PROJECT SUMMARY Diabetic kidney disease (DKD) is a major complication associated with Type 1 and Type 2 diabetes. Approximately 25% of patients with diabetes develop DKD. DKD is the leading cause of kidney failure and accounts for 44% of all end-stage renal disease cases. It is associated with heart disease and heart failure, stroke, and early death. Treatment options for DKD are limited and focus on slowing progression to end-stage renal disease through glycemic control. Current FDA-approved treatments do not address pathogenic mechanisms driving the disease. The pathophysiology of DKD is related to prolonged exposure to high blood sugar, leading to non-specific glycation of proteins and subsequent formation of toxic advanced glycation end-products (AGEs). Evidence suggests that accumulation of AGEs in the kidneys plays a role in the vascular complications associated with diabetes, including DKD. Revel Pharmaceuticals is developing a novel class of therapeutics designed to cleave one of the most abundant AGEs accumulating in DKD, carboxymethyl-lysine (CML), thus removing a significant driver of DKD. Using rational design approaches, we have engineered enzymes that cleave free CML and CML modified peptides. Our goal in this Phase I grant is to optimize lead drug candidates and demonstrate enzyme activity on physiologically relevant DKD substrates. Our specific aims are to (1) engineer CML oxidase to efficiently cleave CML modifications from physiological substrates (2) characterize CML oxidase activity on model proteins & human tissues. Successful completion of this Phase I SBIR project will yield the first therapeutic enzyme designed to remove and repair AGE damage in vivo. In Phase II, we will carry out in vivo efficacy and IND-enabling studies with our collaborators at the National Mouse Metabolic Phenotyping Center to support an IND submission.