Targeting ILK and LIMK: Key modulators of the focal adhesion-actin cytoskeleton signaling axis

Focal adhesions are highly dynamic multi-protein assemblies that physically and functionally connect the extracellular matrix (ECM) to the actin cytoskeleton. These structures regulate essential cellular processes including adhesion, migration, mechano-signal transduction, proliferation, and survival. Central signaling pathways downstream of focal adhesions involve integrins, SRC family kinases, focal adhesion kinase (FAK), RHO GTPases, ROCK/PAK signaling, and LIM kinases (LIMK1/2), which together coordinate cytoskeletal organization and cellular responses to mechanical stimuli. Dysregulation of focal adhesion-actin cytoskeleton signaling has been implicated in numerous diseases, particularly cancer progression and metastasis, as well as neurological disorders, glaucoma, and fibrotic diseases. The work presented in this thesis focused on two distinct but functionally connected components of this signaling network: the pseudokinase Integrin-Linked Kinase (ILK), positioned upstream at focal adhesions, and the actin-regulating kinases LIMK1/2, which function downstream as direct regulators of cofilin phosphorylation and actin dynamics. ILK is a highly conserved pseudokinase that forms the core of the ILK:PINCH:α-parvin (IPP) complex, a central scaffold linking β-integrins to the actin cytoskeleton. Although ILK adopts a canonical kinase fold, it lacks several catalytic residues required for phosphoryl transfer and is therefore classified as a pseudokinase. Despite the absence of catalytic activity, ILK retains ATP-binding capability, which contributes to structural stability and mechano-signal transduction within focal adhesions. ILK has emerged as an important node in cancer, fibrosis, and cardiovascular disease, yet the lack of validated small-molecule ligands has significantly limited mechanistic and therapeutic exploration of this target. In this thesis, a biophysical screening strategy identified the cyano-quinoline scaffold as the first validated small molecule chemotype capable of targeting the ATP binding site of ILK. Differential scanning fluorimetry (DSF) screening of kinase-focused libraries revealed pelitinib and related quinoline derivatives as initial binders of the ILK:α-parvin complex. Subsequent structural and biophysical characterization using surface plasmon resonance (SPR), X-ray crystallography, and mutagenesis demonstrated that these ligands engage the ILK pseudoactive site in a type-I kinase binding mode. Structural studies revealed that ligand selectivity was strongly influenced by the unique architecture of the ILK hinge region, particularly the GK+2 tryptophan residue, which sterically restricted binding of related EGFR inhibitors such as afatinib. Extensive structure–activity relationship (SAR) studies identified key ligand positions required for ILK binding, including the importance of the 7-ethoxy substitution and halogenated aniline back-pocket interactions. To evaluate ligand engagement in a cellular setting, a NanoBRET-based assay was established using the ILK:α-parvin complex. Although the assay functioned primarily in digitonin-permeabilized cells, it provided an important orthogonal platform to validate intracellular target engagement. Kinome-wide selectivity profiling further demonstrated that optimized cyano-quinoline ligands displayed favorable selectivity compared to the parent EGFR inhibitor, pelitinib. Finally, we confirmed the biological activity of compound 1 (DHP) via ILK by demonstrating its effect on the actin cytoskeleton through F-actin staining experiments. Collectively, this work establishes the cyano-quinoline scaffold as a chemical starting point for future ILK ligand optimization and provides the first validated chemical tools for probing ILK biology. Furthermore, the structural insights generated in this study suggest future opportunities for targeting the ILK ATP pocket, the ILK:α-parvin interface, and potentially exploiting ILK for targeted protein degradation strategies.