KINOMEscan assay profile for gilteritinib at 100 nM. This commercially available assay of a kinase inhibitor’s relative potency and selectivity is widely displayed but often misunderstood. This profile displays gilteritinib’s affinity for the different kinases in the human genome at 100 nM. A concentration of 100 nM gilteritinib is a crude approximation of steady-state levels achieved in plasma by patients receiving the standard dose of 120 mg/d. The KINOMEscan assay measures the ability of a test compound, such as gilteritinib, at specific concentrations (100 nM in this case) to displace a reference ligand from the active site of a kinase. Therefore, it is not an in vitro kinase assay but rather a drug-binding assay. The reference ligand is a proprietary compound designed to bind in highly promiscuous fashion to kinase active sites (earlier developmental versions of this assay used staurosporine). Kinases, derived from a library of 468 genes, are individually expressed in bacteriophages for use in the assay. In the case of transmembrane receptor kinases, just the cytoplasmic domain is expressed. The library includes the vast majority of wild-type human kinases, as well as key mutant kinases, such as FLT3-ITD and FLT3-TKD variants, including the F691L gatekeeper mutation. Each small gray circle on a branch represents an individual kinase. A red circle over any point of a branch indicates that gilteritinib competes with the reference compound for binding. “Percent control” refers to the efficacy of the competition. The larger the circle, the more effectively it displaces the reference ligand. A value of “0%” (the largest red circle) indicates the highest-affinity binding, meaning no reference ligand binds to the kinase. Note that these are not dissociation constant (Kd) values, although this assay can be used to calculate such values. Images generated using the TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of DiscoveRx Corporation. © 2010 DiscoveRx Corporation.