SGC-GAK-1

SGC-GAK-1 A chemical probe for GAK

This probe is available from Tocris and Sigma.

Its negative control (SGC-GAK-1N) is available for purchase from Sigma.

  • overview
  • properties
  • references
overview

Cyclin G associated kinase (GAK) is a 160 kDa serine/threonine kinase originally identified and so named as a direct association partner with cyclin G.1 GAK is a member of the numb-associated kinase (NAK) family, which includes AAK1 (adaptor protein 2-associated kinase), STK16/MPSK1 (serine/threonine kinase 16/myristoylated and palmitoylated serine/threonine kinase 1), and BMP2K/BIKE (BMP-2 inducible kinase).2 In addition to its kinase domain, the C-terminus of GAK protein bears high homology to a domain found in auxilin and tensin.3 GAK has ubiquitous tissue expression and within the cell localizes to the Golgi complex, cytoplasm, and nucleus.

NAK family domain structures

Location of GAK on kinome tree

Multiple biological roles for GAK and disease associations have been made despite it being a relatively understudied kinase. GAK is involved in membrane trafficking and sorting of proteins, including as an essential cofactor for HSC70-dependent uncoating of clathrin coated vesicles in the cytoplasm.6-7 GAK is required for maintenance of centrosome maturation and progression through mitosis.8 GAK is over-expressed in osteosarcoma cell lines and tissues where it contributes to proliferation and survival.9 Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms in the GAK gene associated with susceptibility to Parkinson’s disease.10 Emerging evidence suggests that GAK may be a therapeutic target in prostate cancer (PCa): GAK expression levels increase upon prolonged androgen treatment and during the progression of cells to hormone independence, and analysis of patient-derived tissue samples demonstrated that GAK expression levels positively correlated with the Gleason score, a quantitative scale of PCa aggressiveness.11 GAK interacts directly with the androgen receptor (AR) and potentiates its transcriptional activity.12 Further elucidation of the role of GAK in these and other biological processes would be facilitated by access to a potent and selective inhibitor of the kinase.

SGC-GAK-1 is a chemical probe for GAK that potently targets the ATP-binding site (KD = 1.9 nM). Regarding kinase selectivity, no kinases were observed to bind SGC-GAK-1 within 30-fold of the KD of GAK in a KINOMEscan assay (DiscoverX) at 1 μM followed by KD determinations. In a live cell NanoBRET assay (Promega) SGC-GAK-1 had an IC50 of 110 nM against ectopically expressed full-length GAK-Nluc fusion.

A chemically related negative control compound, SGC-GAK-1N, is provided.

Despite weakly binding RIPK2 in weakly binding RIPK2 in vitro (DiscoverX RIPK2 KD = 110 nM; 58-fold of GAK KD), SGC-GAK-1 potently engaged RIPK2 in a live cell NanoBRET assay (RIPK2 IC50 = 360 nM); accordingly, we have identified a control compound that is a highly cell-potent RIPK2 ligand, HY-19764, (IC50 = 2.2 nM) that lacks GAK affinity (IC50 > 10 µM).

properties


SGC-GAK-1


SGC-GAK-1N
(negative control)

Physical and chemical properties for BAY-876
Molecular weight389.25
Molecular formulaC38H36BrF3N4O6
IUPAC name6-Bromo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine
MollogP4.65
PSA40.19
No. of chiral centres0
No. of rotatable bonds5
No. of hydrogen bond acceptors4
No. of hydrogen bond donors1
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM
Physical and chemical properties for BAY-588 (Negative Control)
Molecular weight392.38
Molecular formulaC20H19F3N2O3
IUPAC nameN-methyl-6-(trifluoromethyl)-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine
MollogP5.157
PSA33.85
No. of chiral centres0
No. of rotatable bonds6
No. of hydrogen bond acceptors4
No. of hydrogen bond donors0
Storage-20 °C as DMSO stock
DissolutionSoluble in DMSO at least up to 10 mM

SMILES:
SGC-GAK1-1: BrC1=CC2=C(C=C1)N=CC=C2NC3=CC(OC)=C(OC)C(OC)=C3
SGC-GAK1-1N: CN(C1=CC(OC)=C(OC)C(OC)=C1)C2=C3C(C=CC(C(F)(F)F)=C3)=NC=C2

 

InChI:
SGC-GAK1-1: InChI=1S/C18H17BrN2O3/c1-22-16-9-12(10-17(23-2)18(16)24-3)21-15-6-7-20-14-5-4-11(19)8-13(14)15/h4-10H,1-3H3,(H,20,21)

SGC-GAK1-1N: InChI=1S/C20H19F3N2O3/c1-25(13-10-17(26-2)19(28-4)18(11-13)27-3)16-7-8-24-15-6-5-12(9-14(15)16)20(21,22)23/h5-11H,1-4H3

InChIKey:
SGC-GAK1-1: AUOSKLDNVNGKRR-UHFFFAOYSA-N
SGC-GAK1-1N: PVTQCCFMFWASHK-UHFFFAOYSA-N

selectivity profile
in vitro potency
cell based assay data
references
  1. Kanaoka, Y.; Kimura, S. H.; Okazaki, I.; Ikeda, M.; Nojima, H., GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. FEBS Lett 1997, 402 (1), 73-80.
  2. Sorrell, F. J.; Szklarz, M.; Abdul Azeez, K. R.; Elkins, J. M.; Knapp, S., Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure 2016, 24 (3), 401-11.
  3. Kimura, S. H.; Tsuruga, H.; Yabuta, N.; Endo, Y.; Nojima, H., Structure, expression, and chromosomal localization of human GAK. Genomics 1997, 44 (2), 179-87.
  4. Sato, J.; Shimizu, H.; Kasama, T.; Yabuta, N.; Nojima, H., GAK, a regulator of clathrin-mediated membrane trafficking, localizes not only in the cytoplasm but also in the nucleus. Genes Cells 2009, 14 (5), 627-41.
  5. Thul, P. J.; Akesson, L.; Wiking, M.; Mahdessian, D.; Geladaki, A.; Ait Blal, H.; Alm, T.; Asplund, A.; Bjork, L.; Breckels, L. M.; Backstrom, A.; Danielsson, F.; Fagerberg, L.; Fall, J.; Gatto, L.; Gnann, C.; Hober, S.; Hjelmare, M.; Johansson, F.; Lee, S.; Lindskog, C.; Mulder, J.; Mulvey, C. M.; Nilsson, P.; Oksvold, P.; Rockberg, J.; Schutten, R.; Schwenk, J. M.; Sivertsson, A.; Sjostedt, E.; Skogs, M.; Stadler, C.; Sullivan, D. P.; Tegel, H.; Winsnes, C.; Zhang, C.; Zwahlen, M.; Mardinoglu, A.; Ponten, F.; von Feilitzen, K.; Lilley, K. S.; Uhlen, M.; Lundberg, E., A subcellular map of the human proteome. Science 2017, 356 (6340).
  6. Zhang, C. X.; Engqvist-Goldstein, A. E.; Carreno, S.; Owen, D. J.; Smythe, E.; Drubin, D. G., Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events. Traffic 2005, 6 (12), 1103-13.
  7. 7.         Eisenberg, E.; Greene, L. E., Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 2007, 8 (6), 640-6.
  8. Chaikuad, A.; Keates, T.; Vincke, C.; Kaufholz, M.; Zenn, M.; Zimmermann, B.; Gutierrez, C.; Zhang, R. G.; Hatzos-Skintges, C.; Joachimiak, A.; Muyldermans, S.; Herberg, F. W.; Knapp, S.; Muller, S., Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem J 2014, 459 (1), 59-69.
  9. Susa, M.; Choy, E.; Liu, X.; Schwab, J.; Hornicek, F. J.; Mankin, H.; Duan, Z., Cyclin G-associated kinase is necessary for osteosarcoma cell proliferation and receptor trafficking. Mol Cancer Ther 2010, 9 (12), 3342-50.
  10. Dzamko, N.; Zhou, J.; Huang, Y.; Halliday, G. M., Parkinson's disease-implicated kinases in the brain; insights into disease pathogenesis. Front Mol Neurosci 2014, 7, 57.
  11. Sakurai, M. A.; Ozaki, Y.; Okuzaki, D.; Naito, Y.; Sasakura, T.; Okamoto, A.; Tabara, H.; Inoue, T.; Hagiyama, M.; Ito, A.; Yabuta, N.; Nojima, H., Gefitinib and luteolin cause growth arrest of human prostate cancer PC-3 cells via inhibition of cyclin G-associated kinase and induction of miR-630. PLoS One 2014, 9 (6), e100124.
  12. Ray, M. R.; Wafa, L. A.; Cheng, H.; Snoek, R.; Fazli, L.; Gleave, M.; Rennie, P. S., Cyclin G-associated kinase: a novel androgen receptor-interacting transcriptional coactivator that is overexpressed in hormone refractory prostate cancer. Int J Cancer 2006, 118 (5), 1108-19.
pk properties
co-crystal structures
synthetic schemes
materials and methods
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