Selective Inhibitor of Protein Kinase PKN3 Generated by Conjugation of a Structurally Optimized Bumped N-(2-Aminoethyl)-8-anilinoisoquinoline-5-sulfonamide (H-9) with d-Arginine-Rich Chain

The protein kinase N family belongs to the AGC kinase group and contains three isozymes: PKN1, PKN2, and PKN3. Catalytic domains of PKNs share high sequence similarity, yet the proteins differ in tissue distribution, functions, and involvement in pathological processes. In particular, PKN3 has been implicated in tumor growth and metastatic progression, highlighting the need for isozyme-selective inhibitors as both research tools and therapeutic leads. Here, we report the rational design of selective PKN3 inhibitors based on distinctive structural features of this kinase. Two strategies were applied. First, the smaller threonine gatekeeper residue unique to PKN3 within the AGC group was exploited by derivatization of N-(2-aminoethyl)isoquinoline-5-sulfonamide (H-9) at position C8. Among the resulting compounds, a phenylamino-substituted derivative displayed the highest affinity, with a dissociation constant (KD) of 23 nM and more than 1000-fold selectivity over protein kinase A. Second, bisubstrate-analog design was employed to enhance binding to basophilic AGC kinases through covalent attachment of a (d-Arg)3-containing chain to H-9 derivatives. This approach yielded ARC-2603, which bound PKN3 with a KD value of 0.2 nM and showed 5500-fold selectivity over PKAcα. The selectivity of ARC-2603 was further evaluated in a commercial panel of 397 protein kinases, which supported its utility as a highly selective PKN3 inhibitor.