Protein Engineering by Combined Computational and In Vitro Evolution Approaches

Rosenfeld, Lior; Heyne, Michael; Shifman, Julia M.; Papo, Niv

doi:10.1016/j.tibs.2016.03.002

Cited by 42 publications

(35 citation statements)

References 89 publications

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“…In this work, we engineered four N-TIMP2 mutants with single-digit picomolar K i values for binding to MMP-14, improving affinity toward this target by nearly 900-fold and improving binding specificity relative to other MMP family members by 100 -16,000-fold. These impressive results demonstrate the enormous potential of the combined computational/directed evolution methodology for protein engineering (39). In this study, we used computational methods to reduce the library size to ϳ10 8 of the most promising N-TIMP2 variants, a number that is tractable by the YSD technique.…”

Section: Discussionmentioning

confidence: 99%

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

Arkadash

Yosef

Shirian

et al. 2017

Journal of Biological Chemistry

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101

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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 ( ) 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 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.

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Section: Discussionmentioning

confidence: 99%

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

Arkadash

Yosef

Shirian

et al. 2017

Journal of Biological Chemistry

Self Cite

101

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“…The best variant identified showed 900-fold improvement in MMP-14 affinity with an inhibitory constant of 0.9 pM, greatly enhanced selectivity, and improvements in binding to MMP-14 on the cell surface and inhibition of breast cancer cell invasion [Arkadash et al 2017]. The encouraging results from these studies point to the utility of integrating structural and computational insights with directed evolution approaches [Rosenfeld et al 2016], and we anticipate that this could be a general path forward for developing TIMP-based drugs selectively targeting the different MMPs most strongly implicated as therapeutic targets in breast cancer. Keeping in mind the MMP-independent activities of the natural TIMPs [Chirco et al 2006; Stetler-Stevenson 2008; Brew and Nagase 2010], an additional important aspect of developing TIMP-based drugs will be to better define the TIMP epitopes responsible for some of these activities, and to remove unwanted off-target activities through protein engineering.…”

Section: Developing Mmp Inhibitors For Therapeutic Applicationsmentioning

confidence: 99%

Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

Radisky

Raeeszadeh‐Sarmazdeh

Radisky

2017

J of Cellular Biochemistry

117

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Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell-associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re-evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial-mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold.

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“…Protein engineering methods include (i) random mutagenesis, (ii) rational design, and (iii) semirational design (2,8,(18)(19)(20). It was previously shown that these approaches can be applied separately or in combination to tailor enzymes to enhance their stability in organic solvents (1,9,12,18,21,22).…”

mentioning

confidence: 99%

“…Protein engineering by rational design requires structural information, and the precise regions for mutagenesis are identified usually by computational tools or prior knowledge (17,(22)(23)(24). One recently developed concept for enzyme stabilization in organic solvents is the modification of residues buried in tunnels within the protein structure.…”

mentioning

confidence: 99%

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Gihaz

Kanteev

Pazy

et al. 2018

Appl Environ Microbiol

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Enhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by denaturation in polar alcohols. In this study we focused on selected solvent tunnels in lipase from T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated in order to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. Each solvent tunnel was systematically analyzed with respect to its chemical and structural characteristics. Selected residues were replaced with Phe, Tyr or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed elevated stability up to 81-fold compared with wild-type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" x-ray structure. Combining the Phe substitutions created double mutant A187F/L360F with an increase in T of +7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. Kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates ( ), though mostly their stability properties determined the transesterification capability. Seven crystal structures of different variants were solved disclosing new π-π or CH/π intramolecular interactions emphasizing the significance of aromatic interactions to improved solvent stability. This rational approach could be implemented in other enzymes for stabilization in organic solvents. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields, however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability and we were inspired by this to re-design enzyme voids. Rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. Structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting solvent channels void.

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Protein Engineering by Combined Computational and In Vitro Evolution Approaches

Cited by 42 publications

References 89 publications

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

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

Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

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