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 signaling regulates development, tissue homeostasis, and various human diseases. Signaling via the WNT-Frizzled/LRP receptor complex requires proteins involved in clathrin-mediated endocytosis (CME). However, CME also negatively regulates WNT signaling through the internalization and degradation of the receptor complex. Using gain-of-function screening of the human kinaseome, we found that the known CME enhancer AP2-associated kinase 1 (AAK1) inhibits WNT signaling. Conversely, AAK1 gene silencing or pharmacological inhibition using highly selective inhibitors activates WNT signaling. Mechanistic studies revealed that AAK1 promotes LRP6 clearance from the plasma membrane, thereby inhibiting the WNT pathway. Time-course experiments support a transcriptionally uncoupled WNT-driven negative feedback loop. Long-term WNT signaling treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and LRP6 endocytosis. We propose that after WNT receptor activation, enhanced AAK1 function and clathrin-coated pit endocytosis (CME) limit the duration of WNT signaling. [1] AAK1 is associated with a variety of neurological diseases, including neuropathic pain, Alzheimer's disease, Parkinson's disease, schizophrenia, and amyotrophic lateral sclerosis (Kostich et al., 2016; Shi et al., 2014). AAK1 plays a role in dendritic branching and spinous process development (Ultanir et al., 2012), and is associated with schizophrenia by regulating Neuregulin1/ErbB4 (Kuai et al., 2011). Recently, the AAK1 inhibitor LX9211 has completed a phase I clinical trial for the treatment of neuropathic pain. This article reports the development of a highly effective and selective AAK1 pharmacological inhibitor (SGC-AAK1-1). SGC-AAK1-1 exhibits higher biochemical selectivity than the phase I clinical trial drug LX9211 and has been shown to have cellular activity. Therefore, we report the best chemical tool currently available for studying the AAK1/BMP2K pathway and its associated biology. Similar to lead compound 25A, SGC-AAK1-1 inhibits AAK1-dependent AP2M1 phosphorylation and activates the WNT signaling pathway. Looking ahead, in vitro and in vivo neurotargeting studies of SGC-AAK1-1 are needed, comparing it with LX9211 to evaluate its therapeutic potential. Furthermore, although we have described the role of AAK1 in negatively regulating the WNT signaling pathway, 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 modulate these signaling cascades, potentially playing a therapeutic role in diseases involving dysregulation of the NOTCH or Neuregulin1/ErbB4 signaling pathways. In cancer, mutations and altered expression of multiple genes can promote the stability of WNT receptors on the plasma membrane, leading to overactivation of 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 whether this leads to WNT activation, remains 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.)