Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma.

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.

Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma.

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter.

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation.

Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation.

This chapter describes the development and implementation of three independent imaging assays for the major mitogen-activated protein kinase (MAPK) signaling modules: p38, JNK, and ERK. There are more than 500 protein kinases encoded in the human genome that share an ATP-binding site and catalytic domain conserved in both sequence and structure. The majority of kinase inhibitors have been found to be competitive with ATP, raising concerns regarding kinase selectivity and potency in an environment of millimolar intracellular concentrations of ATP, as well as the potential for off-target effects via the many other cellular proteins that bind and/or utilize ATP.

Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors.

This chapter describes the generation and characterization of a stable MK2-EGFP expressing HeLa cell line and the subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 MAPK inhibitors. Mitogen-activated protein kinase activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase, and p38-induced phosphorylation of MK-2 induces a nucleus to cytoplasm translocation (Engel et al., 1998; Neininger et al.

Secretion of proinflammatory cytokines and chemokines during amphotericin B exposure is mediated by coactivation of toll-like receptors 1 and 2.

Amphotericin B (AmB) is a ligand of toll-like receptor 2 (TLR2). Here, we demonstrate the participation of TLR1 in AmB-induced cell activation that led to the secretion of tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB.