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Tom R Vollmer, Christopher William Wilson, Enhua Zhou, Ernie Hixon, Dennis S Rice, Amy Chen; High-throughput impedance screening of trabecular meshwork cells for novel target identification in glaucoma. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4705.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a multi-factorial disease characterized by retinal ganglion cell death and is often associated with elevated intraocular pressure (IOP). The conventional outflow pathway (trabecular meshwork, Schlemm’s canal) acts as the site of greatest resistance to aqueous outflow. Modulation of trabecular meshwork cell contractility is a validated mechanism to improve outflow. We used a label-free cellular impedance system to screen compounds that potentially affect cell contractility or relaxation.
Assay parameters were optimized using a set of tool compounds (Prostaglandins, Rho kinase inhibitors) that have demonstrated clinical efficacy in lowering IOP via cell contractility or relaxation. A pilot screen of over 4000 compounds with known targets was conducted in immortalized NTM5 trabecular meshwork cells. Compounds were assayed in duplicate at 10 µM and 2 µM. Impedance was measured at 1-minute intervals for the first 4 hours post-treatment and subsequently at 15-minute intervals for a total of 18 hours. Cell viability was assessed at 18 hours using CellTiter-Glo (CTG) reagents. The change in impedance at 30, 60, 120 and 240 minute time points was used for hit selection. Compounds were selected for hit confirmation if the impedance measurement was greater than 3 standard deviations from the median at 3 or more time points and if the compound did not reduce cell viability by more than 25%. Selected hits were further assayed with an 8-point dose curve. Confirmed hits were evaluated in primary bovine and human trabecular meshwork cells.
The Z’ factor in the screen was greater than 0.5, indicating a high quality screen. 4683 compounds were screened and 247 compounds were identified as primary hits. 80 of the primary hit compounds were confirmed with 8-point dose curves. Among them, ROCK inhibitors and PGE1 were identified, providing internal validation of the screen. The impedance effect of these hits in primary cells is under evaluation.
The impedance assay platform was amenable to high throughput screening. Several novel compounds that could potentially modulate cell contraction or relaxation were identified. The translatability of the impedance assay changes in outflow facility and IOP will be further explored.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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