Cyclin G-associated kinase (GAK) is a 160 kDa serine/threonine kinase. Importantly, GAK features high homology outside its kinase domain with auxilin. GAK regulates receptor tyrosine kinases, such as the EGF receptor (EGFR). GAK plays a pivotal role in regulating not only receptor trafficking but also receptor signaling and function. GAK is a member of the numb-associated kinase (NAK) family. NAK family 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).

NAK is a putative serine/threonine kinase. NAK interacts physically with the phosphotyrosine binding (PTB) domain of Numb. NAK plays a role in cell fate determination during asymmetric cell divisions. Moreover, GAK expression increases during prostate cancer progression to androgen independence and is positively correlated with Gleason score in resections from prostate cancer patients.

In order to confirm the selectivity profiles across the NAK subfamily, Asquith CRM, et al evaluated the affinity of SGC-GAK-1 in homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET)-based ligand binding displacement assays. As a result, SGC-GAK-1 is a potent, selective, and cell-active GAK inhibitor. SGC-GAK-1 increases the thermal stability of the isolated GAK kinase domain. ITC reveals potent affinity for GAK (KD=4.5 nM). SGC-GAK-1 displays an impressive profile of over 16,000-fold selectivity relative to the other three NAK family members. Hopefully, SGC-GAK-1 shows strong growth inhibition in LNCaP, VCaP, and 22Rv1 cells at 10 µM, but minimal effect in PC3 and DU145 cells. SGC-GAK-1 shows potent anti-proliferative activity in LNCaP and 22Rv1 cells at a similar dose range that it engages GAK. All in all, SGC-GAK-1 is a chemical probe for GAK.