Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with key roles in extracellular matrix degradation and remodeling. MMP overexpression is long known for its contribution to several diseases such as cancer, neurological disorders, and cardiovascular diseases, making MMPs great targets for developing novel therapeutics. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of MMPs that bind through several flexible interacting loops. TIMPs, a family of four in human, have great similarities in sequence and structure. However, TIMPs have a wide spectrum of binding affinity and selectivity to different MMPs.<br/>Employing yeast surface display and DNA shuffling techniques among the TIMP family, we generated a scrambled TIMP (ScTIMP) library to identify the dominant minimal MMP inhibitory domain and fragments among TIMPs. We first analyzed the levels of TIMP expression and MMP binding of yeast displayed full-length and the inhibitory N-terminal domain of human TIMPs using flow cytometry. Full length TIMPs and N-TIMP proteins showed a spectrum of yeast display and MMP-3 catalytic domain (MMP-3 cd). Additionally, we screened our ScTIMP library for protein expression and MMP-3cd binding via fluorescent-activated cell sorting (FACS). Interestingly, several of the ScTIMP fragments as short as 20 amino acids were isolated after three rounds of FACS screening, with maintained or improved binding to MMP-3cd (up to 4-fold). We also sought to determine the inhibition profile of these isolated clones towards soluble MMP-3cd. Our study allowed us to explore the TIMP motifs that are involved in binding to MMP. We expect this study will shed light on understanding of molecular mechanism of TIMP-MMP binding and inhibition to develop the next class of MMP inhibitors with more flexibility, and tissue-penetration.

Engineering of minimal metalloproteinase inhibitor scaffolds

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.