Targeting new pathogenic biomarkers of neurodegenerative diseases is critical as there are limited efficient therapeutics available to control such diseases. The metzincin superfamily, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), play multifaceted roles in physiological and pathological processes in the central nervous system and therefore are therapeutic targets to limit neurodegeneration in diseases such as Huntington disease, Parkinson’s disease (PD), and Alzheimer’s disease (AD). Given the significance of recognizing enzymes that play a central role in neurodegenerative disease as novel neurodegenerative therapeutics, enzyme inhibitors with high selectivity are desired. Overexpression of MMP-9 plays a significant role in several neurodegenerative disorders, while ADAM-10 helps block progression of AD. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a natural inhibitor of MMP-9 with pico- and subnanomolar affinity. To overcome the challenges of wide multispecificity of TIMP-3 for different classes of MMPs and ADAMs and its interaction with growth factors, a protein engineering approach is desired to tailor a TIMP-3 scaffold to create an outstanding neurodegenerative drug candidate. The overarching goal of this proposal is to engineer protein-based scaffolds that high selectively target a specific metzincin without off-target effects can create novel therapeutics for neurodegenerative diseases. In Aim 1, we propose to engineer protein scaffolds based on TIMPs to improve binding selectivity toward MMP-9 using directed evolution and yeast surface display. In Aim 2, we will study the mechanism of interaction of these TIMP-3 variants in complex with MMP-9 and ADAM-10 using X-ray crystallography to understand the underlying mechanism of inhibition. In Aim 3, we will evaluate these engineered TIMP-3 scaffolds with improved selectivity for MMP-9 in neurodegenerative models. Our long-term goal is to develop selective protein-based therapeutics based on MMP inhibitors or similar scaffolds. These studies will lay the foundation for preclinical in vivo models and novel therapeutic strategies for neurodegenerative and other MMP-related diseases.