-396, BMP2K: 38-345, GAK: 12-347, STK16: 13-305col on a PheraStarFS plate reader. The data was normalized to 0% and 100% inhibition control values and fitted to a four parameter dose-response binding curve in GraphPad Software. 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).

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Kinome screening [1]
The KINOMEscan assay panel was measured at DiscoverX Corporation as previously described (Davis et al., 2011). Data collection can be found in Table S2 and has been deposited on Mendeley Data (https://doi.org/10.17632/cz9bx7d52c.1#file-bd678698-6718-4979-8b9e-1b0f7645884e). Kinases were produced either as fusions to T7 phage3, or were expressed as fusions to NF-κB in HEK293 cells and subsequently tagged with DNA for PCR detection18. 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 nonspecific 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. Primary screen interactions were performed in 384-well plates, whereas KD determinations were performed in 96-well plates. 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. KD values were determined using 11 serial threefold dilutions of test compound and a DMSO control.

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Isothermal Titration Calorimetry [1]
AAK1 and BMP2K proteins were produced as previously described (Sorrell et al., 2016). Isothermal titration calorimetry measurements were made on a Microcal VP-ITC instrument at 25°C. For the interaction of AAK1 with SGC-AAK1-1, the compound was diluted to 22 μM in ITC buffer from a stock at 10 mM in DMSO and loaded directly into the cell. AAK1 was dialyzed at 4°C overnight into ITC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and loaded into the ITC syringe at a final concentration of 218 μM. Following thermal equilibration, AAK1 was titrated into the cell using serial injections of 8 μL until saturation was observed in the thermogram. The same method was repeated for the BMP2K versus SGC-AAK1-1 interaction where protein was loaded into the syringe at a concentration of 288 μM and injected into a 32 μM solution of SGC-AAK1-1. The ITC data was analyzed with NITPIC (Keller et al., 2012) and SEDPHAT (Zhao et al., 2015). The final fitted data values are in Table S4.

Transcriptional reporter assays [1]
All luciferase reporter assays and IncuCyte fluorescent reporter assays were performed as previously described (Walker et al., 2015). Briefly, for loss-of-function assays, cell lines stably expressing the BAR-Firefly luciferase reporter and TK-Ren luciferase were used and transfected with RNAiMAX for 72 hr. For gain of function studies, the BAR-reporter (20 ng), TK-Ren (10 ng), and indicated constructs (70 ng) were transfected with TansIT2020 for 24 hr. For IncuCyte fluorescent reporter assays, stable BAR-mCherry cells were treated and imaged as indicated using the IncuCyte Live Cell Analysis System from Essen BioScience. Luciferase readouts were normalized using co-transfected TK-Ren (Luciferase assays) and IncuCyte Analysis was normalized to internal mCherry control. Conditions were plated in triplicate, and normalized values were averaged across triplicates to yield the data presented and standard error. Each assay was repeated in biological triplicate, unless otherwise stated. Firefly luciferase and the Renilla (Ren) control were detected using the Promega Dual-Luciferase Reporter Assay System per the manufacturer’s protocol. Plates were read on the EnSpire plate reader from PerkinElmer.

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Real-Time Quantitative PCR [1]
Quantitative PCR (qPCR) was performed as described previously (Walker et al., 2015). Briefly, cells were treated as indicated and RNA was collected using PureLink RNA Mini Kit. cDNA was generated from 1ug of RNA using the iScript cDNA Synthesis Kit following kit specifications. qPCR was performed using Fast SYBER Green Master Mix on the Applied Biosystems 7400HT following manufacturer specifications. Samples were run in technical triplicate on a 384-well plate, with biological triplicates run on subsequent plates. Primers were previously published (Walker et al., 2015) and sequences are listed in Table S7.

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Generation of LRP5/6 DKO stable cell lines [1]
HEK293T cells were transfected with Cas9 and sgRNAs targeting LRP5/6. Sequences are listed in Table S7. Deletions were confirmed by immunoblotting and verified via sequencing.

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Surface biotinylation [1]
For surface biotinylation assays, the Pierce Cell Surface Protein Isolation Kit was utilized and manufacturer specifications were followed. Briefly, cells were grown to 70% confluency, washed 3 times with cold PBS, and then biotinylated for 30 mins at 4°C with NHS-SS-sulfo-linked biotin (0.25mg/mL). The free biotin was quenched, and then the samples were washed 3 times with cold TBS prior to lysis and sonication. Lysates were cleared and then incubated with Streptavidin beads for 1 hour at 4°C with nutation. Beads were washed 4 times with cold TBS and then proteins were eluted with LDS protein loading buffer supplemented with DTT at 95°C for 10 mins.

[1]. WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop. Cell Rep. 2019 Jan 2;26(1):79-93.e8.

β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity. [1]

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AAK1 contributes to several neurological disorders, including neuropathic pain, Alzheimer’s disease, Parkinson’s disease, schizophrenia, and amyotrophic lateral sclerosis (Kostich et al., 2016, Shi et al., 2014). AAK1 has a described role in dendrite branching and spine development (Ultanir et al., 2012), and through the regulation of Neuregulin1/ErbB4, AAK1 has been linked to schizophrenia (Kuai et al., 2011). Just recently, the LX9211 AAK1 inhibitor passed phase 1 clinical trials for neuropathic pain. Here we report the development of a potent and selective AAK1 pharmacological inhibitor (SGC-AAK1-1). SGC-AAK1-1 demonstrates improved biochemical selectivity over the phase 1 clinical agent LX9211 and is confirmed to be cell active. Thus, we report the best available chemical tool to study AAK1/BMP2K pathways and related biology. Like the lead compound 25A, SGC-AAK1-1 inhibited AAK1-dependent phosphorylation of AP2M1 and activated WNT signaling. Looking forward, in vitro and in vivo neural-directed studies of SGC-AAK1-1 and its comparison with LX9211 are needed to evaluate the therapeutic potential of SGC-AAK1-1. Furthermore, while we describe a role for AAK1 in negatively regulating WNT signaling, AAK1 also inhibits Neuregulin1/ErbB4 and positively regulates the NOTCH pathway (Gupta-Rossi et al., 2011, Kuai et al., 2011). Therefore, AAK1 inhibitors may also regulate these signaling cascades and consequently prove therapeutically beneficial in diseases with misregulated NOTCH or Neuregulin1/ErbB4 signaling. In cancer, mutation and altered expression of several genes promote WNT receptor stability on the plasma membrane, resulting in hyperactive WNT signaling and tumorigenesis (e.g., ZNRF3/RNF43 and USP6; Hao et al., 2012, Koo et al., 2012, Madan et al., 2016, Ruffner et al., 2012, Schmid, 2017). Whether AAK1 expression or activity is suppressed in cancer and if this contributes to WNT activation remain to be determined.[1]

C21H25N5O3S

427.519902944565

427.17

C, 59.00; H, 5.89; N, 16.38; O, 11.23; S, 7.50

2247894-32-0

134812845

Light brown to khaki solid powder

2.8

3

6

8

30

704

0

O=C(C1CC1)NC2=NNC3=C2C=CC(C4=CC=CC(NS(=O)(N(CC)CC)=O)=C4)=C3

UCBIQZUJJSVQHL-UHFFFAOYSA-N

InChI=1S/C21H25N5O3S/c1-3-26(4-2)30(28,29)25-17-7-5-6-15(12-17)16-10-11-18-19(13-16)23-24-20(18)22-21(27)14-8-9-14/h5-7,10-14,25H,3-4,8-9H2,1-2H3,(H2,22,23,24,27)

N-[6-[3-(diethylsulfamoylamino)phenyl]-1H-indazol-3-yl]cyclopropanecarboxamide

SGC-AAK1-1; SGC AAK1-1; SGC-AAK1 1; 2247894-32-0; SGC-AAK1-1; N-(6-(3-((N,N-diethylsulfamoyl)amino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide; CHEMBL4452939; N-[6-[3-(diethylsulfamoylamino)phenyl]-1H-indazol-3-yl]cyclopropanecarboxamide; AAK1 inhibitor 1; SCHEMBL26677931; SGC AAK1 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~116.95 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)