Design of Novel Rho Kinase Inhibitors Using Energy Based Pharmacophore Modeling, Shape-Based Screening, in Silico Virtual Screening, and Biological Evaluation

Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound 8 exhibited an IC50 value of 1.5 μM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI50 value of 0.27 μM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound 8 with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 μM CC50 (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor.

[1]  P. Yogeeswari,et al.  Potential role of Rho kinase inhibitors in combating diabetes-related complications including diabetic neuropathy--a review. , 2013, Current diabetes reviews.

[2]  J. Plumb.  Cell sensitivity assays: the MTT assay. , 2004, Methods in molecular medicine.

[3]  K. Roy,et al.  On Two Novel Parameters for Validation of Predictive QSAR Models , 2009, Molecules.

[4]  P. McGuire,et al.  Diabetic Retinopathy and Inflammation: Novel Therapeutic Targets , 2012, Middle East African journal of ophthalmology.

[5]  Hyun Kook Cho,et al.  Curcumin inhibits hepatitis C virus replication via suppressing the Akt‐SREBP‐1 pathway , 2010, FEBS letters.

[6]  M. Zweig,et al.  Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. , 1993, Clinical chemistry.

[7]  R. Biondi,et al.  Regulation of Protein Kinase C-related Protein Kinase 2 (PRK2) by an Intermolecular PRK2-PRK2 Interaction Mediated by Its N-terminal Domain* , 2012, The Journal of Biological Chemistry.

[8]  J. Pintor,et al.  Potential role of Rho-associated protein kinase inhibitors for glaucoma treatment. , 2012, Recent patents on endocrine, metabolic & immune drug discovery.

[9]  Sara E. Nichols,et al.  Identification of Potential Small Molecule Binding Pockets on Rho Family GTPases , 2012, PloS one.

[10]  M. Fleming,et al.  The Structure of Dimeric ROCK I Reveals the Mechanism for Ligand Selectivity* , 2006, Journal of Biological Chemistry.

[11]  Neerja Kaushik-Basu,et al.  2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-nucleoside inhibitors of HCV NS5B polymerase. , 2013, European journal of medicinal chemistry.

[12]  C. Ayata,et al.  Targeting cerebrovascular Rho-kinase in stroke. , 2008, Expert opinion on therapeutic targets.

[13]  R. Komers.  Rho kinase inhibition in diabetic nephropathy , 2011, Current opinion in nephrology and hypertension.

[14]  W. Guida,et al.  Fragment-based and structure-guided discovery and optimization of Rho kinase inhibitors. , 2012, Journal of medicinal chemistry.

[15]  G. Reynolds,et al.  University of Birmingham Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells. , 2009 .

[16]  S. Izumo,et al.  Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure. , 2011, Toxicology and applied pharmacology.

[17]  Huanxiang Liu,et al.  Molecular modeling studies of Rho kinase inhibitors using molecular docking and 3D-QSAR analysis. , 2010, European journal of medicinal chemistry.

[18]  J. Haveman,et al.  Clonogenic assay of cells in vitro , 2006, Nature Protocols.

[19]  Patrisha Joseph Therese,et al.  Multiple e-Pharmacophore Modeling, 3D-QSAR, and High-Throughput Virtual Screening of Hepatitis C Virus NS5B Polymerase Inhibitors , 2014, J. Chem. Inf. Model..

[20]  M. Jacobs,et al.  ROCK enzymatic assay. , 2008, Methods in molecular biology.

[21]  K. Nakao,et al.  ROCK‐I and ROCK‐II, two isoforms of Rho‐associated coiled‐coil forming protein serine/threonine kinase in mice , 1996, FEBS letters.

[22]  Anupama Munshi,et al.  Clonogenic cell survival assay. , 2005, Methods in molecular medicine.

[23]  N. Sakamoto,et al.  Inhibition of hepatitis C virus NS5B polymerase by S-trityl-L-cysteine derivatives. , 2012, European journal of medicinal chemistry.

[24]  A. Takashima,et al.  Methylmercury exposure downregulates the expression of Racl and leads to neuritic degeneration and ultimately apoptosis in cerebrocortical neurons. , 2009, Neurotoxicology.

[25]  S. Kim,et al.  Suppression of hepatitis C virus replication by protein kinase C‐related kinase 2 inhibitors that block phosphorylation of viral RNA polymerase , 2009, Journal of viral hepatitis.

[26]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[27]  J. Irwin,et al.  Benchmarking sets for molecular docking. , 2006, Journal of medicinal chemistry.

[28]  P. Yogeeswari,et al.  Multiple e‐Pharmacophore Modeling Combined with High‐Throughput Virtual Screening and Docking to Identify Potential Inhibitors of β‐Secretase(BACE1) , 2013, Molecular Informatics.

[29]  K. Kaibuchi,et al.  Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. , 2001, Trends in pharmacological sciences.

[30]  D. Ingber,et al.  Role of RhoA, mDia, and ROCK in Cell Shape-dependent Control of the Skp2-p27kip1 Pathway and the G1/S Transition* , 2004, Journal of Biological Chemistry.

[31]  E. Eruslanov,et al.  Identification of ROS using oxidized DCFDA and flow-cytometry. , 2010, Methods in molecular biology.

[32]  C. Stief,et al.  Activated RhoA/Rho kinase impairs erectile function after cavernous nerve injury in rats. , 2010, The Journal of urology.

[33]  A. Wirth.  Rho kinase and hypertension. , 2010, Biochimica et biophysica acta.

[34]  F. Lombardo,et al.  Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. , 2001, Advanced drug delivery reviews.