Purpose: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein.Methods: The kinase inhibitory activity of netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a commercially available kinase assay kit. Disruption of actin stress fibers was measured in primary porcine TM cells and disruption of focal adhesions in transformed human TM (HTM) cells. Induction of fibrosis markers after exposure to transforming growth factor-β2 (TGF-β2) was conducted in primary HTM cells. Ocular hypotensive activity and tolerability of topical formulations were evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted rabbits.Results: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 and 16 nM, respectively, and blocked the profibrotic effects of TGF-β2 in HTM cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that were sustained for at least 24 h after once daily dosing, with transient, mild hyperemia observed as the only adverse effect.Conclusion: Netarsudil is a novel ROCK/NET inhibitor with high potency in biochemical and cell-based assays, an ability to produce large and durable IOP reductions in animal models, and favorable pharmacokinetic and ocular tolerability profiles.

SignificanceGlaucoma is the leading cause of irreversible blindness worldwide. The primary and only modifiable risk factor for the development of glaucoma is elevated intraocular pressure (IOP), and lowering IOP effectively slows glaucomatous disease progression. Unfortunately, the majority of available treatments do not target, or intentionally bypass, the diseased and stiffened glaucomatous outflow tissues responsible for IOP elevation. We recently established that conventional outflow tissue stiffness reflects tissue function. Therefore, detection of outflow tissue stiffness using noncontact, noninvasive optical coherence tomography, as we here demonstrate in an animal model of glaucoma, represents a valuable tool for assessing outflow tissue functional status. Such technology has the potential to monitor recently approved treatments targeting the outflow tissues, and to inform glaucoma surgery decisions.

The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is an important issue for the treatment and prevention of severe acute respiratory syndrome. Recently, SARS-CoV has been demonstrated to induce cell apoptosis in Vero-E6 cells. The possible role of SARS-CoV 3C-like protease (3CLpro) in virus-induced apoptosis is characterized in this study. Growth arrest and apoptosis via caspase-3 and caspase-9 activities were demonstrated in SARS-CoV 3CLpro -expressing human promonocyte cells. The fluorescence intensity of dihydrorhodamine 123 staining indicated that cellular reactive oxygen species were markedly increased in SARS-CoV 3CLpro -expressing cells. Moreover, in vivo signalling pathway assay indicated that 3CLpro increased the activation of the nuclear factor-kappa B-dependent reporter, but inhibited activator protein-1-dependent transcription. This finding is likely to be responsible for virus-induced apoptotic signalling.

(17,23,25). The flavivirus E protein has approximately 500 amino acids with six conserved disulfide bonds to maintain its conformational structure (20,29). The E protein structures have been determined for the tick-borne encephalitis (TBE) virus by using X-ray crystallography (24) and recently have been used for a low-resolution map of whole dengue virions (13). The E protein is a head-to-tail homodimer with three structurally distinct domains: a central ␤-barrel (domain I), an elongated dimerization region (domain II), and a C-terminal immunoglobulin (Ig)-like module (domain III) (24). The E protein is associated with viral attachment, fusion, hemagglutination, cellular tropism, viral virulence, and the induction of protective immune response (17).The domain III protein contains one disulfide bond to maintain its conformational structure and can be independently folded as a trypsin-resistant core protein (16,20,29). Neutralizing epitopes on the lateral surface of domain III have been identified, including residues 333 (1), 373 to 399 (28), and 306, 331, and 387 of JEV (30); 308, 310, and 311 of louping ill virus (8); 384 and 386 of TBE (6, 7); 305 of yellow fever virus (27); and 383 to 389 (5), 333 to 351 (26), and 307 (15) of the dengue viruses. However, most studies have not provided detailed molecular information about the spatial configuration of the epitopes on domain III of the flavivirus E proteins, where the epitopes are defined as a few contact residues that are energetically important for binding affinity in the antibody-antigen complex (9, 21). In this study, domain III of the JEV E protein was expressed in Escherichia coli to investigate the functional epitope determinants for a JEV-specific neutralizing monoclonal antibody (MAb), E3.3 (30). The alteration of proteins with single or double site-directed mutagenesis was conducted at positions 306 (Glu3Gly), 331 (Ser3Arg), and 387 (Met3Arg). Also, mutations at or near position 331 of the JEV E protein were replaced with alanine or other hydrophobic and charged amino acids. By using complementary structural modeling (4,14,22), the structure of the domain III MAb E3.3 complex was constructed. Specific interactions of the functional epitope determinants on domain III were confirmed by further mutating the combining sites of MAb E3.3 Fab antibody fragment. This study elucidates the detailed molecular structures of the neutralizing epitope determinants on the JEV domain III protein, which can provide useful information for designing new vaccines. MATERIALS AND METHODSVirus, cells, and media. The JEV attenuated variant CH2195LA was plaque purified in Vero cells from a Taiwanese isolate (30) and was propagated in Vero cells by using M199 medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen). MAb E3.3-producing hybridoma cells were grown in Iscove's modified Dulbecco's medium (Invitrogen) with 10% FBS.Cloning, expression, purification, and site-directed mutagenesis of E and domain III fusion protein. Viral RNAs were extracted from the cult...