Matrix metalloproteinases (MMPs) play a pivotal role in diagnosis and progression of several diseases such as cancer and fibrosis, which makes them promising targets for developing protein therapeutics. The challenge in using MMP inhibitors as drugs in MMP-associated diseases is lack of high selectivity for binding to a specific MMP. Therefore, development of selective MMP inhibitors is a critical goal. Tissue inhibitor of metalloproteinases (TIMPs), as natural MMP inhibitors, offer an ideal scaffold for engineering highly selective MMP-targeted therapeutics. We have previously shown a key role for MMP-3 in the pathogenesis of pulmonary fibrosis.<br/><br/>TIMP-1 is a natural inhibitor MMP-3 with picomolar affinity; it also binds to other MMPs with a spectrum of affinities. I have used directed evolution and yeast surface display toward development of selective MMP-3-targeted biologic agents through protein engineering of TIMP-1. I have screened a library of TIMP-1 mutants generated by random mutagenesis of residues comprising the MMP-interacting loops of TIMP-1 to select for enhanced MMP-3 binding on the yeast surface. Diversified residues have been chosen based on structural studies of TIMP-1/MMP-3 interactions. TIMP-1 mutants isolated after rounds of Fluorescent-Activated Cell Sorting (FACS) showed substantial improvement in MMP-3 binding compared to the wild-type TIMP-1. Mutations found in mutants with enhanced binding affinity toward MMP-3 were further characterized using soluble proteins. I have also developed a counter-selection strategy to screen TIMP-1 mutants that bind selectively to MMP-3 in the presence of MMP-10, an MMP with the most structural similarity to MMP-3. The isolated TIMP-1 mutants after five rounds of competitive screening showed up to 20-fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single-MMP 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 tissue inhibitor of metalloproteinase-1 (TIMP-1) as a selective inhibitor of matrix metalloproteinase-3 (MMP-3)

255th National Meeting (2018)

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