Summaryβ-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.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.The human protein Ser/Thr kinases Adaptor protein 2-Associated Kinase 1 (AAK1) and BMP-2-Inducible Kinase (BMP2K/BIKE) play critical roles in mediating endocytosis and other key signaling pathways. Both are broadly expressed and are members of the NAK family of human kinases, which also includes Cyclin G-Associated Kinase (GAK) and Myristoylated and Palmitoylated Serine/Threonine Kinase 1 (MPSK1/STK16). The family shares little homology outside of their kinase domains. 1 AAK1 and BMP2K are the most closely related, with overall sequence identity of 50% and kinase domain sequence identity of 74%. 2 A key function of AAK1 is regulation of receptor-mediated endocytosis via binding directly to clathrin and phosphorylating the medium subunit of AP2 (adaptor protein 2), which stimulates binding to cargo proteins. [3][4][5] AAK1 also modulates the Notch pathway, partially through its phosphorylation of Numb. 6, 7 BMP2K plays a role in osteoblast differentiation, is a clathrin-coated vesicle-associated protein, and, like AAK1, also associates with Numb. 8, 9 Due to their many functions, AAK1 and BMP2K have been implicated as potential drug targets for diverse conditions. AAK1 has been linked to diseases affecting the brain such as schizophrenia, Parkinson's disease and amyotrophic lateral sclerosis as well as implicated as a potential anti-viral target for the treatment of Hepatitis C. 5, 10, 11 BMP2K has been associated with myopia and evaluated as a potential treatment for HIV. 12, 13 A dual AAK1/BMP2K small molecule inhibitor was recently reported as a novel therapeutic to treat neuropathic pain. 14 X-ray crystal structures for the kinase domains of all NAK family members have been solved and reported. 2, 15, 16 Published and novel high-resolution crystal structures of AAK1 and BMP2K reveal target-specific structural features that have enabled our design of specific chemical probes and allowed further

Potent, selective, and cell active small molecule kinase inhibitors are useful tools to help unravel the complexities of kinase signaling. As the biological functions of individual kinases become better understood, they can become targets of drug discovery efforts. The small molecules used to shed light on function can also then serve as chemical starting points in these drug discovery efforts. The Nek family of kinases has received very little attention, as judged by number of citations in PubMed, yet they appear to play many key roles and have been implicated in disease. Here we present our work to identify high quality chemical starting points that have emerged due to the increased incidence of broad kinome screening. We anticipate that this analysis will allow the community to progress towards the generation of chemical probes and eventually drugs that target members of the Nek family.