Abstract
Purpose :
We previously demonstrated that optic nerve injury induces neuronal endoplasmic reticulum (ER) stress and inhibiting its downstream detrimental effectors ATF4 and CHOP achieves significant neuroprotection in mouse glaucoma models. This study focuses on the discovery of small molecule neuroprotectants for glaucoma by targeting ATF4 and CHOP and evaluate their in vivo neuroprotection efficacies in glaucomatous neurodegeneration.
Methods :
We generated a stable cell line in HEK293T to drive luciferase expression by CHOP promoter, by which we performed cell-based high throughput screening (HTS) with several chemical libraries to identify chemical inhibitors of CHOP expression induced by ER stress inducer Tunicamycin (Tm) and Thapsigargin (Tg). Some of the “hit” compounds were further verified and selected for structure-activity relationship (SAR) analysis. Newly designed and synthesized chemical analogs were further evaluated by cell-based luciferase assay, western blot (WB) and qPCR to determine CHOP levels of targeted molecule and its upstream ATF4. Finally, the “lead” compounds will be further tested in mouse glaucoma models to determine their in vivo efficacies in neuroprotection and visual function preservation.
Results :
Among the 446 CHOP inhibitors identified through HTS assay, we established 6 chemical series based on their common scaffolds. We further selected and verified representative compounds from each series using the cell-based luciferase assay, by which we narrowed down to 4 compounds from Chemical series 3-6. Next, we selected Compound 5a as the lead compounds for SAR studies due to its superb CHOP inhibition and neuroprotection effect in optic nerve crush mouse model. With two rounds of extensive SAR analysis, we determined the key scaffold of 5a that is required for its CHOP inhibition activity, and identified 10 analog as top CHOP inhibitors. Lastly, the qPCR and WB cell-based analysis allow us to further narrow down to five Compound 5a analogs, including T-12, T-22, T-27, T-2029 and T-2054, for in vivo neuroprotection testing in mouse glaucoma models.
Conclusions :
This study successfully identified multiple novel chemical series of CHOP/ATF4 inhibitors for further development. Compounds 5a and its most potent analogs were established as promising neuroprotectant candidates for glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.