In vitro activity: CNX-774 is a potent, irreversible/covalent, orally bioactive, and highly selective BTK (Bruton’s tyrosine kinase) inhibitor with IC50 of<1 nM. It forms a ligand-directed covalent bond with Cys-481, a non-conserved amino acid within the active site of the enzyme.

Kinase Assay: In biochemical assays, CNX-774 has demonstrated potent inhibition of Btk with an IC50 of<1nM in a continuous-read assay. The covalent bonding of CNX-774 to Btk was confirmed by incubating recombinant Btk protein with a 10-fold molar excess of CNX-774 for 1 hour at room temperature and analysis by MALDI-TOF MS. A shift of protein mass corresponding to the molecular weight of CNX-774 confirmed the covalent bonding of CNX-774 to Btk. Digestion of the covalently bonded Btk with pepsin followed by MSMS analysis established the bonding of CNX-774 to Cys-481.

Cell Assay: In cellular assays, CNX-774 demonstrates potent inhibitory activity towards BTK with IC50 of 1-10 nM by targeting Cys-481 residue within the ATP binding site of the enzyme. Cellular potency as well as prolonged duration of action of CNX-774 was demonstrated in Ramos cells by using a biotinylated covalent probe that targets the same Cysteine residue as CNX-774. CNX-774 was found to be>90% extractable after 2 hrs of incubation in both rat and human whole blood. These results demonstrate that CNX-774 has potent inhibitory activity towards the intended target, Btk, while achieving remarkable specificity in a variety of assays designed to assess off-target reactivity towards abundant cellular thiols and blood proteins.