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

Shirian, Jason; Arkadash, Valeria; Cohen, Itay; Sapir, Tamila; Radisky, Evette S.; Papo, Niv; Shifman, Julia M.

doi:10.1101/241372

Cited by 5 publications

(13 citation statements)

References 55 publications

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“…The naturally occurring inhibitor TIMP‐2 has provided the starting point for the design of other selective MMP inhibitors. Using yeast surface display technology, fluorescently labelled catalytic domains of MMP‐14 and MMP‐9 can be used to identify mutant TIMP‐2 that has greater specificity for either one MMP or the other . The mutant TIMP‐2 molecules inhibit the catalytic function of MMP‐14 and MMP‐2 in vitro , and reduce cell migration in a breast cancer cell line .…”

Section: Recent Advances In the Development Of Mmp Inhibitorsmentioning

confidence: 99%

Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis

Conlon

Murray

2019

The Journal of Pathology

146

101

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This review aims to provide an overview of recent developments regarding the roles of MMPs in tumour invasion and metastasis. Much of the mortality burden belonging to cancer relates to its ability to invade adjacent tissue and form metastases at distant sites. This would not be possible without remodelling of the ECM, a process which is enabled by the functions of MMPs. Recent studies provide a better understanding of the importance of the biophysical nature of the ECM, how this influences cancer cell motility, and how MMPs act to modify matrix stiffness. The regulation of MMPs and the role of immune cell generated MMPs has also become better understood. All of this provides a framework for the therapeutic targeting of MMPs and recent advances in the development of selective MMPs inhibitors are also reviewed.

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Section: Recent Advances In the Development Of Mmp Inhibitorsmentioning

confidence: 99%

Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis

Conlon

Murray

2019

The Journal of Pathology

146

101

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“…Here, the greater challenge becomes evolving selectivity among MMPs, since native TIMPs broadly inhibit most MMPs. Earlier studies employed the isolated N-terminal domain of TIMP-2 (N-TIMP-2) as a scaffold for engineering selective inhibitors of MMP-14 and MMP-9 (17,18). In addition to the core inhibitory epitope that binds in the enzyme active site, this domain includes the TIMP AB-loop, which contacts the MMP catalytic domain at a nonconserved surface exosite, giving opportunities for evolution of specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Prior work has focused primarily on engineering the N-terminal domain of TIMPs, an independently folding and stable domain that retains MMP inhibitory capability (15,16). Yeast surface display of N-TIMP-2 was used successfully as a directed evolution platform to develop inhibitors of MMP-9 and MMP-14 with enhanced affinity and selectivity (17)(18)(19)(20). Recently, we demonstrated that full-length TIMPs could offer further advantages as scaffolds for engineering inhibitors with improved binding toward target MMPs.…”

Section: Introductionmentioning

confidence: 99%

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10

Raeeszadeh‐Sarmazdeh

Coban

Mahajan

et al. 2022

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…We and others have engineered N-TIMP2 with the goal of converting it into a highly specific inhibitor of one MMP type (28)(29)(30)(31)(32). Our group first explored how various single mutants in N-TIMP2 affect its affinity and specificity to various MMPs (29).…”

Section: Introductionmentioning

confidence: 99%

A broad matrix metalloproteinase inhibitor with designed loop extension exhibits ultrahigh specificity for MMP-14

Bonadio

Wenig

Hockla

et al. 2022

Preprint

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Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. While several antibodies against MMPs are in development, our goal is to construct therapeutic anti-MMP inhibitors based on a natural broad MMP inhibitor, tissue inhibitor of metalloproteinases-2 (N-TIMP2). To confer high binding specificity toward one MMP type, we extend one of the N-TIMP2 loops, allowing it to interact with the non-conserved MMP surface. Multiple computational designs of the loop were used to design a focused library for yeast surface display, which was sorted for high binding to the target MMP-14 and low binding to off-target MMP-3. Deep sequencing of the two selected populations followed by comparative data analysis was used to identify the most promising variants, which were expressed, purified, and tested for inhibition of MMP-14 and off-target MMPs. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 μM affinity to MMP-3, 7500-fold more specific than WT N-TIMP2. Furthermore, the variant inhibited cell invasion with increased potency relative to WT N-TIMP2 in two breast cancer cell lines. We obtained the engineered variant high-accuracy model by including NGS data as input to AlphaFold multiple sequence alignment (MSA). Modeling results together with experimental mutagenesis demonstrate that the loop packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates that introduction of loop extensions into inhibitors to stretch to the non-conserved surface of the target proteins is an attractive strategy for conferring high binding specificity in design of MMP inhibitors and other therapeutic proteins.

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

Cited by 5 publications

References 55 publications

Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis

Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10

A broad matrix metalloproteinase inhibitor with designed loop extension exhibits ultrahigh specificity for MMP-14

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