SUMMARY Limited efficient therapeutics are available to control Alzheimer's disease (AD), so there is a strong demand to target new pathogenic biomarkers of AD. The metzincin superfamily, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), play a multifaceted role in physiological and pathological processes in the central nervous system and therefore are therapeutic targets to limit neurodegeneration in AD. For instance, upregulation of MMP-9 promotes apoptosis, blood-brain barrier disorder, and demyelination and is involved in the amyloid-β processing pathway through proteolytic degradation of extracellular matrix components and tissue remodeling, and increasing tau accumulation which play key roles in AD. But on the other hand, upregulation of ADAM-10 increases soluble amyloid-β formation, which helps control AD. Thus, using highly selective inhibitors to tailor the proteolytic activity of metzincin- induced pathological changes in AD patients’ brains may be a promising therapeutic strategy. Protein inhibitors offer higher selectivity to target MMP-9 compared to smaller synthetic inhibitory molecules with broad-spectrum targets. Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous inhibitors of MMPs and ADAMs with varying degrees of binding affinity. Among all TIMPs, TIMP-3 has the lowest association with the cell signaling molecules triggering undesired off-target effect such as the ones responsible in tumorigenesis, suggesting that it has a low off-target therapeutic effect in MMP-dependent diseases. This makes TIMP-3 an ideal protein scaffold for engineering improved inhibitors for MMP-9. However, TIMP-3 also binds to other MMPs, ADAMs, and growth factors. Here, we propose to use a combination of rational and random combinatorial approaches to develop TIMP-3-based therapeutics to control neurodegenerative disease progression by targeting MMP-9 with high selectivity. These engineered TIMP-based probes will avoid binding to ADAM-10, which is neuroprotective in AD, to decrease off-target effects. The overarching goal of this project is to engineer and design a new molecularly targeted therapy based on natural MMP inhibitors that target MMP-9 with high selectivity, understand their mechanism of inhibition using structural studies and cell culture models to improve outcomes in neurodegenerative diseases with inadequate treatment options.