Access Type

Open Access Thesis

Date of Award

January 2024

Degree Type

Thesis

Degree Name

M.S.

Department

Pharmacology

First Advisor

Ahmad Ibrahim

Abstract

Purpose: Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly population, characterized by the degeneration of the retinal pigment epithelium (RPE). The link between endoplasmic reticulum (ER) stress and AMD is well established, however FDA-approved medications for the treatment of AMD caused by unregulated ER stress are limited and have potential side effects. The purpose of this study was to conduct a comprehensive screening of the FDA-approved drug library in order to identify potential candidates that could be repurposed in order to protect against the degeneration and death of RPE cells due to ER stress.

Methods: The FDA Pharmakon compound library (containing 1760 medicines) was acquired from MicroSource Discovery Systems, Inc. (USA). These compounds were received in microplates at 10 mM, dissolved in DMSO, and stored at -80°C until needed. A human retinal pigment epithelium cell line (ARPE-19) was exposed to tunicamycin (TM) with or without various FDA-approved drugs to induce ER stress. Lactate dehydrogenase (LDH) release into the media was measured over 24, 48 and 72 hours to assess cellular toxicity. Statistical analysis was performed using a two-way ANOVA test followed by post hoc tests.

Results: Treatment of ARPE-19 cells with tunicamycin (TM) resulted in cell death, as evidenced by LDH leakage after 48 and 72 hours of treatment. Significantly, medications containing beta-lactam compounds, including penicillin and cephalosporin derivatives, emerged as the most effective drug category in mitigating TM-induced RPE cell death. Furthermore, a detailed exploration of the structure-activity relationship was undertaken to identify the specific chemical groups responsible for this observed protective effect. Based on these results, increasing the hydrophilicity of the benzene ring reduces the protective effect, similar to amoxicillin. As well, converting the primary amino group in ampicillin into a secondary amino group, such as those found in piperacillin and azlocillin, eliminates its activity. Replacing the primary amino group with a carboxylic group extends the protective effect of ampicillin as seen in carbenicillin. Deletion of the primary amino or carboxyl group at position 2 of the side chain, as in Penicillin G, resulted in partial protection of RPE cells against ER stress-induced cell death. The protective effect could be enhanced by adding chlorine atoms like in cloxacillin and dicloxacillin or methoxy groups like in methicillin. Similarly, the presence of a six-membered ring adjacent to the beta-lactam ring enhances the protective effect, as seen in cephalosporin. A number of moieties can be added to cephalosporin derivatives' R1 side chains to enhance their protection against TM-induced RPE cell death. Specifically, incorporating methyl (CH₃), chlorine (Cl), ester groups, thiadiazole rings, and tetrazole rings resulted in complete (100%) protection, highlighting the importance of these structural modifications in improving therapeutic efficacy. As a result of these modifications, the drug is likely to interact more effectively with retinal cells. This will reduce the possibility of damage caused by ER stress and related degenerative processes in the retina.

Conclusion: The study concludes that beta-lactam medications, including penicillin and cephalosporin derivatives, can serve as potent protections against RPE cell degeneration and death as a result of ER stress. A number of these compounds, particularly those with specific structural modifications, are highly effective at preserving the viability of RPE cells. These findings offer promising avenues for developing therapeutic strategies to combat AMD associated with unregulated ER stress.

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