PROJECT SUMMARY Advanced glycation end product (AGE) accumulation is a hallmark of mammalian aging. AGEs play causative roles in various age-related diseases by interfering with cell signaling pathways and forming adducts on cellular macromolecules, contributing to their inactivation and pathophysiology in many tissues/organs. Nε-carboxymethyl-lysine (CML), a maillard reaction product of lysine, is the best characterized AGE and is used clinically for predictive screening of diabetic kidney disease and as a biomarker for diabetic retinopathy and macular oedema. Despite the importance of CML as a biomarker of and contributor to age related disease, there are currently no techniques available for rapid, quantitative detection of CML in patient samples. Therefore, improved methods for measuring CML would be highly significant for advancing the study and treatment of diseases of aging. Revel Pharmaceuticals proposes to develop enzymes designed to cleave CML, thus enabling reagents for enzyme-based detection of one of the most important AGEs in aging, diabetes and glycobiology. Development of a convenient enzymatic method for measurement of CML in clinical samples would be an important step forward in diagnostic medicine. Revel has discovered a CML oxidase enzyme, capable of oxidizing CML to lysine. The newly discovered CML oxidase provides an opportunity to develop enzymes for diagnostic use in monitoring CML-modifications. The goal of this Phase I SBIR is to develop an enzymatic assay for rapid measurement of CML. The proposed assay will be developed analogous to the enzymatic HbA1c test which is used to assess patient blood sugar levels and diagnose diabetes. In Phase II, we will develop a workflow for CML detection in patient samples.