In Vivo Evaluation of Validated Targets from High Throughput Screening Models for Sulfur Mustard Ocular Injury

Abstract

This work was conducted in an effort to develop medical countermeasures to treat exposure to toxicants that can be utilized in a terrorism or warfare scenario. Chemical injuries to the eyes are true ocular emergencies because of their high potential to inflict rapid and enduring tissue damage. The cornea and other ocular surface tissues are particularly susceptible to chemical exposures such as sulfur mustard (SM), and long‐term prognosis can be poor due to progressive corneal pathologies. Chronic and delayed complications include persistent corneal epithelial defects, dry eye, corneal opacification, and corneal neovascularization. Such long‐term complications can lead to permanent discomfort and vision deficits or blindness despite the best current treatment regimens. To identify novel therapeutic targets, we had carried out siRNA‐mediated high throughput screening (HTS) using human corneal epithelial cell models of SM exposure. Over 3000 genes identified as differentially regulated after SM ocular exposure by previous microarray studies were tested for their functions involved in cell viability and IL‐8 production. The current study transitioned eight validated targets from the HTS to a mouse model of SM‐induced ocular injury. Interestingly, four out of the eight targets, PSMA1, PSMA2, PSMD7 and FBXO5, were subunits of proteasomes or E3 ubiquitin ligase complexes. The ubiquitin‐proteasome pathway maintains protein homeostasis and therefore important cellular functions through the degradation of misfolded, redundant, and damaged proteins. Treatment with the proteasome inhibitor bortezomib in the first two weeks after SM ocular exposure greatly promoted the rate and number of corneal neovascularization, which is a key feature of a delayed injury phase. Such outcomes were also observed with treatment with another proteasome inhibitor, carfilzomib. In addition to proteasome subunits, GABRR1 (GABA type A receptor subunit rho‐1) was among the validated targets from the HTS. We learned that muscimol, a GABA type A receptor agonist, significantly decreased corneal neovascularization during 8‐week observation periods. Taken together, these outcomes demonstrated the effectiveness of our systemic approach to identify novel molecular signaling pathways implicated in toxicant‐induced ocular injuries and future studies for potential drugs. For example, our future studies include investigating proteasome‐mediated protein regulation of angiogenic factors in response to SM ocular exposure and testing the therapeutic potential of proteasome agonists. Some small molecule proteasome agonists such as chlorpromazine and imidazoline were identified in recent years and have been studied for their efficacy in treating different diseases such as neurodegeneration, cancer, and autoimmunity.