Efficacy and Safety of Small Molecule Proteostasis Modulators in vitro and in the eye

Abstract

Purpose : The Unfolded Protein Response (UPR) and Integrated Stress Response (ISR) are important protein quality control mechanisms. Both are pathomechanisms in many eye diseases such as glaucoma and retinitis pigmentosa. Modulation of these pathways may provide new strategies to prevent or treat eye diseases. PERK, ATF6, and IRE-XBP1 are key UPR/ISR regulators. Recently, small molecules have been found to regulate these pathways. However, the bioactivity and safety of UPR/ISR small molecule proteostasis agents have not been tested after intraocular injections in mouse eyes, which was the purpose of this project.

Methods : Five UPR and ISR small molecule modulators were evaluated: ISRIB, an ISR inhibitor, Salubrinal, an ISR activator; CeapinA7, an ATF6 inhibitor; AA147, an ATF6 activator; and IXA4, an IRE1/XBP1 activator. Tunicamycin or Thapsigargin, nonspecific UPR/ISR activators, were used as controls. Compounds' efficacy and safety were tested in HEK293 cells. The drugs were intravitreally injected in C57Bl6/J mice at postnatal day 30. Retinas were evaluated at 12 hrs, 24 hrs, 3 days and 7 days after injection (n=48 per group). Electroretinography, retinal histology, and RNA-seq of retinal lysates were used to evaluate the efficacy of these compounds and their potential adverse impacts. Targeted gene panels regulated by PERK/ISR, ATF6, and IRE1-XBP1 were quantified. Gene Ontology (GO) analysis of RNA-seq datasets was used to find significantly altered molecular pathways.

Results : Tunicamycin and Thapsigargin, significantly modulated all targeted gene panels, and ER stress and cell death processes by GO analysis. ISRIB and Salubrinal significantly modulated PERK/ISR gene panel (Wilcoxon, P<0.0001; One sample t test P<0.0001). GO found significant modulation of ER stress terms after treatment with both compounds (Wilcoxon, P<0.003). CeapinA7 significantly modulated ATF6 gene panel (Wilcoxon, P<0.02), but GO found no significant impact. AA147 treatment did not significantly modulate ATF6 gene panel or ER stress terms in GO analysis. Cell death and programmed cell death were not significantly changed with all UPR/ISR compounds tested (Wilcoxon, P<0.45).

Conclusions : ISRIB, Salubrinal, and CeapinA7 were found to be effective in modulating UPR/ISR in our study. The compounds and delivery conditions were safe and effective at modulating UPR/ISR may be further investigated in ocular disease models for therapeutic effects.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.