Design, synthesis, and biological evaluation of urea-based ROCK2 inhibitors.

A series of urea-based ROCK2 inhibitors were design and synthesized. The inhibitory activity on ROCK2 was screened by enzyme-linked immunosorbent assay (ELISA). The study results showed that the urea derivatives exhibited certain ROCK2 inhibitory activity. The most potent compound 10p showed ROCK2 inhibitory activity with the IC50  value of 0.03 µM. A preliminary structure-activity relationship was then summarized. The molecular docking studies showed that further optimization needs to conduct to obtain more potent ROCK inhibitors.

Forsythoside A and Forsythoside B Contribute to Shuanghuanglian Injection-Induced Pseudoallergic Reactions through the RhoA/ROCK Signaling Pathway.

In recent years, hypersensitivity reactions to the Shuanghuanglian injection have attracted broad attention. However, the componential chief culprits inducing the reactions and the underlying mechanisms involved have not been completely defined. In this study, we used a combination of approaches based on the mouse model, human umbilical vein endothelial cell monolayer, real-time cellular monitoring, immunoblot analysis, pharmacological inhibition, and molecular docking. We demonstrated that forsythoside A and forsythoside B contributed to Shuanghuanglian injection-induced pseudoallergic reactions through activation of the RhoA/ROCK signaling pathway. Forsythoside A and forsythoside B could trigger dose-dependent vascular leakage in mice. Moreover, forsythoside A and forsythoside B slightly elicited mast cell degranulation. Correspondingly, treatment with forsythoside A and forsythoside B disrupted the endothelial barrier and augmented the expression of GTP-RhoA, p-MYPT1, and p-MLC2 in a concentration-dependent manner. Additionally, the ROCK inhibitor effectively alleviated forsythoside A/forsythoside B-induced hyperpermeability in both the endothelial cells and mice. Similar responses were not observed in the forsythoside E-treated animals and cells. These differences may be related to the potential of the tested compounds to react with RhoA-GTPγS and form stable interactions. This study innovatively revealed that some forsythosides may cause vascular leakage, and therefore, limiting their contents in injections should be considered.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

Derivation of Self-inhibitory Helical Peptides to Target Rho-kinase Dimerization in Cerebrovascular Malformation: Structural Bioinformatics Analysis and Peptide Binding Assay.

Rho-kinase dimerization is essential for its kinase activity and biological function; disruption of the dimerization has recently been established as a new and promising therapeutics strategy for cerebrovascular malformation (CM). Based on Rho-kinase dimer crystal structure we herein combined in silico analysis and in vitro assay to rationally derive self-inhibitory peptides from the dimerization interface. Three peptides namely Hlp1, Hlp2 and Hlp3 were successfully designed that have potential capability to rebind at the dimerization domain of Rho-kinase. Molecular dynamics (MD) simulations revealed that these peptides are helically structured when bound to Rho-kinase, but exhibit partially intrinsic disorder in unbound state. Binding free energy (BFE) analysis suggested that the peptides have a satisfactory energetic profile to interact with Rho-kinase. The computational findings were then substantiated by fluorescence anisotropy assays, conforming that the helical peptides can bind tightly to Rho-kinase with affinity KD at micromolar level. These designed peptides are considered as lead molecular entities that can be further modified and optimized to obtain more potent peptidomimetics as self-competitors to disrupt Rho-kinase dimerization in CM.

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

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.

High-throughput screening against ~6.1 million structurally diverse, lead-like compounds to discover novel ROCK inhibitors for cerebral injury recovery.

Rho-associated protein kinase (ROCK) has been recognized as an attractive therapeutic target to promote neurogenesis, neuroregeneration, and neurorecovery after cerebral injury. Here, a high-throughput screening protocol was described to discover novel ROCK inhibitors from a large chemical library containing ~6.1 million structurally diverse, lead-like compounds. The protocol employed empirical rules such as ADMET evaluation and chemical similarity analysis to exclude those of drug-unlike candidates, and then molecular docking and binding affinity predictions were performed to suggest few promising candidates with high scores. Consequently, five compounds were successfully identified to have satisfactory activity profile with IC50 values at nanomolar level. In order to elucidate the molecular mechanism of inhibitor binding to target, the complex structures of ROCK kinase domain with the five identified compounds were modeled and examined in detail. A number of polar chemical forces such as hydrogen bonds and cation-π interactions as well as nonpolar contacts such as π-π stacking and hydrophobic forces were revealed at the complex interface, conferring high affinity and strong specificity to inhibitor binding. In addition, several key residues around the kinase active site, including Val90, Lys105, Asn203, and Phe368, were found to play an important role in binding.

Design, synthesis, and biological evaluation of novel, highly active soft ROCK inhibitors.

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.

Development and evaluation of an integrated virtual screening strategy by combining molecular docking and pharmacophore searching based on multiple protein structures.

In this study, we developed and evaluated a novel parallel virtual screening strategy by integrating molecular docking and complex-based pharmacophore searching based on multiple protein structures. First, the capacity of molecular docking or pharmacophore searching based on any single structure from nine crystallographic structures of Rho kinase 1 (ROCK1) to distinguish the known ROCK1 inhibitors from noninhibitors was evaluated systematically. Then, the naïve Bayesian classification or recursive partitioning technique was employed to integrate the predictions from molecular docking and complex-based pharmacophore searching based on multiple crystallographic structures of ROCK1, and the integrated protocol yields much better performance than molecular docking or complex-based pharmacophore searching based on any single ROCK1 structure. Finally, the well-validated integrated virtual screening protocol was applied to identify potential inhibitors of ROCK1 from traditional chinese medicine (TCM). The obtained potential active compounds from TCM are structurally novel and diverse compared with the known inhibitors of ROCK1, and they may afford valuable clues for the development of potent ROCK1 inhibitors.