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

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.

Download Full-text

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

10.1101/241372 ◽

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.

Download Full-text

Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

10.1101/2021.04.07.438849 ◽

2021 ◽

Author(s):

Jimmy D Gollihar ◽

Jason S McLellan ◽

Daniel R Boutz ◽

Jule Goike ◽

Andrew Horton ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Surface Display ◽

Functional Characterization ◽

Yeast Surface Display ◽

Spike Protein ◽

Emerging Pathogens ◽

Effective Interventions ◽

Yeast Surface

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

Download Full-text

Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

Chemistry - A European Journal ◽

10.1002/chem.201803485 ◽

2018 ◽

Vol 24 (57) ◽

pp. 15195-15200 ◽

Cited By ~ 12

Author(s):

Lukas Deweid ◽

Lara Neureiter ◽

Simon Englert ◽

Hendrik Schneider ◽

Jakob Deweid ◽

...

Keyword(s):

Directed Evolution ◽

Surface Display ◽

Microbial Transglutaminase ◽

Yeast Surface Display ◽

Bond Forming ◽

Yeast Surface

Download Full-text

Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models

Applied and Environmental Microbiology ◽

10.1128/aem.00054-19 ◽

2019 ◽

Vol 85 (10) ◽

Cited By ~ 3

Author(s):

Seth C. Ritter ◽

Benjamin J. Hackel

Keyword(s):

Protein Stability ◽

Clostridium Perfringens ◽

High Throughput Sequencing ◽

Surface Display ◽

High Specificity ◽

Classification Performance ◽

Yeast Surface Display ◽

Content Type ◽

Cell Wall Binding ◽

Yeast Surface

ABSTRACT Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C. IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

Download Full-text

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

BioMed Research International ◽

10.1155/2019/6051870 ◽

2019 ◽

Vol 2019 ◽

pp. 1-22 ◽

Cited By ~ 1

Author(s):

Biancamaria Cembrola ◽

Valentino Ruzza ◽

Fulvia Troise ◽

Maria Luisa Esposito ◽

Emanuele Sasso ◽

...

Keyword(s):

High Throughput Sequencing ◽

Surface Display ◽

Affinity Maturation ◽

Yeast Surface Display ◽

Antigen Concentration ◽

Selection Scheme ◽

High Affinity ◽

Novel Approach ◽

Yeast Surface

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Download Full-text

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01466-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5732-5742 ◽

Cited By ~ 17

Author(s):

Michael L. Burns ◽

Thomas M. Malott ◽

Kevin J. Metcalf ◽

Benjamin J. Hackel ◽

Jonah R. Chan ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Neurotrophic Factor ◽

Surface Display ◽

Brain Derived Neurotrophic Factor ◽

Binding Activity ◽

Yeast Surface Display ◽

Site Directed Mutagenesis ◽

Secretion Systems ◽

Yeast Surface

ABSTRACTBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employingSaccharomyces cerevisiaedisplay and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

Download Full-text