LIMK2

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the 'x-Φ-G-Φ' motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

The therapeutic potential of small molecule kinase inhibitors in cancer treatment is well recognized. However, achieving selectivity remains a formidable challenge, primarily due to the structural similarity of ATP binding pockets among kinases. Allosteric inhibition, which involves targeting binding pockets beyond the ATP-binding site, provides a promising alternative to overcome this challenge. In this study, a meticulous approach was implemented to prioritize type 3 inhibitors for LIMK2, employing a range of techniques including Molecular Dynamics (MD) simulations, e-pharmacophore-guided High Throughput Virtual Screening (HTVS), MM/GBSA and ADMETox analyses, Density Functional Theory (DFT) calculations, and MM/PBSA investigations. The e-pharmacophore model identifies a hypothesis featuring five essential pharmacophoric elements (RRRAH). Through virtual screening of the ZINC compound database, we identified only five compounds that align with all four pharmacophoric features: ZINC1044382792, ZINC1433610865, ZINC1044109145, ZINC952869440, and ZINC490621334. These compounds not only exhibit higher binding affinity but also demonstrate favorable ADME/Tox profiles. Molecular dynamics simulations underscore the stability of hydrogen bond interactions with critical cryptic LIMK2 pocket residues, Asp469 and Arg474, only for two compounds: ZINC143361086 and ZINC1044382792. These compounds also exhibit superior occupancy interactions, as indicated by HOMO-LUMO analysis. Additionally, binding free energy calculations highlight the significant affinities of these two compounds when complexed with LIMK2: -83.491 ± 1.230 kJ/mol and -90.122 ± 1.248 kJ/mol for ZINC1044382792 and ZINC1433610862, respectively. Hence, this comprehensive investigation identifies ZINC1433610862 and ZINC1044382792 as prospective hits, representing promising leads for targeting LIMK2 in cancer therapeutics.Communicated by Ramaswamy H. Sarma.

Breast cancer is frequently the most diagnosed female cancer in the world. The experimental studies on cancer seldom focus on the relationship between the central nervous system and cancer. Despite extensive research into the treatment of breast cancer, chemotherapy resistance is an important issue limiting the efficacy of treatment. Novel biomarkers to predict prognosis or sensitivity to chemotherapy are urgently needed. This study examined nervous-system-related genes. The profiling of differentially expressed genes indicated that high-LET radiation, such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of tumorigenicity in human breast epithelial cells. Bioinformatic tools allowed us to analyze the genes involved in breast cancer and associated with the nervous system. The results indicated that the gene expression of the Ephrin A1 gene (EFNA1), the roundabout guidance receptor 1 (ROBO1), and the kallikrein-related peptidase 6 (KLK6) was greater in T2 and A5 than in the A3 cell line; the LIM domain kinase 2 gene (LIMK2) was greater in T2 than A3 and A5; the kallikrein-related peptidase 7 (KLK7), the neuroligin 4 X-linked gene (NLGN4X), and myelin basic protein (MBP) were greater than A3 only in T2; and the neural precursor cell expressed, developmentally down-regulated 9 gene (NEDD9) was greater in A5 than in the A3 and E cell lines. Concerning the correlation, it was found a positive correlation between ESR1 and EFNA1 in BRCA-LumA patients; with ROBO1 in BRCA-Basal patients, but this correlation was negative with the kallikrein-related peptidase 6 (KLK6) in BRCA-LumA and -LumB, as well as with LIMK2 and ROBO1 in all BRCA. It was also positive with neuroligin 4 X-linked (NLGN4X) in BRCA-Her2 and BRCA-LumB, and with MBP in BRCA-LumA and -LumB, but negative with KLK7 in all BRCA and BRCA-LumA and NEDD9 in BRCA-Her2. The differential gene expression levels between the tumor and adjacent tissue indicated that the ROBO1, KLK6, LIMK2, KLK7, NLGN4X, MBP, and NEDD9 gene expression levels were higher in normal tissues than in tumors; however, EFNA1 was higher in the tumor than the normal ones. EFNA1, LIMK2, ROBO1, KLK6, KLK7, and MBP gene expression had a negative ER status, whereas NEDD9 and NLGN4X were not significant concerning ER status. In conclusion, important markers have been analyzed concerning genes related to the nervous system, opening up a new avenue of studies in breast cancer therapy.