Combinatorial engineering of N-TIMP2 variants that selectively inhibit MMP9 and MMP14 function in the cell

Oncotarget
Valeria ArkadashNiv Papo

Abstract

Developing selective inhibitors for proteolytic enzymes that share high sequence homology and structural similarity is important for achieving high target affinity and functional specificity. Here, we used a combination of yeast surface display and dual-color selective library screening to obtain selective inhibitors for each of the matrix metalloproteinases (MMPs) MMP14 and MMP9 by modifying the non-specific N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP2). We generated inhibitor variants with 30- to 1175-fold improved specificity to each of the proteases, respectively, relative to wild type N-TIMP2. These biochemical results accurately predicted the selectivity and specificity obtained in cell-based assays. In U87MG cells, the activation of MMP2 by MMP14 was inhibited by MMP14-selective blockers but not MMP9-specific inhibitors. Target specificity was also demonstrated in MCF-7 cells stably expressing either MMP14 or MMP9, with only the MMP14-specific inhibitors preventing the mobility of MMP14-expressing cells. Similarly, the mobility of MMP9-expressing cells was inhibited by the MMP9-specific inhibitors, yet was not altered by the MMP14-specific inhibitors. The strategy developed in this study for ...Continue Reading

References

Jun 1, 1997·Nature Biotechnology·E T Boder, K D Wittrup
May 13, 1999·Proceedings of the National Academy of Sciences of the United States of America·M C KiekeD M Kranz
Jul 10, 1999·The Journal of Biological Chemistry·G S ButlerG Murphy
Mar 29, 2000·Proceedings of the National Academy of Sciences of the United States of America·Z ZhouK Tryggvason
Jul 11, 2000·Nature Biotechnology·E V ShustaK D Wittrup
Sep 14, 2000·Proceedings of the National Academy of Sciences of the United States of America·E T BoderK D Wittrup
May 8, 2002·Nature Reviews. Molecular Cell Biology·Constance E Brinckerhoff, Lynn M Matrisian
Jun 8, 2002·Journal of Molecular Biology·Siân RowsellRichard A Pauptit
Dec 3, 2002·Nature Structural Biology·James J Havranek, Pehr B Harbury
Mar 23, 2004·Nature Structural & Molecular Biology·Tanja KortemmeDavid Baker
Apr 30, 2004·Protein Engineering, Design & Selection : PEDS·Christilyn P GraffK Dane Wittrup
Sep 7, 2004·The International Journal of Developmental Biology·Alicia R FolguerasCarlos López-Otín
Nov 9, 2004·Protein Engineering, Design & Selection : PEDS·Paul H BessettePatrick S Daugherty
Dec 1, 2004·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Johanna M PellikainenVeli-Matti Kosma
Jun 7, 2005·Protein Science : a Publication of the Protein Society·Patrick J Fleming, George D Rose
May 9, 2006·Cancer Metastasis Reviews·Elena I Deryugina, James P Quigley
Jun 17, 2006·Breast Cancer Research : BCR·Bernard TêtuDominique Trudel
Apr 5, 2007·Nature Protocols·Ginger ChaoK Dane Wittrup
Sep 14, 2007·Nature Reviews. Cancer·Carlos López-Otín, Lynn M Matrisian
May 3, 2008·Cancer Research·Christina BendrikCharlotta Dabrosin
Jun 17, 2008·Cell·Jeffrey M SkerkerMichael T Laub
Jul 22, 2008·Journal of Molecular Biology·Magdalena WisniewskaWolfram Bode
Sep 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·Casim A SarkarAndreas Plückthun
Nov 29, 2008·Journal of Molecular Biology·Adam P SilvermanJennifer R Cochran
Feb 4, 2009·Breast Cancer Research : BCR·Xiao-Jun MaDennis C Sgroi
Apr 7, 2010·Journal of Molecular Biology·Sanjib DuttaAmy E Keating

❮ Previous
Next ❯

Citations

Sep 15, 2020·Expert Opinion on Drug Discovery·Thomas Fischer, Rainer Riedl
Apr 4, 2021·International Journal of Molecular Sciences·Vincent MariauleMoez Rhimi

❮ Previous
Next ❯

Methods Mentioned

BETA
protein-library display
X-ray
fluorescence-activated cell sorting
FACS
Flow cytometry
size-exclusion chromatography
zymography
light microscopy

Software Mentioned

Geneious
GraphPad Prism
GraphPad
Prism
WebLogo
ImageJ

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.