Rho-associated Coiled-coil Protein Kinase Inhibitor Research Articles

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.

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.

Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival

Corneal allograft survival is mediated by the variety of immunological reactions and wound healing process. Our aim was to explore the effects of topical administration of ripasudil, a selective Rho-associated coiled-coil protein kinase inhibitor, on corneal allograft survival. Ripasudil was administered to mice thrice a day after allogeneic corneal transplantation. Corneal graft survival, opacity, neovascularization, re-epithelization, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in the grafted cornea and draining lymph nodes (dLNs) were evaluated with slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. Graft survival was significantly prolonged with lower graft opacity and neovascularization scores in 0.4% and 2.0% ripasudil-treated groups, and mRNA levels of angiogenic and pro-inflammatory factors in ripasudil-treated grafted corneas were reduced. Moreover, 0.4% and 2.0% ripasudil reduced CD45+-infiltrated leukocyte frequency, Cd11b and Cd11c mRNA levels, and the frequencies of mature dendritic cells, IFNγ-, and IL-17- producing CD4+T cells in the dLNs of recipients. Re-epithelization rate of the grafted cornea was significantly higher in the 0.4% and 2.0% ripasudil groups than in the control. Topically applied ripasudil prolonged graft survival by downregulating neovascularization and inflammation factors, while promoting corneal re-epithelization, suggesting that ripasudil may be useful for suppressing immunological rejection in corneal transplantation.

Design of novel ROCK inhibitors using fragment-based de novo drug design approach.

Rho-associated coiled-coil protein kinase (ROCK) is playing a vital role in the regulation of key cellular events and also responsible for causing several pathological conditions such as cancer, hypertension, Alzheimer's, cerebral vasospasm, and cardiac stroke. Therefore, it has attracted us to target ROCK protein as a potential therapeutic target for combating various diseases. Consequently, we investigated the active site of ROCK I protein and designed novel leads against the target using the de novo evolution drug design approach. Caffeic acid (an aglycone of acteoside) as a scaffold and fragments from 336 reported ROCK inhibitors were used for the design of novel leads. Multiple copy simultaneous search docking was used to identify the suitable fragments to be linked with the scaffold. Basic medicinal chemistry rules, coupled with structural insights generated by docking, led to the design of 7a, 8a, 9a, and 10a as potential ROCK I inhibitors. The designed leads showed better binding than the approved drug fasudil and also interacted with the key hinge region residue Met156 of ROCK I. Further, molecular dynamics (MD) simulation revealed that the protein-ligand complexes were stable and maintained the hydrogen bond with Met156 throughout the MD run. The promising in silico outcomes suggest that the designed compounds could be suitable anti-cancer leads that need to be synthesized and tested in various cancer cell lines. Graphical abstract.

Rho-kinase ROCK inhibitors reduce oligomeric tau protein

Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3β or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.

Abstract 9614: Ca 2+ -Independent Di-phosphorylation of Myosin Light Chain by Rho-associated Coiled-Coil Protein Kinase (ROCK) in the Subjunctional Regions of Cell-Cell Contact Mediates Thrombin-Induced Barrier Dysfunction in Vascular Endothelial Cells

A disruption of endothelial barrier function is a predisposing factor for the development of atherosclerosis. The phosphorylation of myosin light chain (MLC) is a key signal of barrier dysfunction. MLC phosphorylation is thought to take place sequentially at S19 and then T18, induces actin stress fiber formation and generates traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier dysfunction. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier dysfunction. Thrombin (1 u/mL) increased the cytosolic Ca 2+ concentration and decreased the transendothelial electrical resistance (TEER) with a peak at 1 and 3-5 min, respectively, in porcine aortic endothelial cells (PAEC). A new SDS-PAGE method using a Phos-tag TM compound, which specifically binds to a phosphate group, allowed separate quantification of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP under resting conditions. Thrombin stimulation only slightly increased MLC-P at 3 min, while it showed up on stress fibers. MLC-PP increased to a peak of 35% at 3-5 min, and thereafter declined to the resting level within 15 min. MLC-PP was localized in the subjunctional region of cell-cell contact in addition to the stress fibers at 3 min. ROCK inhibitors, Y27632 and H1152, inhibited the thrombin-induced decrease in TEER, the increase in MLC-PP and the subjunctional localization of MLC-PP, while having no effect on the Ca 2+ response. The MLC-P level remained unaffected by Y27632, while the stress fiber localization was inhibited. Gαq inhibitor, YM254890, inhibited the Ca 2+ response and the decrease in TEER, but it had no effect on the increase in MLC-PP. The introduction of dominant negative mutants of Gα12, Gα13 and Gαq using a cell-penetrating peptide inhibited the thrombin-induced decrease in TEER. The present study provides the first evidence that MLC-P and MLC-PP are differentially regulated in endothelial cells, and that Ca 2+ -independent, ROCK-mediated MLC-PP in the subjunctional regions plays a critical role in the thrombin-induced barrier dysfunction. Inhibition of MLC-PP is thus necessary to restore endothelial barrier dysfunction.

Effect of ROCK Inhibitor Y-27632 on Normal and Variant Human Embryonic Stem Cells (hESCs) In Vitro: Its Benefits in hESC Expansion

The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.