(534d) Design and Engineering Protein Therapeutics for Targeting Metalloproteinases

The metalloproteinase (MP) family of zinc-dependent proteases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteases (ADAMs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) plays a crucial role in the extracellular matrix (ECM) remodeling and degradation activities, and thus responsible in several diseases. Specific MPs has shown to play a significant role in progression of several cancers and neurodegenerative disease such as Alzheimer’s disease (AD), while other MMPs and ADAMs found to be anti-tumorigenic and neuroprotective. Therefore, it is important to target specific MPs with pathological roles with high selectivity. Protein engineering techniques such as directed evolution can be used to tailor the binding selectivity of protein binders toward pathological MPs. Tissue inhibitor of metalloproetainses (TIMPs) as natural inhibitors of MPs with a wide range of binding affinity offer great scaffolds for developing therapeutics. Engineering and design of TIMP-based scaffolds to improve the selectivity and eliminate the off-target effects requires a comprehensive understanding of the protein structures combined with library design and high throughput screening. We have previously developed a counter-selection strategy to screen TIMP-1mutants that bind selectively to MMP-3 in the presence of MMP-10, an MMP with the greatest sequence and structural similarity to MMP-3. This strategy has improved TIMP-1 mutants with up to 20-fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single-MP selectivity. In ongoing studies, we combine rational design and directed evolution to develop more efficient TIMP-based scaffolds to target specific MPs with high affinity and selectivity. These studies and methodology developed will lay the foundation for developing novel protein engineering strategies for generating therapeutics for MP-related diseases and understanding protein-protein interaction between MPs and their inhibitors.