Targeting PAK1 using Adhiron reagents to inform drug design

Cancer remains a leading cause of death worldwide, with the dysregulation of the cell cycle being a hallmark of all human cancers. p21-activated kinase 1 (PAK1) plays a crucial role in cell cycle regulation and is often overexpressed in certain cancers. Due to its involvement in multiple oncogenic pathways, PAK1 has emerged as a promising therapeutic target. However, as a member of the PAK family, PAK1 has distinct as well as overlapping functions and structures with other isoforms, making selective targeting challenging. This study explores the use of non-antibody engineered proteins, known as Adhirons, to selectively inhibit PAK1, highlighting their potential for novel cancer treatment strategies.
Using phage display and high-throughput screening, three Adhirons (P60, P66, and P73) were identified with inhibitory effects on PAK1, exhibiting 99.74%, 91.19%, and 21.08% inhibition, respectively. Structural studies revealed distinct modes of inhibition among Adhirons. P60 functions as an ATP-competitive inhibitor, occupying the ATP-binding site of PAK1, whilst P66 and P73 act as allosteric inhibitors, binding away from ATP-binding pocket. Cross-reactivity and surface plasmon resonance studies showed, 1) P66 exhibited high affinity and broad binding across the PAK family, 2) P60 showed also high affinity but interacted with PAK1, PAK2, and PAK3, and 3) P73 showed lowest affinity but had highest specificity for PAK1. Differences in PAK1 inhibitory and specificity activities were related to variations in Adhiron-PAK1 interaction and conformational effects on each PAK.
Our strategy has enabled the successful identification of PAK1 inhibiting Adhirons which will offer valuable insights for the development of PAK1 specific small molecules.