Search

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.

LIMKs are serine/threonine and tyrosine kinases responsible for controlling cytoskeletal dynamics as key regulators of actin stability, ensuring synaptic health through normal synaptic bouton structure and function. However, LIMK1 overactivation results in abnormal dendritic synaptic development that characterizes the pathogenesis of Fragile X Syndrome (FXS). As a result, the development of LIMK inhibitors represents an emerging disease-modifying therapeutic approach for FXS. We report the discovery of MDI-114215 ( 85 ), a novel, potent allosteric dual-LIMK1/2 inhibitor that demonstrates exquisite kinome selectivity. 85 reduces phospho-cofilin in mouse brain slices and rescues impaired hippocampal long-term potentiation in brain slices from FXS mice. We also show that LIMK inhibitors are effective in reducing phospho-cofilin levels in iPSC neurons derived from FXS patients, demonstrating 85 to be a potential therapeutic candidate for FXS that could have broad application to neurological disorders or cancers caused by LIMK1/2 overactivation and actin instability.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

High rates of recurrence and treatment resistance in the most common malignant adult brain cancer, glioblastoma (GBM), suggest that monotherapies are not sufficiently effective. Combination therapies are increasingly pursued, but the possibility of adverse drug-drug interactions may preclude clinical implementation. Developing single molecules with multiple targets is a feasible alternative strategy to identify effective and tolerable pharmacotherapies for GBM. Here, we report the development of a novel, first-in-class, dual aurora and lim kinase inhibitor termed F114. Aurora kinases and lim kinases are involved in neoplastic cell division and cell motility, respectively. Due to the importance of these cellular functions, inhibitors of aurora kinases and lim kinases are being pursued separately as anti-cancer therapies. Using in vitro and ex vivo models of GBM, we found that F114 inhibits GBM proliferation and invasion. These results establish F114 as a promising new scaffold for dual aurora/lim kinase inhibitors that may be used in future drug development efforts for GBM, and potentially other cancers., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

LIMK1 and LIMK2 are involved in the regulation of cellular functions that depend on the dynamics of actin cytoskeleton. Disregulation of LIM kinases has been associated with diseases, such as tumor progression and metastasis, viral infection, and ocular diseases. Motivated by this, numerous studies have been carried out to discover small organic molecules capable of inhibiting LIM kinase effectively and selectively. In this review, a comprehensive survey of small organic molecules for LIM kinase inhibitors is reported, together with SAR study results, and the synthesis of these inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom., Competing Interests: The authors have declared that no competing interests exist.

Lung cancer is the leading cause of cancer-related deaths. LIM domain kinase (LIMK) 1 is a member of serine/threonine kinase family and highly expressed in various cancers. Luteolin, a polyphenolic plant flavonoid, has been reported to suppress tumour proliferation through inducing apoptosis and autophagy via MAPK activation in glioma. However, the mechanism of luteolin on suppressing lung cancer growth is still unclear. We found that luteolin targeted LIMK1 from the in silico screening and significantly inhibited the LIMK1 kinase activity, which was confirmed with pull-down binding assay and computational docking models. Treatment with luteolin inhibited lung cancer cells anchorage-independent colony growth and induced apoptosis and cell cycle arrest at G1 phase. Luteolin also decreased the expression of cyclin D1 and increased the levels of cleaved caspase-3 by down-regulating LIMK1 signalling related targets, including p-LIMK and p-cofilin. Furthermore, luteolin suppressed the lung cancer patient-derived xenograft tumour growth by decreasing Ki-67, p-LIMK and p-cofilin expression in vivo. Taken together, these results provide insight into the mechanism that underlies the anticancer effects of luteolin on lung cancer, which involved in down-regulation of LIMK1 and its interaction with cofilin. It also provides valuable evidence for translation towards lung cancer clinical trials with luteolin., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Peritoneal metastasis is a common form of metastasis among advanced gastric cancer patients. In this study, we reported the identification of LIM domain kinase 1 (LIMK1) as a promoter of gastric cancer peritoneal metastasis, and its potential to be a therapeutic target of dabrafenib (DAB). Using transcriptomic sequencing of paired gastric cancer peritoneal metastasis, primary tumors, and normal gastric tissues, we first unveiled that LIMK1 is selectively up-regulated in metastatic tumors. Increased LIMK1 in gastric cancer peritoneal metastasis was validated by immunohistochemistry analysis of an independent patient cohort. In vitro functional studies demonstrated that LIMK1 knockout or knockdown significantly inhibited cell migration and invasion of gastric cancer cells. LIMK1 knockout also abrogated peritoneal and liver metastases of gastric cancer cells in nude mice in vivo. Dabrafenib, a small molecule targeting LIMK1, was found to decrease cell migration and invasion of gastric cancer cells in vitro and abolish peritoneal and liver metastasis formation in vivo. Mechanistically, either LIMK1 knockout or Dabrafenib inhibited LIMK1 expression and phosphorylation of its downstream target cofilin. Taken together, our results demonstrated that LIMK1 functions as a metastasis promoter in gastric cancer by inhibiting LIMK1-p-cofilin and that Dabrafenib has the potential to serve as a novel treatment for gastric cancer peritoneal metastasis.

Primary Objective : In an ischemic stroke, the damage spreads from the infarction core to surrounding tissues. The present work was aimed at the search of effective neuroprotectors that restrict injury propagation. Research Design : We studied possible protective effects of inhibitors of protein kinases LIMK2 (T56-LIMKi), DYRK1A (harmine), and tryptophan hydroxylase (4-chlorophenylalanine) on infarction size and morphology of peri-infarct area after photothrombotic stroke (a model of ischemic stroke) in mouse brain. Methods and Procedures : Photothrombotic stroke was induced by laser irradiation of mouse cortex after administration of photosensitizer Bengal Rose, which does not penetrate cells and remains in blood vessels. Under light exposure, it induces vessel occlusion. Infarct volume and histological changes in the cerebral cortex were evaluated 3, 7 and 14 days after photothrombotic impact. Main Outcomes and Results : Harmine and 4-chlorophenylalanine did not influence infarct volume and morphology of peri-infarct area in the mouse brain cortex after photothrombotic stroke. However, LIMK2 inhibitor T56-LIMKi significantly reduced infarct volume 7 and 14 days after photothrombotic stroke. It also increased the percent of normochromic neurons and decreased the fraction of altered cortical cells (hypochromic, hyperchromic and pyknotic neurons). Conclusions : T56-LIMK2i may be considered as a promising anti-stroke agent.

Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ AML., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Depression is one of the most common forms of mental illness and also a leading cause of disability worldwide. Developing novel antidepressant targets beyond the monoaminergic systems is now popular and necessary. LIM kinases, including LIM domain kinase 1 and 2 (LIMK1/2), play a key role in actin and microtubule dynamics through phosphorylating cofilin. Since depression is associated with atrophy of neurons and reduced connectivity, here we speculate that LIMK1/2 may play a role in the pathogenesis of depression., Methods: In this study, the chronic unpredictable mild stress (CUMS), chronic restraint stress (CRS), and chronic social defeat stress (CSDS) models of depression, various behavioral tests, stereotactic injection, western blotting, and immunofluorescence methods were adopted., Results: CUMS, CRS, and CSDS all significantly enhanced the phosphorylation levels of LIMK1 and LIMK2 in the medial prefrontal cortex (mPFC) but not the hippocampus of mice. Administration of fluoxetine, the most commonly used selective serotonin reuptake inhibitor in clinical practice, fully reversed the effects of CUMS, CRS, and CSDS on LIMK1 and LIMK2 in the mPFC. Moreover, pharmacological inhibition of LIMK1 and LIMK2 in the mPFC by LIMKi 3 infusions notably prevented the pro-depressant effects of CUMS, CRS, and CSDS in mice., Conclusions: In summary, these results suggest that LIMK1/2 in the mPFC has a role in chronic stress-induced depressive-like effects in mice and could be a novel pharmacological target for developing antidepressants., (© The Author(s) 2020. Published by Oxford University Press on behalf of CINP.)

LIM kinase 1 (LIMK1) and p21-activated kinase 4 (PAK4) are often over-expressed in breast tumors, which causes aggressive cancer phenotypes and unfavorable clinical outcomes. In addition to the well-defined role in regulating cell division, proliferation and invasion, the two kinases promote activation of the MAPK pathway and cause endocrine resistance through phosphorylating estrogen receptor alpha (ERα). PAK4 specifically phosphorylates LIMK1 and its functional partners, indicating possible value of suppressing both kinases in cancers that over-express PAK4 and/or LIMK1. Here, for the first time, we assessed the impact of combining LIMK1 inhibitor LIMKi 3 and PAK4 inhibitor PF-3758309 in preclinical breast cancer models. LIMK1 and PAK4 pharmacological inhibition synergistically reduced the survival of various cancer cell lines, exhibiting specific efficacy in luminal and HER2-enriched models, and suppressed development and ERα-driven signals in a BT474 xenograft model. In silico analysis demonstrated the cell lines with reliance on LIMK1 were the most prone to be susceptible to PAK4 inhibition. Double LIMK1 and PAK4 targeting therapy can be a successful therapeutic strategy for breast cancer, with a unique efficiency in the subtypes of luminal and HER2-enriched tumors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.

Arsenic, an environmental contaminant in drinking water worldwide is well-established to increase cardiovascular diseases (CVDs) in humans. Of these, thrombotic events represent a major adverse effect associated with arsenic exposure, for which an abundance of epidemiological evidence exists. Platelet aggregation constitutes a pivotal step in thrombosis but arsenic alone doesn't induce aggregation and the mechanism underlying arsenic-induced thrombosis still remains unclear. Here we demonstrated that arsenic induces morphological changes of platelets, i.e., contraction and pseudopod projection, the primal events of platelet activation, which can increase platelet reactivity. Arsenite induced prominent platelet shape changes in a dose-dependent manner in freshly isolated human platelets. Of note, arsenite suppressed focal adhesion kinase (FAK) activity, which in turn activated RhoA, leading to altered actin assembly through LIMK activation, and subsequent cofilin inactivation. Arsenic-induced platelet shape change appeared to increase the sensitivity to thrombin and ADP-induced aggregation. Supporting this, latrunculin A, an inhibitor of actin-dynamics abolished it. Taken together, we demonstrated that arsenic induces cytoskeletal changes and shape changes of platelets through FAK-mediated alteration of actin dynamics, which renders platelets reactive to activating stimuli, ultimately contributing to increased thrombosis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

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., (© 2019 The Author(s).)

Actin is highly abundant in platelets, and its function is dependent on its structure. Actin filaments (F-actin) are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and its phosphatase slingshot (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that the inhibition of LIMK activity may prevent the changes in platelet shape and their function during activation by controlling the dynamics of F-actin. Our results demonstrate that in platelet, inhibition of LIMK by small LIMK inhibitors controls the level of filamentous actin leading to decreased platelet adhesion and aggregation. These findings encourage further studies on controlling platelet function via the cytoskeleton., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg -1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg -1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg -1 LIMK2i and 20.0 mg kg -1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement., Competing Interests: None

Objective: To determine if combined administration of LIMK2 and JNK inhibitors in a rat model of erectile dysfunction induced by cavernosal nerve (CN) injury could restore erectile function by suppressing both cavernosal apoptosis and fibrosis via rectification of molecular pathways related to the structural alterations., Methods: Sixty 12-week-old male Sprague-Dawley rats were categorized into 4 groups: (1) Sham-surgery (Sham) group, (2) CN-crush-injury (CNCI), (3) CNCI group (CNCI+L+1.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and low-dose (1.0 mg/kg) JNK-inhibitors, and (4) CNCI group (CNCI+L+10.0J) treated with a combination of 10.0 mg/kg LIMK2-inhibitors and a high dose (10.0 mg/kg) of JNK-inhibitors. Ten days after surgery, erectile response, histological-studies, and Western-blot was investigated., Results: The CNCI group showed a reduced maximal ICP/MAP or AUC/MAP, decreased immunohistochemical-staining of α-SMA, decreased SM/collagen ratio, increased phospho-cJun-positive apoptotic cells, increased phospho-LIMK2-positive fibroblasts, increased cJun-phosphorylation, increased LIMK2/Cofilin-phosphorylation, decreased Bcl-2/Bax ratio, and increased protein-expression of fibronectin, compared to the Sham group. Both the CNCI+L+1.0J and CNCI+L+10.0J groups showed improvements in erectile-responses, content of cavernosal α-SMA, number of phospho-cJun-positive apoptotic cells, Bcl-2/Bax ratio and cJun phosphorylation. Their improvements in the CNCI+L+10.0J group showed a tendency to be greater than those in the CNCI+L+1.0J group. Also, in the 2 treatment groups, rectification of SM/collagen ratio, number of phospho-LIMK2-positive fibroblasts, LIMK2/Cofilin-phosphorylation, and protein-expression of fibronectin was observed., Conclusion: This study suggests that combined inhibition of JNK and LIMK2 may improve erectile function by suppressing cavernosal apoptosis and fibrosis via restoration of cJun/Bcl-2/Bax and LIMK2/Cofilin pathways at 10 days after CN injury., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Actin is a highly abundant cytoskeletal protein that is essential for all eukaryotic cells and participates in many structural and functional roles. It has long been noted that estrogen affects cellular morphology. However, recent studies observed that both estrogen and tamoxifen induce a remarkable cytoskeletal remodeling independent of ER. In addition to ER, G protein-coupled estrogen receptor 1 (GPER, also known as GPR30) also binds to estrogen with high affinity and mediates intracellular estrogenic signaling. Here, we show that activation of GPER by its specific agonist G-1 induces re-organization of F-actin cytoskeleton. We further demonstrate that GPER acts through PLCβ-PKC and Rho/ROCK-LIMK-Cofilin pathway, which are upstream regulators of F-actin cytoskeleton assembly, thereby enhancing TAZ nuclear localization and activation. Furthermore, we find that LIMK1/2 is critical for GPER activation-induced breast cancer cell migration. Together, our results suggest that GPER mediates G-1-induced cytoskeleton assembly and GPER promotes breast cancer cell migration via PLCβ-PKC and Rho/ROCK-LIMK-Cofilin pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) therapies predominantly focus on β-amyloid (Aβ), but Aβ effects may be maximal before clinical symptoms appear. Downstream of Aβ, dendritic spine loss correlates most strongly with cognitive decline in AD. Rho-associated kinases (ROCK1 and ROCK2) regulate the actin cytoskeleton, and ROCK1 and ROCK2 protein abundances are increased in early AD. Here, we found that the increased abundance of ROCK1 in cultured primary rat hippocampal neurons reduced dendritic spine length through a myosin-based pathway, whereas the increased abundance of ROCK2 induced spine loss through the serine and threonine kinase LIMK1. Aβ 42 oligomers can activate ROCKs. Here, using static imaging studies combined with multielectrode array analyses, we found that the ROCK2-LIMK1 pathway mediated Aβ 42 -induced spine degeneration and neuronal hyperexcitability. Live-cell microscopy revealed that pharmacologic inhibition of LIMK1 rendered dendritic spines resilient to Aβ 42 oligomers. Treatment of hAPP mice with a LIMK1 inhibitor rescued Aβ-induced hippocampal spine loss and morphologic aberrations. Our data suggest that therapeutically targeting LIMK1 may provide dendritic spine resilience to Aβ and therefore may benefit cognitively normal patients that are at high risk for developing dementia., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.

Purpose: Retraction of the axon terminals of rod photoreceptors after retinal detachment breaks the first synapse in the visual pathway, resulting in visual impairment. Previous work showed that the mechanism of axonal retraction involves RhoA signaling and its downstream effector LIM Kinase (LIMK) activation. We examined the response of the downstream component cofilin, a direct binding protein of actin filaments, as well as the regulation by RhoA-LIMK-Cofilin signaling of actin assembly/disassembly, in the presynaptic ribbon terminal of injured rod cells., Methods: Injury was produced by retinal detachment or rod cell isolation. Detached porcine retina was probed for levels and localization of phosphorylated cofilin with Western blots and confocal microscopy, whereas rod cell cultures of dissociated salamander retina were examined for filamentous actin assembly/disassembly with a barbed end assay and phalloidin staining., Results: A detachment increased phosphorylation of cofilin in retinal explants; phosphorylation occurred in rod terminals in sections of detached retina. Isolation of rod cells resulted in axon retraction accompanied by an increase in actin barbed ends and a decrease in net filament labeling. All changes were significantly reduced by either Rho kinase (ROCK) or LIMK inhibition, using Y27632 or BMS-5, respectively. Cytochalasin D also reduced retraction and stabilized filaments in isolated rod cells., Conclusions: These results indicate that actin depolymerization via activation of RhoA downstream kinases and cofilin contributes to axon retraction. Preventing depolymerization, in addition to actomyosin contraction, may stabilize ribbon synapses after trauma.

We evaluated whether chronic administration of LIMK2-inhibitors could improve erectile function by alleviating CVOD through suppressing cavernosal fibrosis in a rat model of cavernosal nerve crush-injury (CNCI). Forty-two 12-week-old rats were equally categorized into the three groups: sham-surgery (S), CNCI (I), and CNCI treated with LIMK2-inhibitors (L). The L-group was treated with daily intraperitoneal injection of LIMK2-inhibitors (10.0 mg/kg) for 30-days after surgery. Erectile function was assessed using dynamic-infusion-cavernosometry (DIC). Penile tissue was processed for Masson's-trichrome staining, Western-blotting, and double immunofluorescence. The I-group showed significantly higher maintenance and drop rates as well as lower papaverine response, compared to the S-group. Chronic inhibition of LIMK2 in the L-group significantly improved the DIC parameters compared to those in the I-group, although the parameters were not completely restored to normal control values. Also, the I-group showed a reduced smooth muscle (SM)-to-collagen ratio, decreased immunohistochemical staining for α-SM-actin, increased number of fibroblasts positive for phosphorylated Cofilin, increased LIMK2/Cofilin phosphorylation and increased protein expression of Collagen-1 or Fibronectin, compared to the S-group. The L-group showed significant improvements in SM/collagen ratio and the deposition of Collagen-1 or Fibronectin compared to the I-group, although not completely normalized. According to the densitometry and confocal microscopy results, the L-group showed restoration of LIMK2/Cofilin phosphorylation and amount of fibroblasts positive for phosphorylated Cofilin to the normal control value. In conclusion, chronic inhibition of LIMK2 can improve CVOD and ED by alleviating cavernosal fibrosis via normalizing the LIMK2/Cofilin pathway., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Esophageal squamous cell carcinoma (ESCC) represents an aggressive malignancy often accompanied with a poor prognosis. Owing to the poor mortality and morbidity rates associated with this malignancy, a deeper understanding of the finer molecular changes that occur in ESCC is required in order to identify novel potential targets for early detection and therapy. At present the mechanism by which ESCC functions on a molecular level is not fully understood. Hence, the aim of the present study was to ascertain as to whether microRNA-384 (miR-384) influences the progression of ESCC., Material and Methods: Bioinformatics analysis was initially conducted to identify ESCC-related differentially expressed genes and predict regulatory miRs. After the target relationship between miR-384 and LIMK1 had been verified, the expression of miR-384 and LIMK1 in the EC9706 cell line was altered in an attempt to investigate the regulatory roles of miR-384 in the expression of the LIMK1/cofilin signaling pathway-related genes, cell proliferation, invasion, cell cycle distribution and apoptosis, in addition to lymph node metastasis (LNM) and tumor growth in nude mice., Results: Microarray-based gene expression profiling indicated that miR-384 affected the progression of ESCC through the LIMK1-mediated LIMK1/cofilin signaling pathway. Furthermore, miR-384 and Bax were observed to be poorly expressed, while LIMK1, cofilin and Bcl-2 were highly expressed in ESCC. The obtained evidences indicating that miR-384 targeted and negatively regulated LIMK1. Upregulation of miR-384 or LIMK1 inhibition was determined to block the LIMK1/cofilin signaling pathway, repress cell proliferation, invasion, cell cycle, LNM and tumor growth, while promote cell apoptosis in ESCC., Conclusion: Collectively, based on the key findings of the study, miR-384 could sequester LIMK1, which acts to suppress activation of the LIMK1/cofilin signaling pathway, thus ultimately inhibiting the development and progression of ESCC., (Copyright © 2018. Published by Elsevier Masson SAS.)

LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM kinases controls cofilin actin-severing activity. Consequently, LIM kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer treatments. LIMK has also been reported to be involved in pathways that are deregulated in hematologic malignancies, with little information regarding cofilin phosphorylation status. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Platelets activation/aggregation with subsequent thrombus formation is the main event in the pathophysiology of acute coronary syndrome. Once activated, platelets show an extensive cytoskeleton rearrangement that leads to recruitment of additional platelets to finally cause haemostatic plug formation. Thus, the cytoskeleton plays a pivotal role in this phenomenon. Colchicine (COLC) is an anti-inflammatory drug proven to reduce major cardiovascular events in patients with coronary artery disease. The molecular mechanisms by which COLC exerts these protective effects remain partially still unknown. Since COLC causes disruption of tubulin, a component of cell cytoskeleton, we investigated whether this drug might interfere with platelet aggregation by acting on cytoskeleton rearrangement., Methods and Results: Platelets isolated from healthy volunteers were activated with Adenosine Diphosphate (ADP, 20 μM) Collagen (COLL, 60 μg/ml) and Thrombin Activating Receptor Peptide (TRAP 25 μM) with/without COLC 10 μM pretreatment. After stimulus, aggregation was measured by light aggregometry overtime. Microtubules structure was assessed by immunohistochemistry and key proteins involved in regulation of actin-filament assembly and contractility such as Myosin Phosphatase Targeting subunit (MYPT), LIM domain kinase 1(LIMK1) and cofilin were evaluated by Western Blot analysis. Colchicine pretreatment significantly blunted ADP/COLL/TRAP-induced platelet aggregation (up to 40%). COLC effects appeared mediated by microtubules depolymerization and cytoskeleton disarrangement associated to inactivation of MYPT and LIMK1 that finally interfered with cofilin activity., Conclusions: Our data indicate that colchicine exerts anti-platelet effects in vitro via inhibition of key proteins involved in cytoskeleton rearrangement, suggesting that its beneficial cardiovascular properties may be due, at least in part, to an inhibitory effect of platelet activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

LIM kinases are involved in various pathophysiological processes that depend on actin organization. Alteration of microtubule dynamics by LIMK dysregulation is in fact related to tumor progression and metastasis, viral infection, and ocular diseases, such as glaucoma. As a consequence, many efforts have been done in recent years to rationally design small molecules able to inhibit LIMK activity selectively, without affecting other kinases. As a result, compounds optimized in terms of binding affinity and pharmacokinetic parameters have been discovered, that however failed to access clinical trials. In this review, a comprehensive survey of recent LIMK inhibitors is reported, together with SAR considerations and optimization processes., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg -1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg -1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway., Competing Interests: All authors declared no competing interests

Background and Purpose: In men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction., Experimental Approach: Human prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments., Key Results: Real-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 μM) and LIMKi3 (1 μM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α 1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA 2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability., Conclusions and Implications: Smooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors., (© 2018 The British Pharmacological Society.)

Background: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions., Results: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy., Conclusion: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.

Long-lasting changes in dendritic spines provide a physical correlate for memory formation and persistence. LIM kinase (LIMK) plays a critical role in orchestrating dendritic actin dynamics during memory processing, since it is the convergent downstream target of both the Rac1/PAK and RhoA/ROCK pathways that in turn induce cofilin phosphorylation and prevent depolymerization of actin filaments. Here, using a potent LIMK inhibitor (BMS-5), we investigated the role of LIMK activity in the dorsal hippocampus during contextual fear memory in rats. We first found that post-training administration of BMS-5 impaired memory consolidation in a dose-dependent manner. Inhibiting LIMK before training also disrupted memory acquisition. We then demonstrated that hippocampal LIMK activity seems to be critical for memory retrieval and reconsolidation, since both processes were impaired by BMS-5 treatment. Contextual fear memory extinction, however, was not sensitive to the same treatment. In conclusion, our findings demonstrate that hippocampal LIMK activity plays an important role in memory acquisition, consolidation, retrieval, and reconsolidation during contextual fear conditioning.

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.

Aims: Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation., Main Methods: E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling., Key Findings: Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5., Significance: Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Central sensitization represents a key mechanism mediating chronic pain, a major clinical problem lacking effective treatment options. LIM-domain kinases (LIMKs) selectively regulate several substrates, e.g. cofilin and cAMP response element-binding protein (CREB), that profoundly affect neural activities, such as synaptogenesis and gene expression, thus critical in the consolidation of long-term synaptic potentiation and memory in the brain. In this study, we demonstrate that LIMK deficiency significantly impaired the development of multiple forms of chronic pain. Mechanistic studies focusing on spared nerve injury (SNI) model reveal a pivotal role of LIMKs in the up-regulation of spontaneous excitatory synaptic transmission and synaptogenesis after pain induction. Depending on the pain induction methods, LIMKs can be transiently activated with distinct time courses. Accordingly, pharmacological inhibition of LIMKs targeting this critical period remarkably attenuated central sensitization in the spinal cord and alleviated pain behaviors. We propose selectively targeting LIMKs during their activation phase as a potential therapeutic strategy for clinical management of chronic pain, especially for chronic pain with predictable onset and development time courses, such as chronic post-surgical pain (PSP)., (Copyright © 2017. Published by Elsevier Ltd.)

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs. IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs., (Copyright © 2017 Yi et al.)

A sulfated saponin called "Frondoside A" (FRA) from sea cucumber and ingredients from Okinawa propolis (OP) have been previously shown to suppress the PAK1-dependent growth of A549 lung cancer as well as pancreatic cancer cells. However, the precise molecular mechanism underlying their anti-cancer action still remains to be clarified. In this study, for the first time, we found that both FRA and OP directly inhibit PAK1 in vitro in a selective manner (far more effectively than two other oncogenic kinases, LIMK and AKT). Furthermore, at least two major anti-cancer ingredients of OP, nymphaeols A and C, also directly inhibit PAK1 in vitro in a selective manner. To the best of our knowledge, FRA is the first marine compound that selectively inhibits PAK1. Likewise, these nymphaeols are the first propolis ingredients that selectively inhibit PAK1.

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 ( FMR1 ) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics., (Copyright © 2017, American Association for the Advancement of Science.)

Aim: To investigate the molecular mechanisms by which long-term exposure to cigarette smoke extract (CSE) contributes to ovarian cancer metastasis., Materials and Methods: Western blot analysis for diverse p110 isoforms of phosphoinositide 3-kinase (PI3K)-related signaling pathway and epithelial-mesenchymal transition (EMT) markers was performed to analyze the underlying mechanisms. Migratory activity of CSE-exposed ovarian cancer cells was determined by transendothelial migration and invasion assay., Results: After exposure to CSE for four weeks, CaOV3 (primary) and SKOV3 (metastatic) ovarian cancer cells showed enhanced mesenchymal characteristics and produced EMT-related cytokines [intwerleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF-β1)]. CSE exposure activated the Src-p110δ-p21 protein-activated kinase 1 (PAK1) in CaOV3 cells and the Lyn-p110β-Rho-associated kinases 1/2 (ROCK1/2) in SKOV3 cells, which led to the stimulation of LIM kinase 1 (LIMK1) phosphorylation and TGF-β1 release. LIMK1 knockdown efficiently blocked the migratory activity and suppressed the mesenchymal phenotypes of CSE-treated ovarian cancer cells. Reactive oxygen species (ROS) initiated the CSE-mediated EMT processes in ovarian cancer cells., Conclusion: Characterization of the p110 isotypes of PI3K is critical for regulating cancer metastasis; LIMK1 could be a common therapeutic target of ovarian cancer metastasis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

LIM kinase 1 (LIMK1) activity is essential for cell migration and cell cycle progression. Little is known about LIMK1 expression and function in mammalian oocytes. In the present study we assessed LIMK1 protein expression, subcellular distribution and function during mouse oocyte meiosis. Western blot analysis revealed high and stable expression of LIMK1 from the germinal vesicle (GV) to MII stage. In contrast, activated LIMK1 (i.e. LIMK1 phosphorylated at threonine 508 (pLIMK1 Thr508 )) was only detected after GV breakdown, with levels increasing gradually to peak at MI and MII. Immunofluorescence showed pLIMK1 Thr508 was colocalised with the microtubule organising centre (MTOC) components pericentrin and γ-tubulin at the spindle poles. A direct interaction between γ-tubulin and pLIMK1 Thr508 was confirmed by co-immunoprecipitation. LIMK inhibition with 1μM BMS3 damaged MTOC protein localisation to spindle poles, undermined the formation and positioning of functional MTOC and thus disrupted spindle formation and chromosome alignment. These effects were phenocopied by microinjection of LIMK1 antibody into mouse oocytes. In summary, the data demonstrate that LIMK activity is essential for MTOC organisation and distribution and so bipolar spindle formation and maintenance in mouse oocytes.

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research., (Copyright © 2015 Elsevier Inc. All rights reserved.)

PAK1 (RAC/CDC42-activated kinase 1) is the major oncogenic kinase, and a number of herbal PAK1-blockers such as propolis and curcumin have been shown to be anti-oncogenic and anti-melanogenic as well as anti-alopecia (promoting hair growth). Previously, we found several distinct PAK1-inhibitors in Okinawa plants including Alpinia zerumbet (alpinia). Thus, here, we tested the effects of these herbal compounds and their derivatives on the growth of cancer or normal hair cells, and melanogenesis in cell culture of A549 lung cancer, hair follicle dermal papilla cell, and B16F10 melanoma. Among these herbal PAK1-inhibitors, cucurbitacin I from bitter melon (Goya) turned out to be the most potent to inhibit the growth of human lung cancer cells with the IC50 around 140 nM and to promote the growth of hair cells with the effective dose around 10 nM. Hispidin, a metabolite of 5,6-dehydrokawain from alpinia, inhibited the growth of cancer cells with the IC50 of 25 μM as does artepillin C, the major anti-cancer ingredient in Brazilian green propolis. Mimosine tetrapeptides (MFWY, MFYY, and MFFY) and hispidin derivatives (H1-3) also exhibited a strong anti-cancer activity with the IC50 ranging from 16 to 30 μM. Mimosine tetrapeptides and hispidin derivatives strongly suppressed the melanogenesis in melanoma cells., (Copyright © 2015 John Wiley & Sons, Ltd.)

Lim kinase (Limk), a proline/serine-rich sequence, can regulate the polymerization of the actin filaments by phosphorylating, and it is found to be highly involved in various human diseases. In this paper, 47 reported Limk1 inhibitors with bis-aryl urea scaffold were used to design potent and selective Limk inhibitors by computational approaches. Firstly, the structure-Limk1 activity relationship models (3D-QSAR) and structure-Limk1/ROCK2 selectivity relationship models (3D-QSSR) were developed and both 3D-QSAR and 3D-QSSR models showed good correlative and predictive abilities. Then, the molecular docking and molecular dynamics (MD) simulations were employed to validate the optimal docking conformation and explore the binding affinities. Finally, five new compounds were designed and all of them exhibited good Limk1 inhibition and Limk1/ROCK2 selectivity after synthesis and biological evaluation, which demonstrated that the obtained information from computational studies were valuable to guide Limk inhibitors' design., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy.

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

Inhibitors of LIM kinases are considered of interest for several indications, including elevated intraocular pressure (IOP), cancer, or infection by HIV-1. LX-7101 (Lexicon Pharmaceuticals) was advanced to Phase-I clinical trials as an IOP-lowering agent for treatment of glaucoma. We here discuss the design, synthesis and evaluation of LIMK inhibitors based on a pyrrolopyrimidine scaffold, which represent close analogs of LX-7101. Exploration of structure-activity relationships revealed that many of such compounds, including LX-7101, cause potent inhibition of LIMK1 and LIMK2, and also ROCK2 and PKA. Molecular variations around the various structural elements of LX-7101 were attempted. Substitution on position 6 of the pyrrolopyrimidine scaffold led to the identification of LX-7101 analogs displaying good selectivity versus ROCK, PKA and Akt., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microwave irradiation of 2-halobenzonitriles and methyl thioglycolate in the presence of triethylamine in DMSO at 130 °C provides rapid access to 3-aminobenzo[b]thiophenes in 58-96% yield. This transformation has been applied in the synthesis of the thieno[2,3-b]pyridine core motif of LIMK1 inhibitors, the benzo[4,5]thieno[3,2-e][1,4]diazepin-5(2H)-one scaffold of MK2 inhibitors and a benzo[4,5]thieno[3,2-d]pyrimidin-4-one inhibitor of the PIM kinases.

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (>400-fold), potent inhibition of cofilin phosphorylation in A7r5, PC-3, and CEM-SS T cells (IC50 < 1 μM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors.

Prostate cancer affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining prostate cancer tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate Cofilin were found to be associated with poor outcome and reduced survival in patients with nonmetastatic prostate cancer. A LIMK-selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential prostate cancer therapy. LIMKi reduced prostate cancer cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent prostate cancer cells more effectively than in androgen-independent prostate cancer cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased αTubulin acetylation and decreased AR interactions with αTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of prostate cancer both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting prostate cancer cell dissemination., (©2014 American Association for Cancer Research.)