Lessons from LIMK1 enzymology and their impact on inhibitor design

Figure 1.. The ADF cascade regulates actin cytoskeleton dynamics. (A) The ADF cascade regulates actin cytoskeleton dynamics. Several pathways converge in LIMK1/2. In both ALS and FXS, the cascade is deregulated. (B) Chemical structure of LIMKi3, a widely used LIMK1 inhibitor characterized by high LIMK1 affinity, but unfavourable off-target activity [17]. (C) Chemical structure of LX-7101, a dual inhibitor targeting LIMK1 and ROCK1 [20].

Figure 2.. The rock-and-poke mechanism of catalysis. (A) LIMK1 is composed of two N-terminal LIM domains, the PDZ domain and the kinase domain. The expression construct LIMK1KD comprised the C-terminus only. (B) LIMK1KD (blue) in complex with ATPγS, bound to its physiological substrate CFL1 (green). In CFL1, the serine residue subjected to phosphorylation was mutated to cysteine, in order to trap the enzyme-substrate complex (PDB ID 5L6W). A previously published LIMK1-CFL1 structure (PDB ID 5HVK) showed the complex after the reaction (ADP and Phospho-CFL1 in white, LIMK1 omitted because its conformation was identical with the one shown in blue). The CFL1 binding angles differed by 12°. This rotation resulted in the CFL1 N-terminus poking into the LIMK1 active site (rock-and-poke mechanism). (C) The CFL1 anchor helix was firmly attached to the LIMK1 docking interface. The CFL1 residue K112 was co-ordinated by three LIMK1 residues. (D) LIMK1 bound its substrate ATPγS in the way typical for kinases. There were two hinge hydrogen bonds to hold the adenine in position. K368 and N465 were adjusting the triphosphate. The CFL1 C3 side chain was oriented for catalysis. For comparison, the…

Figure 3.. LIMK1 is a dual-specificity kinase. (A) CFL1 variants were tested as LIMK1KD substrates. AXG is the amino acid sequence of the CFL1 N-terminus. The X stands for S (CFL1 wt), T and Y, respectively. Please note M1 was cleaved during expression in bacteria. (B) Deconvoluted mass spectra of the reaction mixtures containing either CFL1 wt, S3T or S3Y. Phosphorylation induced an 80 Da shift to the CFL1 mass as indicated by the red arrows. (C) Comparison of the CFL1 phosphorylation grades. Both CFL1 wt and S3Y were good LIMK1 substrates, which makes LIMK1 a dual-specificity kinase. CFL1 S3T was not phosphorylated by LIMK1. The experiment was performed in triplicate.

Figure 4.. Identification and confirmation of LIMK1 inhibitors. (A) The effect of 407 conventional kinase inhibitors on the LIMK1 thermal stability was determined in DSF assay. PF-477736 induced the highest shift with 11 K, staurosporine stabilised LIMK1 by 5.1 K. (B) The inhibitory potential of the best DSF hits was probed in the RapidFire kinase activity assay. The dose-response curves for PF-477736 and staurosporine are plotted in blue and red, respectively. (C) The results obtained from DSF and RapidFire assays were in excellent agreement. Several inhibitors stabilised LIMK1, but not LIMK2. These inhibitors were isoform-specific (see also Supplementary Table S1).

Figure 5.. Examples of inhibitors bound to LIMK1. (A) Binding properties and chemical structures of PF-477736 (blue) and staurosporine (red). (B) The LIMK1-PF-477736 complex (PDB ID 5NXC). Beside the hinge interactions including the gatekeeper residue T413, the inhibitor bridged the DFG motif, the β3 sheet and the P loop. Parts of the model were removed for clarity. (C) The LIMK1-staurosporine complex (PDB ID 3S95). In addition to the two salt bridges to the LIMK1 hinge, there is only the interaction to H464 prior to the β7 sheet. This explains the low affinity of the compound. (D) Overlay of the PF-477736 (blue) and staurosporine (red) structures, as seen from the active site in the direction of the N-lobe. Staurosporine bound LIMK1 in an active-like conformation with intact K-E salt bridge, while PF-477736 binding induced an inactive conformation, characterised by a 9.6 Å outwards shift of αC helix, with no K-E salt bridge formed. The dark cloud represents the inhibitor binding site.

Figure 6.. Modulating the LIMK1 enzyme and the CFL1 substrate properties. (A) Recombinant PAK1KD phosphorylated recombinant LIMK1KD, while PAK1FL and PAK1KD D389N were not active. Please note the basal LIMK1 autophosphorylation. (B) Phosphorylation increased LIMK1 kinase activity by more than factor 10. PAK1KD did not phosphorylate CFL1 at all. (C) Binding of the CFL1 anchor helix to the LIMK1 docking interface. Residues important for binding and residues subjected to mutagenesis are highlighted. (D) Impact of amino acid exchanges in the CFL1 anchor helix on the CFL1 substrate properties. CFL1 K112 was indispensable for substrate recognition. The introduction of tryptophan in position 118 made CFL1 an even better LIMK1 substrate. Breaking the anchor helix in position 118 did not significantly alter substrate recognition. All experiments were performed in triplicate.