Allosteric regulation of REDD1 as a therapeutic target for diabetic retinopathy

Project Summary Diabetic retinopathy (DR) is a significant ocular complication caused by diabetes that can progress to blindness. Diabetes causes an increased abundance of the stress response protein Regulated in Development and DNA Damage 1 (REDD1) in the retina, which has been implicated in visual function deficits in both preclinical models and diabetic patients. In fact, patients with diabetic macular edema had dose-dependent improvement in best- corrected visual acuity with intravitreal administration of an siRNA targeting the REDD1 mRNA (PF-04523655). However, the approach was abandoned over a decade ago due to its failure to outperform vascular endothelial growth factor (VEGF) blockade. Our laboratory recently made an important discovery that revealed a key weakness in the prior approach used to inhibit REDD1. Specifically, we discovered that formation of a redox- sensitive disulfide bond acts allosterically to prevent the normally rapid degradation of REDD1 protein in the context of diabetes. The observation suggests that REDD1 mRNA knockdown may only be partially effective for reducing REDD1 protein abundance in DR, due to the blockade of REDD1 protein degradation. The objective of this F31 pre-doctoral fellowship is to address important knowledge gaps in the field by characterizing the molecular mechanisms that promote retinal REDD1 protein abundance in response to diabetes and exploring alternative intervention strategies for preventing the retinal complications that are caused by REDD1. The central hypothesis is that diabetes-induced allosteric regulation of REDD1 contributes to increased REDD1 protein abundance, leading to loss of retinal homeostasis and the development of retinal pathology. The proposed studies will employ a newly developed REDD1 CRISPR knockin mouse that expresses a REDD1 variant that continues to be rapidly degraded even after its redox-modification. Aim 1 will characterize the mechanism through which diabetes acts to alter REDD1 proteolysis. The proposed studies will evaluate a role for competitive binding of REDD1 to thioredoxin interacting protein (TXNIP) versus heat shock cognate 70 kDa (HSC70) in the suppressive effect of diabetes on REDD1 proteolysis. To complement this genetic approach, artificial intelligence (AI) molecular docking, Microscale Thermophoresis (MST), and biochemical techniques will also be used to validate small molecule inhibitors of REDD1 allostery. Aim 2 will evaluate a role for REDD1 allosteric regulation in the development of diabetes-induced retinal defects. Through the proposed training plan, I will develop new laboratory skills to evaluate REDD1 protein allostery (e.g., biotin-switch assay, MST) and develop the technical expertise to determine its impact on retinal pathophysiology [e.g., optical coherence tomography (OCT), FITC- BSA perfusion, electroretinography (ERG), behavioral optometry]. This project has significant implications for addressing visual dysfunction in diabetic patients, as it may facilitate the development of improved therapies that more effectively address the specific molecular events that contribute to early disease progression.