Development of enzymatic tools for rapid measurement of Advanced Glycation and Lipoxidation End Product-protein adducts

PROJECT SUMMARY Revel Pharmaceuticals is developing an innovative, cost-effective, and user-friendly enzymatic method to measure advanced glycation and lipoxidation end products (AGE/ALEs). AGE/ALEs are molecular hallmarks of aging and their accumulation in the body is linked to many age-related diseases. Research progress in AGE/ALE biology has been hindered by the absence of dependable and accessible measurement techniques and has consequently delayed advancements in scientific and clinical interventions aimed at AGE/ALEs. Current methods for measurement of AGE/ALEs are inaccessible to many researchers or unreliable. Liquid chromatography mass spectrometry (LC-MS) based methods are expensive (costing over $120 per sample), labor-intensive, and demand specialized equipment, making them inaccessible to many researchers. ELISA methods suffer from poor specificity, poor reproducibility and show poor correlation to LC-MS. Introduction of an efficient and accurate measurement method for significant AGE/ALEs would democratize the research tools for studying AGEs, serving as a pivotal technology for scientific, clinical, and therapeutic research targeting these key hallmarks of aging and disease. Revel's innovation is an enzymatic method to detect AGE/ALEs, providing a highly specific, rapid, and inexpensive assay. In Phase I, we developed a prototype enzymatic detection assay for one of the most well characterized AGEs, carboxymethyl lysine (CML). Revel’s proprietary CML oxidase is the first enzyme known to catalyze removal of the CML modification from lysine. We used the engineered CML oxidase enzyme to develop a prototype assay for quantifying CML in model proteins and human tissues (Phase I, Aim 2), and showed correlation of over 0.98 with the gold-standard of isotope dilution LC-MS. We met our Phase I milestones and demonstrated strong proof-of-concept for our approach to generate detection reagents for other AGE/ALE modifications. In Phase II, we aim to broaden our array of enzyme-based detection assays to include other critical AGE/ALE modifications and complete development, manufacturing, and external testing activities necessary to support launch of research-use assay kits. In Aim 1, we will engineer and optimize three additional oxidase enzymes to detect scientifically significant AGE/ALE modifications. In Aim 2, we will validate five ENZYMATIC AGE/ALE DETECTION ASSAY kits with a range of sample types, including cell lysates, serum, tissues, and food. Finally, in Aim 3, we will generate commercial-scale lots of Revel’s five ENZYMATIC AGE/ALE DETECTION ASSAY kits to support internal reagent stability, lot-to-lot performance studies, and external validation testing. Together, these Aims will result in commercial launch of Revel’s ENZYMATIC AGE/ALE DETECTION ASSAY kits.