Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe.

Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), was originally identified as a tumor cell autocrine motility factor and was found to be identical to plasma lysophospholipase D, which is the predominant contributor to lysophosphatidic acid (LPA) production from lysophospholipids. ATX is therefore considered to regulate the physiological and pathological roles of LPA, including angiogenesis, lymphocyte trafficking, tissue fibrosis, and cancer cell invasion and metastasis. Thus, it is a potential therapeutic target. Here, we first developed a sensitive and specific ATX fluorescence probe, TG-mTMP, and used it to screen ATX inhibitors in a large chemical library. This probe, which is superior to previously available probes FS-3 and CPF4 in terms of sensitivity or specificity, enabled us to identify several novel ATX inhibitor scaffolds. We solved the crystal structures of ATX complexes with the hit compounds at high resolution (1.75-1.95 Å) and used this information to guide optimization of the structure of a selected inhibitor. The optimized compounds, 3BoA and its derivatives, exhibited potent ATX-inhibitory activity both in vitro and in vivo. These inhibitors are expected to be useful tools to understand the roles of ATX in vitro and in vivo and may also be candidate anti-ATX therapeutic agents.

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors.

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.