Matrix metalloproteinases (MMPs) have important functions in extracellular matrix remodeling and degradation, and play central roles in development of many pathologies including cancer, pulmonary fibrosis, and neurological disorders. The main challenge in developing therapeutics targeting MMPs is the lack of selectivity for individual target MMPs and consequent undesired off-target effects. To overcome this challenge, engineering highly selective protein-based MMP inhibitors is desired. Tissue inhibitors of metalloproteinases (TIMPs), natural MMP inhibitors, could offer novel avenues for developing such selective therapeutics. TIMP-1 is an endogenous inhibitor of MMP-3 with picomolar affinity that also binds to other MMPs with a spectrum of affinities. I have used directed evolution and yeast surface display to identify highly selective inhibitors of MMP-3, a pivotal mediator in pathogenesis of pulmonary fibrosis and cancer, through protein engineering of TIMP-1. I have recruited a counter-selection strategy to screen a library of TIMP-1 mutants using fluorescence-activated cell sorting (FACS) and identify variants that bind selectively to MMP-3 in the presence of incremental amounts of a competitor ligand, MMP-10. TIMP-1 mutants isolated after five rounds of competitive screening showed up to 20-fold improvement in binding selectivity toward MMP-3 versus MMP-10 compared to the wild type TIMP-1. DNA sequencing of TIMP-1 clones with improved MMP-3 binding selectivity revealed a repeatedly occurring pair of adjacent mutations that appear to be responsible for improvements in selectivity. I have obtained crystal structures of the TIMP-1 variant with the highest MMP-3 binding selectivity in complex with both MMP-3 and MMP-10 enzymes using X-ray crystallography. These structures offer insights into the structural basis for improvements in selectivity. This work provides a promising strategy to develop protein therapeutics based on natural enzyme inhibitors, critically improving selectivity to eliminate off-target effects.

Directed evolution of the tissue inhibitor of metalloproteinases-1 (TIMP-1) scaffold for developing selective therapeutic agents

2019 Spring National Meeting

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