Drug Design And Molecular Modeling Studies Of Rho Kinase

Rho-associated kinases(ROCK1 and ROCK2) belong to the AGC family of serine-threonine kinases, and they are originally discovered as the downstream effectors of Rho. ROCKs display their biological activities by phosphorylating a series of downstream targets, including myosin light chain(MLC), Lin-11, Isl-1, Mec-3 kinase,(LIMK), ezrin/radixin/moesin(ERM), myristoylated alanine-rich C-kinase substrate(MARCKS), collapsin response mediator protein-2(CRMP-2), etc. Extensive studies have revealed that ROCKs are involved in a wide range of diseases, including cardiovascular diseases, cancers and neurological diseases, and is therefore considered as essential therapeutic targets. Although a lot of ROCK inhibitors have been identified, most of them are not the ideal lead compounds. Currently, only fasudil has been approved in Japan for the treatment of cerebral vasospasm and ischemia. Thus, identification and design of novel ROCKs inhibitors with good potency and less side effects are quite demanding.First, molecular docking was utilized to virtually screen ChemBridge and Specs databases and identify molecules that can interact with ROCK1. 174 virtual hits was purchased and submitted to a series of experimental assays. The in vitro enzyme-based and cell-based assays reveal that 12 compounds have good inhibitory activity against ROCK1 in micromolar regime(IC50 values between about 7 and 28 μM/L) and antitumor activity against lung cancer, breast cancer or/and myeloma cell lines. The structural analysis shows that two active compounds present novel scaffolds and are potential lead compounds for the development of novel anti-cancer drugs. In addition, the pharmacological effect of the two ROCK1 inhibitors with novel scaffolds on atorvastatin-induced cerebral hemorrhage was evaluated by using zebrafish, and one compound candidate is able to prevent atorvastatin-induced cerebral hemorrhage effectively.Then, the interactions between ROCK1 and a series of triazine/pyrimidine-based inhibitors and those between pyrrolopyrimidine derivatives and LIMK2 were studied by using an integrated computational protocol that combines molecular docking, MD simulations, and binding free energy calculations and decomposition. The dynamic binding processes of the studied inhibitors, the impact of the essential residues of the receptor and the important substituents of the inhibitors on ligand binding were analyzed. Based on the molecular modeling studies obtained above, a series of derivatives were designed and confirmed by theoretical predictions. According to the computational results, these derivatives may become potent drug leads.In the fifth chapter of this thesis, in vitro enzyme-based assays revealed that a new compound(FPND) with novel scaffold, identified from docking-based virtual screening, can inhibit ROCK1 specifically at low micromolar concentration. Molecular modeling studies showed that FPND forms more favorable interactions with ROCK1 than ROCK2 and the difference between the binding affinities of FPND toward ROCK1 and ROCK2 is primarily contributed from the non-polar contributions. Then, FPND was found to prevent atorvastatin-induced cerebral hemorrhage effectively in zebrafish model. In addition, in vitro studies of xCELLigence RTCA System, immunofluorescence and Western blotting revealed that FPND prevents the atorvastatin-induced cerebral hemorrhage by the enhanced endothelial cell-cell junctions via ROCK and Src/VEC signaling pathway. Furthermore, the extreme low toxicity of FPND in rat suggests that FPND is safe and has potential to prevent cerebral hemorrhage stroke.In the last chapter of this thesis, parallel virtual screening, similarity searching and in vitro bioassays were utilized to identify novel promising inhibitors of ROCK1. As the results, 79 hits were selected for the further study. The in vitro enzyme-based assays revealed that 21 compounds, identified from parallel virtual screening and similarity searching, can inhibit ROCK1 in the micromolar regime, and including four compounds with the IC50 values below 0.5 μM. Molecular modeling studies including MD simulations, MM/GBSA free energy calculations and free energy decomposition analysis showed that the scaffold of 4-Phenyl-1H-pyrrolo [2,3-b] pyridine is quite necessary for their binding in the active site of ROCK1. Furthermore, compounds TS-15 and TS-40 were found to efficacies in inhibition of phosphorylation of downstream target in the ROCK singling pathway in human umbilical vein endothelial cells and prevention of chemically-induced cerebral hemorrhage in zebrafish model. Overall, the parallel virtual screening provides a new strategy for drug design.