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Irene Tom, Johnny Gutierrez, Kenneth J Katschke, Isabel Figueroa, Victoria Pham, Helen S Booler, Linda Rangell, Sheila Ulufatu, Guy Salvesen, Jennie Lill, Menno Van Lookeren Campagne, Amos Baruch; Determining ocular HtrA1 activity and inhibition using novel activity based probe. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2441.
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HtrA1 is a serine protease expressed in the eye and implicated, through genome-wide association studies, in age-related macular degeneration (AMD). Since the endogenous substrates of HtrA1 are unknown, it is challenging to determine its activity within the eye. Here, we report the design and characterization of a HtrA1 activity-based probe (ABP) that specifically binds to the active form of HtrA1.
A specific HtrA1 ABP was developed and characterized in vitro and in vivo. To determine HtrA1 activity in the retina, HtrA1 ABP was administered to Dutch-Belted rabbit eyes via intravitreal (ITV) injection at a dose of 10uM/eye and retinas were subjected to immunohistochemical analysis. Pharmacodynamic (PD) analysis using the HtrA1 ABP was performed on vitreous humor from rabbits that were treated with Anti-HtrA1 mAb or control by bilateral ITV injection dosing.
In vitro, the HtrA1 ABP covalently modified recombinant WT HtrA1 but did not bind a mutated S328A inactive enzyme. Furthermore, ABP-labeling of recombinant HtrA1 was blocked in the presence of Anti-HtrA1 mAb inhibitor (Anti-HtrA1). Using the ABP we were able to detect HtrA1 activity directly in rabbit vitreous but not in aqueous humor. Moreover, direct administration of the ABP intravitreally confirmed activity of HtrA1 in vivo. We next used the HtrA1 ABP as a PD tool to determine HtrA1 inhibition in vitreous humor from rabbits administered with anti-HtrA1. HtrA1 activity was detected in controls but was markedly inhibited in a dose-related manner following IVT injection of Anti-HtrA1.
This activity-based approach provides information on the spatial and temporal patterns of HtrA1 activity within its natural environment and can further shed light on its role in AMD disease initiation and progression.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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