Rho-associated Coiled-coil Kinase Research Articles

Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adultmice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/β-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Hydrogen Sulfide Protects against Rat Ischemic Brain Injury by Promoting RhoA Phosphorylation at Serine 188.

The protective role of hydrogen sulfide against cerebral ischemia-reperfusion injury involves the inhibition of the RhoA-/Rho-associated coiled-coil kinase (ROCK) pathway. However, the specific mechanism remains elusive. This study investigates the impact of hydrogen sulfide on RhoA phosphorylation at serine 188 (Ser188) in vivo, aiming to test the hypothesis that hydrogen sulfide exerts neuroprotection by enhancing RhoA phosphorylation at Ser188, subsequently inhibiting the RhoA/ROCK pathway. Recombinant RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids were constructed and administered via stereotaxic injection into the rat hippocampus. A rat global cerebral ischemia-reperfusion model was induced by bilateral carotid artery ligation to elucidate the neuroprotective mechanisms of hydrogen sulfide. Both RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids expressed RhoAwild and RhoAS188A proteins, respectively, in rat hippocampal tissues, alongside the intrinsic RhoA protein. Systemic administration of the exogenous hydrogen sulfide donor sodium hydrosulfide led to an increase in Ser188 phosphorylation of transfected RhoAwild and intrinsic RhoA protein within the hippocampus. However, this effect was not observed in tissues transfected with RhoAS188A. Sodium hydrosulfide-mediated RhoA phosphorylation correlated with decreased RhoA and ROCK2 activity in rat hippocampal tissues. Furthermore, sodium hydrosulfide administration reduced cerebral ischemia-reperfusion-induced neuronal damage and apoptosis in rat hippocampal tissues transfected with RhoAwild. However, this neuroprotective effect was attenuated in rats transfected with RhoAS188A. These findings suggest that the neuroprotective mechanism of hydrogen sulfide against cerebral ischemia/reperfusion injury involves increased RhoA phosphorylation at Ser188. Promoting this phosphorylation may represent a potential intrinsic therapeutic target for ischemic stroke.

Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans.

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.

Reduction of Phosphorylated Tau in Alzheimer's Disease Induced Pluripotent Stem Cell-Derived Neuro-Spheroids by Rho-Associated Coiled-Coil Kinase Inhibitor Fasudil.

Alzheimer's disease (AD) is the most predominant form of dementia. Rho-associated coiled coil kinase (ROCK) inhibitor, fasudil, is one of the candidate drugs against the AD progression. We aimed to investigate possible changes of AD associated markers in three-dimensional neuro-spheroids (3D neuro-spheroids) generated from induced pluripotent stem cells derived from AD patients or healthy control subjects (HC) and to determine the impact of pharmacological intervention with the ROCK inhibitor fasudil. We treated 3D neuro-spheroids with fasudil and tested the possible effect on AD markers by ELISA, transcriptomic and proteomic analyses. Transcriptomic analysis revealed a reduction in the expression of AKT serine/threonine-protein kinase 1 (AKT1) in AD neuro-spheroids, compared to HC. This decrease was reverted in the presence of fasudil. Proteomic analysis showed up- and down-regulation of proteins related to AKT pathway in fasudil-treated neuro-spheroids. We found an evident increase of phosphorylated tau at four different residues (pTau181, 202, 231, and 396) in AD compared to HC-derived neuro-spheroids. This was accompanied by a decrease of secreted clusterin (clu) and an increase of intracellular clu levels in AD patient-derived neuro-spheroids. Increases of phosphorylated tau in AD patient-derived neuro-spheroids were suppressed in the presence of fasudil. Fasudil modulates clu protein levels and enhances AKT1 that results in the suppression of AD associated tau phosphorylation.

Effect and mechanism of Poria cocos polysaccharides on myocardial cell apoptosis in rats with myocardial ischemia-reperfusion injury by regulating Rho-ROCK signaling pathway

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.

Unraveling the mechanistic details of rho-associated coiled-coil kinase1 (ROCK1) inhibition: Implications from pharmacophore mapping and free energy analysis

Rho-associated coiled-coil kinases (ROCKs) are essential for various cellular processes and play a crucial role in neurological and inflammatory disorders. The two ROCK isoforms (ROCK1 and ROCK2) are involved in the pathogenesis of multiple diseases. Increased levels of ROCK1 are observed in patients with Alzheimer's disease (AD), making it a promising drug target. This work presents the mechanism of inhibitor binding at the ROCK1 active site using a combination of ligand and structure-based drug design techniques. Ligand-based pharmacophore studies were performed on previously reported ROCK1 inhibitors to unravel the 3D steric and electronic features essential for ROCK1 inhibition. Molecular docking and molecular dynamics-based free energy analysis studies were performed to recognize the amino acids assisting in inhibitor binding by stabilizing the identified pharmacophore features. The crucial 3D pharmacophore features of ROCK1 inhibitors included hydrophobic (HYD), hydrogen-bond acceptor (HBA), and hydrogen-bond donor (HBD) characteristics. These features were oriented next to the complementary amino acids in the ROCK1 active site, as evident from the detailed interaction analysis. The free energy calculations provided a quantifiable estimate of the favorable energetic contribution of Gly83, Val90, Lys105, Asp160, Leu205, and Asp216 to the total binding energy. These amino acids significantly contributed to the ROCK1-inhibitor complexes' overall binding free energy, indicating their importance in inhibitor binding.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Culture methods focusing on bile canalicular formation using primary human hepatocytes in a short time.

The development of models for predicting hepatotoxicity is warranted, as the hepatotoxicity risk of 38-51% of compounds is undetectable in nonclinical studies. Cholestatic drug-induced liver injury (DILI) is a condition in which bile acids are abnormally excreted into the capillary bile canaliculi and are accumulated in hepatocytes, caused by the inhibition of bile salt export pump (BSEP), a transporter that is mainly associated with excretion of bile acids. Although laboratory animals are used as models, the use of human-derived cells is required owing to species differences. Unfortunately, primary human hepatocytes (PHHs) show rapid loss of function in culture and difficulties in forming bile canaliculi. Therefore, we aimed to develop an in vitro culture method for the efficient formation of bile canaliculi and for assessing the function of BSEP in PHHs. Here, PHHs were cultured from 1h after thawing to day 2 with Z-VAD-FMK, a total caspase inhibitor, and RevitaCell™ supplement, an irreversible Rho-associated coiled-coil forming kinase (ROCK) inhibitor, in combination with RM-101. The PHHs formed bile canaliculi and showed BSEP function on day 6 of culture. Our findings suggest that cultured PHHs may improve the prediction accuracy of the risks of cholestatic DILI-contained toxicity on bile canaliculi.

Recent advances in the development of Rho kinase inhibitors (2015-2021).

Rho-associated coiled-coil kinases (ROCKs) are key downstream effectors of small GTPases. ROCK plays a central role in diverse cellular events with accumulating evidence supporting the concept that ROCK is important in tumor development and progression. Numerous ROCK inhibitors have been investigated for their therapeutic potential in the treatment of cancers. In this article, we review recent research progress on ROCK inhibitors, especially those with potential for the treatment of cancers, reported in the literature from 2015 to 2021. Most ROCK inhibitors show potent in vitro and in vivo antitumor activities and have potential in the treatment of cancers.

Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds-AR-13324 (Netarsudil) and its active metabolite, AR-13503-and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Discovery of indoline-based derivatives as effective ROCK2 inhibitors for the potential new treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disease with a median survival of only 3–5 years. Due to the lack of effective therapy, IPF threatens human health. Recently, increasing reports have indicated that Rho-associated coiled-coil protein kinases (ROCKs) play important roles in the development of IPF and might represent a novel target for the treatment of IPF. Herein, a new series of selective ROCK2 inhibitors based on indoline were designed and synthesized. Structural modification resulted in optimized compound 9b with an IC50 value of 6 nM against ROCK2 and the inhibition of collagen gel contraction. Cellular assays demonstrated that 9b could significantly suppress the expression of collagen I and α-SMA, and inhibited ROCK signaling pathway. Oral administration of compound 9b (10 mg/kg) exerted more significant anti-pulmonary fibrosis effects than nintedanib (100 mg/kg) and KD025 (100 mg/kg) in a bleomycin-induced IPF rat model, suggesting that 9b could serve as a potential lead compound for the treatment of IPF.

An Overview of Adding Rho-Associated Coiled-Coil Kinase and Knockout Serum Replacement with Trehalose to a Low Glycerol Tris-Based Semen Extender

It is known that livestock animal semen is very sensitive to cold shock during freezing processes, and this sensitivity directly affects post-thaw sperm qualities which are progressive motility, mitochondrial membrane potential, sperm nuclear DNA integrity and in vitro spermatological parameters such as plasma membrane and acrosome integrity, and sperm fertility. In addition, with the sudden decrease in the total antioxidant level of the semen after thawing, the sperm cells are insufficient to tolerate their damage. Consequently, significant losses occur in sperm fertility. For this reason, researches on freezing the semen of livestock animals include semen processing; cryopreservation/cryogenic damage – thawing methods - sperm extenders, added antioxidants, the mechanisms of action and metabolic pathways of these antioxidants and physiological and metabolic parameters such as sperm fertility. It has been explained that low dose glycerol (trehalose added to increase the cryoprotectant effect) added to the extender in the freezing of livestock animal semen, knockout serum replacement (KSR) and Rho-associated coiled-coil kinase (ROCK), which are antioxidant additives, can increase the in vitro quality parameters of frozen thawed semen.

Opportunities and Challenges for the Development of MRCK Kinases Inhibitors as Potential Cancer Chemotherapeutics.

Cytoskeleton organization and dynamics are rapidly regulated by post-translational modifications of key target proteins. Acting downstream of the Cdc42 GTPase, the myotonic dystrophy-related Cdc42-binding kinases MRCKα, MRCKβ, and MRCKγ have recently emerged as important players in cytoskeleton regulation through the phosphorylation of proteins such as the regulatory myosin light chain proteins. Compared with the closely related Rho-associated coiled-coil kinases 1 and 2 (ROCK1 and ROCK2), the contributions of the MRCK kinases are less well characterized, one reason for this being that the discovery of potent and selective MRCK pharmacological inhibitors occurred many years after the discovery of ROCK inhibitors. The disclosure of inhibitors, such as BDP5290 and BDP9066, that have marked selectivity for MRCK over ROCK, as well as the dual ROCK + MRCK inhibitor DJ4, has expanded the repertoire of chemical biology tools to study MRCK function in normal and pathological conditions. Recent research has used these novel inhibitors to establish the role of MRCK signalling in epithelial polarization, phagocytosis, cytoskeleton organization, cell motility, and cancer cell invasiveness. Furthermore, pharmacological MRCK inhibition has been shown to elicit therapeutically beneficial effects in cell-based and in vivo studies of glioma, skin, and ovarian cancers.

Discovery of unglycosylated indolocarbazoles as ROCK2 isoform-selective inhibitors for the treatment of breast cancer metastasis

Breast cancer metastasis is a major challenge in clinical therapy because of the absence of effective treatments. Rho-associated coiled-coil kinase (ROCK), which is essential for cell invasion and migration, has recently been suggested as a potential target for the treatment of cancer metastasis. Herein, we report the structure−activity relationships (SAR) of indolocarbazoles against ROCK2 and reveal the crucial role of the C-3 hydroxyl for ROCK2 inhibition. The most potent unglycosylated aglycone THK01 was demonstrated to bind to and stabilize ROCK2 with potent anti-metastatic effects in breast cancer in vitro and in vivo with no obvious toxicities. Further mechanistic studies revealed that the anti-metastatic effect of THK01 was closely related to the suppression of STAT3Y705 activation. Moreover, THK01 exhibited excellent selectivity over the isoform protein ROCK1 (>100-fold). Taken together, with low toxicity, the ROCK2 inhibitor THK01 potently inhibited breast cancer metastasis through the ROCK2-STAT3 signaling pathway, which offers a new opportunity for the treatment of metastatic breast cancer.

Effect of the Rho-Kinase/ROCK Signaling Pathway on Cytoskeleton Components.

The mechanical properties of cells are important in tissue homeostasis and enable cell growth, division, migration and the epithelial-mesenchymal transition. Mechanical properties are determined to a large extent by the cytoskeleton. The cytoskeleton is a complex and dynamic network composed of microfilaments, intermediate filaments and microtubules. These cellular structures confer both cell shape and mechanical properties. The architecture of the networks formed by the cytoskeleton is regulated by several pathways, a key one being the Rho-kinase/ROCK signaling pathway. This review describes the role of ROCK (Rho-associated coiled-coil forming kinase) and how it mediates effects on the key components of the cytoskeleton that are critical for cell behaviour.

Characterization of Real-World Chronic Graft-Versus-Host-Disease Patients Receiving Belumosudil

Introduction Chronic graft-versus-host-disease (cGVHD) is a common complication of hematopoietic stem cell transplant (HSCT). cGVHD is characterized by inflammation and fibrosis that negatively impact multiple organ systems, resulting in substantial morbidity and mortality. While many systemic therapies are available to treat cGVHD, these therapies are associated with adverse events that may limit treatment adherence or lead to discontinuation. In July 2021, the Rho-associated coiled-coil kinase 2 (ROCK2) inhibitor belumosudil was approved by the United States Food and Drug Administration for the treatment of cGVHD for patients 12 years and older after failure of at least two prior lines of systemic therapy based on the efficacy observed in a randomized, open-label, multicenter trial (NCT03640481). Characteristics of patients being treated with belumosudil outside of a clinical trial have not been described previously. The objective of this study is to understand the real-world, non-clinical trial use of belumosudil for patients with cGVHD. Methods This retrospective study describes cGVHD patients recorded in the Komodo closed claims dataset between January 2016 and March 2022. The final cohort of interest included patients who were diagnosed with cGVHD (≥1 medical claim with the International Classifications of Disease Version 10 [ICD-10] diagnosis code D89.811), received belumosudil (≥1 claims with National Drug Code 79802020030), and were 12 years of age or older in the year of belumosudil initiation. Patients were required to have at least 36 months of continuous medical and pharmacy enrollment prior to their initial belumosudil prescription to ensure a complete medical diagnosis, treatment, and healthcare utilization history. Results A total of 151 patients were identified [median (interquartile range) age of 53 (40-62) years old; 54% female]. Sixty percent (92/151) of the patients had an HSCT within the 36-month observation period, and 73% (67/92) of the patients with an observable HSCT also had a diagnosis for a hematologic cancer in the 3 months prior to their HSCT. The median times from HSCT (in those with a recorded event) and cGVHD diagnoses (in all patients available) to belumosudil initiation were 3.2 (1.7-4.0) and 1.5 (0.5-3.1) years, respectively. During the 36 months pre-belumosudil treatment, common diagnoses included dyspnea (81%, 123/151), cytopenia (79%, 119/151; includes anemia, neutropenia, and thrombocytopenia), and "complications of stem cell transplant" (76%, 115/151; ICD-10 code T86.5). In the 12 months immediately prior to belumosudil initiation, the mean (standard deviation) Charlson Comorbidity Index score was 3.6 (2.2); a median of 9 (4-20) cGVHD-related healthcare encounters occurred; and nearly half of all patients had a hospitalization (all-cause 48%, 73/151; cGVHD-related, 27%, 41/151), with the most frequent primary diagnoses for admission attributed to respiratory illnesses and transplant complications. The majority of patients had received ≥3 lines of cGVHD treatment (85%, 128/151) in the 36 months pre-belumosudil, with a median of 5 (4-6) systemic therapies. Combination therapies containing either calcineurin inhibitors (17%; 25/151) or ruxolitinib (15% 23/151) were the most common lines of therapy immediately prior to belumosudil prescription. Use of strong Cytochrome P450 Family 3 Subfamily A (CYP3A) inducers was not observed during the observation period. In the 12 months prior to belumosudil initiation, 55% (83/151) patients were prescribed proton pump inhibitors. For the first belumosudil prescription that a patient received, 74% (111/151) of patients received once-daily dosing, and 26% (40/151) of patients received twice-daily dosing. Conclusions In this real-world data, prior to receiving belumosudil, patients received a median of 5 lines of systemic therapies at a median of 1.5 years from cGVHD diagnosis. Patients displayed multiple comorbidities and frequent hospitalizations that could complicate clinical management with existing cGVHD therapies. There is an unmet need for safe and effective treatment for cGVHD patients. Further research is needed to describe outcomes for patients being treated with belumosudil.

Rho-associated coiled-coil kinase contributes an important role in Xingnaojing injection-induced adverse drug reactions

Xingnaojing injection (XNJI) is widely used for acute cerebral hemorrhage. However, the adverse drug reactions (ADRs) caused by XNJI have attracted broad attention. In this study, mice were intravenously administrated with a single dose of XNJI (10 or 20 mL/kg) to evaluate the vascular permeability. Then, pathological examinations on ears and lungs were performed to further determine the characteristics of ADRs induced by XNJI. In addition, human umbilical vein endothelial cells (HUVECs) were stimulated with XNJI (1/128, 1/64, and 1/32 XNJI pure product dilution) and stained with rhodamine-phalloidin to analyze the alterations on cytoskeleton. In addition, endothelial monolayer was incubated with XNJI and the effect on permeability was tested. Western blot testing was conducted to measure the involvement of Rho-associated coiled-coil kinase (ROCK) in XNJI-induced ADRs and fasudil hydrochloride was applied to additionally confirm the contribution of ROCK. The results indicated that XNJI may lead to dose-associated vascular leakage. None visibly abnormal symptom was noticed in the mice treated with XNJI at 10 mL/kg, while inflammation in ears and lungs was observed in the mice administrated with 20 mL/kg XNJI. In in vitro experiment, stimulation with high concentration of XNJI (1/32 XNJI pure product dilution) could enhance the permeability of confluent endothelial monolayer and induce rearrangement of actin cytoskeleton. Activation of ROCK, which was shown as augmented expressions of p-MLC2 and p-MYPT1, was observed in cells and mice treated with XNJI. ROCK inhibitor could attenuate XNJI-induced dysfunction of endothelial barrier in in vitro experiment and alleviate the vascular leakage and inflammation noticed in mice treated with XNJI. This study indicates that overdose of XNJI can destroy vascular endothelial function and induce increased vascular permeability, which may cause ADRs in clinical practice. Meanwhile, ROCK plays an important role in XNJI-induced ADRs.

Fasudil, a ROCK inhibitor, preserves limb integrity in a mouse model of unilateral critical limb ischemia: Possible interplay of inflammatory and angiogenic signaling pathways

Peripheral arterial diseases (PAD) had a great attention owing to devastating consequences of disability and cardiovascular morbidity and mortality. Yet, current therapeutic options are limited to surgical revascularization with no effective pharmacotherapy available. Excessive activity of Rho-associated coiled-coil protein kinase (ROCK) is implicated with several vascular diseases, rendering ROCK inhibition as a potential therapeutic strategy for patients suffering vascular disorders. AimThe current study was dedicated to investigating the vascular protective potential of Fasudil, a ROCK inhibitor, on an experimentally induced unilateral critical limb ischemia (CLI) model in mice and demonstrated the possible underlying mechanisms. MethodsUnilateral CLI was induced by ligation and excision of femoral artery followed by daily i.p. injection of Fasudil (10 mg/kg or 25 mg/kg) up to two weeks post-surgery. Key findingsMice underwent CLI showed decreased antioxidant capacity and increased inflammatory signal, evident by elevation of ERK1/2 in both serum and GC muscles that coincided with increases in VEGFA, HIF-1α and CD34+ cells of GC muscles. CLI resulted in structural damage of GC muscle fibers, with marked apoptosis, declined proliferation and deteriorated peripheral limb function. Treatment with Fasudil restored antioxidant capacity and attenuated VEGFA, HIF-1α, CD34+ cells and inflammatory markers in ischemic limbs. Furthermore, Fasudil preserved histological integrity of ischemic GC muscles, with amelioration of apoptosis, preserved proliferation rate and improvement in peripheral limb function. SignificanceFasudil could protect against experimentally induced unilateral CLI, in a dose-dependent manner, which could pave the way for future clinical application of Fasudil in patients suffering PAD.