In vitro activity: Asciminib is a potent and selective BCR-ABL inhibitor that maintains activity across most mutations, including T315I, with a distinct, allosteric mechanism of action. Asciminib binds at a regulatory site typically occupied by a myristoyl group in wild-type ABL and inhibits ABL kinase activity through a mechanism distinct from catalytic site inhibitors. It binds to a pocket on the BCR-ABL kinase domain that is normally occupied by the myristoylated N-terminus of ABL1. Upon fusion with BCR, this myristoylated N-terminus that serves to autoregulate ABL1 activity is lost. Asciminib functionally mimics the role of the myristoylated N-terminus by occupying its vacant binding site and restores the negative regulation of the kinase activity. Asciminib selectively inhibits the growth of chronic myelogenous leukemia (CML) and Ph+ ALL cells with potencies ranging from 1-10 nM range while BCR-ABL-negative cell lines remained unaffected at concentrations 1000-fold higher. NMR and biophysical studies confirm that Asciminib binds potently (dissociation constant (Kd) = 0.5-0.8 nM) and selectively to the myristoyl pocket of ABL1 and induces the inactive C-terminal helix conformation. Asciminib lacks activity against more than 60 kinases, including SRC and is similarly inactive against G-protein-coupled receptors, ion channels, nuclear receptors and transporters.

Kinase Assay: Asciminib is a potent and selective allosteric inhibitor of BCR-ABL1with dissociation constant (Kd) of 0.5-0.8 nM and selectivity to the myristoyl pocket of ABL1.

Cell Assay: KCL-22 cells are treated with a range of concentrations of ABL001, nilotinib or dasatinib for 1 hour. Cells are harvested, protein lysates generated and analyzed with Western Blots.