scholarly journals Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14

2017 ◽  
Author(s):  
Jason Shirian ◽  
Valeria Arkadash ◽  
Itay Cohen ◽  
Tamila Sapir ◽  
Evette S. Radisky ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Surface Display ◽  
Specific Inhibitor ◽  
Computational Design ◽  
Yeast Surface Display ◽  
Physiological Conditions ◽  
Mmp Inhibitor ◽  
Single Target ◽  
Display Technology ◽  
Yeast Surface

AbstractMMP-14 and MMP-9 are two well established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a non-specific MMP inhibitor, N-TIMP2. The engineered purified N-TIMP2 variants showed enhanced specificity towards MMP-14 and MMP-9 relative to a panel of off-target MMPs. MMP-specific N-TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N-TIMP2 interactions. Our MMP-9 inhibitor exhibited 1000-fold preference for MMP-9 vs. MMP-14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single-target specificities.

scholarly journals Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

2017 ◽  
Vol 292 (8) ◽  
pp. 3481-3495 ◽  
Author(s):  
Valeria Arkadash ◽  
Gal Yosef ◽  
Jason Shirian ◽  
Itay Cohen ◽  
Yuval Horev ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Surface Display ◽  
High Specificity ◽  
Computational Design ◽  
Computational Method ◽  
Yeast Surface Display ◽  
Mmp Inhibitor ◽  
Starting Point ◽  
Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

scholarly journals A Decade of Yeast Surface Display Technology: Where Are We Now?

2008 ◽  
Vol 11 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Eric Shusta ◽  
Lauren Pepper ◽  
Yong Cho ◽  
Eric Boder
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Display Technology ◽  
Yeast Surface

Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology

2016 ◽  
Vol 120 (12) ◽  
pp. 1609-1622 ◽  
Author(s):  
Brandt Bertrand ◽  
María R. Trejo-Hernández ◽  
Daniel Morales-Guzmán ◽  
Luis Caspeta ◽  
Ramón Suárez Rodríguez ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Surface Display ◽  
Functional Expression ◽  
Yeast Surface Display ◽  
Display Technology ◽  
Yeast Surface

scholarly journals Molecular evolution of peptides by yeast surface display technology

MedChemComm ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 1569-1580 ◽  
Author(s):  
Sara Linciano ◽  
Stefano Pluda ◽  
Arianna Bacchin ◽  
Alessandro Angelini
Keyword(s):  
Molecular Evolution ◽  
Detailed Analysis ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Display Technology ◽  
Yeast Surface

This review provides a detailed analysis of the diverse genetically encoded peptides that have been evolved by using yeast surface display technology.

scholarly journals Selection of novel human scFvs against cancer antigen IL1RAP by phage and yeast surface display technology

2020 ◽  
Vol 34 (1) ◽  
pp. 287-293
Author(s):  
Shixin Duan ◽  
Yanrong Jia ◽  
Debao Xie ◽  
Shenglin Xiao ◽  
Cheng Zhou ◽  
...  
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Cancer Antigen ◽  
Display Technology ◽  
Yeast Surface ◽  
Selection Of

scholarly journals Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy

2021 ◽  
Author(s):  
Gal Yosef ◽  
Hezi Hayun ◽  
Niv Papo
Keyword(s):  
Cell Surface ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Catalytic Domain ◽  
Surface Display ◽  
Specific Inhibitor ◽  
Yeast Surface Display ◽  
Great Promise ◽  
Mmp Inhibitor ◽  
Neoplastic Process

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44–MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

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