[ 3 H]BMT-046091 a potent and selective radioligand to determine AAK1 distribution and target engagement

Abstract Adaptor-associated kinase 1 ( AAK1), a member of the Ark1/Prk1 family of serine/threonine kinases, plays a role in modulating clatherin coated endocytosis of specific surface receptors. We have demonstrated that AAK1inhibitors are efficacious in rodent models of neuropathic pain(Kostich et al., 2016). Here we have characterized the binding properties and distribution pattern of the tritiated AAK1 radioligand, [ 3 H]BMT-046091, in rodents and cynomolgus monkeys, and used the radioligandto measure the brain target occupancy following systemic administration of AAK1inhibitors. We have found that [ 3 H]BMT-046091 is potent and selective AAK1inhibitor. It inhibits AAK1phosphorylation of a peptide derived from a physiologic substrate, the μ2 subunit of the adaptor protein complex, with an IC 50 value of 2.8 nM, and is inactive at >5 μM in a panel of functional or binding assays for receptors, transporters and enzymes. [ 3 H]BMT-046091 binding in the brain is absent in the AAK1 knockout mouse, and is displaceable with a high concentration of AAK1inhibitors in wild type mice. Specific [ 3 H]BMT-046091 binding is widespread in the brain and spinal cord with the highest density in the cortex, hippocampus, amygdala, striatum and thalamus. In the spinal cord, [ 3 H]BMT-046091 binding appears enriched in the dorsal horn superficial layers. Oral administration of LP-935509, an AAK1inhibitor, results in a dose-dependent occupation of AAK1binding sites in the brain and spinal cord. The increase in AAK1binding site occupancy by LP-935509 correlates with the decrease in antinociceptive responses in the rat chronic constriction injury model of neuropathic pain.