Lessons from LIMK1 enzymology and their impact on inhibitor design

Abstract

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique ‘rock-and-poke’ mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.

Structural Aspects of LIMK Regulation and Pharmacology

“…Only in the DFG-in conformation, the kinase is capable of binding to ATP and of catalysing the phosphoryl transfer reaction. The LIMK kinase domain is structurally well explored [ 8 , 9 , 10 ]. To date, nine structure models have been deposited to the PDB ( Table 1 ).…”

“…The binding of small-molecule inhibitors often distorts the kinase fold, stabilizing inactive kinase conformations instead. All LIMK inhibitors reported to date bind to the ATP pocket [ 8 , 10 ] and reshape neighbouring structure elements, mainly in the kinase N lobe. This is particularly evident for the G-rich loop, which can appear rotated or even detached upon inhibitor binding ( Figure 1 D).…”

“…This is particularly evident for the G-rich loop, which can appear rotated or even detached upon inhibitor binding ( Figure 1 D). Several inhibitors displace the αC helix and push it outwards [ 8 , 10 ]. As a consequence, the aforementioned K-E salt bridge is broken ( Figure 1 E).…”

“…Some inhibitors occupy the empty back pocket, thus stabilizing the LIMKs in the DFG-out conformation (type-1, type-2 and type-3 inhibitors will be discussed in a later section). Notably, even in the absence of inhibitors, the LIMK kinase domains can adopt both, the DFG-in and the DFG-out conformation, suggesting a low energy barrier between both conformations [ 10 ]. We believe that the existing LIMK kinase structure models cover most of its conformational space.…”

Progress in the therapeutic inhibition of Cdc42 signalling

“…PAK and MRCK pathways converge further downstream on LIM kinases. Obtaining selectivity between LIMK1 and LIMK2 with Type I and II inhibitors has posed a significant challenge, due to their 71% kinase domain sequence identity [ 75 ]. However several compounds have been developed, with high binding affinities and desirable pharmacokinetic properties [ 76 ].…”