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Atsuko Kasai, Yoko Yoshida, Kei Hasegawa, Hiroyoshi Hidaka; Elucidation of molecular mechanism of H-1337, an anti-glaucoma agent. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5695.
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© ARVO (1962-2015); The Authors (2016-present)
Our novel isoquinoline sulfonamide, H-1337, produces long-lasting reduction of intraocular pressure (IOP) (durations were over 14 h and 24 h in rabbits and monkeys, respectively, ARVO2015 Hidaka et al.). To elucidate molecular mechanism by which H-1337 reduces IOP, we conducted kinase inhibition profile of H-1337. We focused especially on the molecular mechanism, Leucine-rich repeat kinase 2 (LRRK2), which is inhibited most effectively by H-1337, is involved.
The kinase inhibition profile of H-1337 was conducted in vitro by Cerep Corporation. Gene expression was analyzed by RT-PCR. Expression and localization of LRRK2 and arrangement of F-actin were evaluated with immunocytochemistry in human trabecular meshwork (TM) cells. In the combination study, IOP reduction following the topical administration of H-1337 together with another IOP-lowering agent was compared with the individual agents in either cynomolgus monkey or rabbit eyes.
LRRK2 was inhibited most effectively by H-1337 (IC50: 0.045 μM). H-1337 also inhibited PKN2, PKD1, and PKA (IC50: 0.047, 0.052, and 0.091 μM, respectively). Its main metabolite, H-1337M1, which declined more slowly in aqueous humor than H-1337, also inhibited effectively same kinases, including LRRK2. Expression of LRRK2 was observed in eye tissues including TM cells. Staining of LRRK2 was widely distributed in TM cells in a scaffold-like pattern and treatment of TM cells with H-1337 altered the appearance of this staining indicating H-1337 directly affects LRRK2 within these cells. Staining with phalloidin revealed that H-1337 also affected actin cytoskeleton. H-1337 induced morphological change of TM cells in a dose dependent manner. The combination of H-1337 and other IOP-lowering agents showed significant hypotensive effect compared with other agents alone.
H-1337 inhibited LRRK2 most effectively, and also inhibited PKN2, PKD1, and PKA, which have been reported to regulate cytoskeletal reorganization. H-1337 induced morphological change of TM cells, which can facilitate aqueous humor outflow via TM. Additionally, H-1337M1, which effectively inhibits same kinases, including LRRK2, may play an important role for long-lasting IOP reduction. These results suggest that H-1337 is a new class of drug that effectively lowers IOP through inhibiting multiple kinases, including LRRK2, and can be effectively combined with other IOP-lowering drugs.
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