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

Raeeszadeh‐Sarmazdeh, Maryam; Coban, Matt; Mahajan, Shivansh; Hockla, Alexandra; Sankaran, Banumathi; Downey, Gregory P.; Radisky, Derek C.; Radisky, Evette S.

doi:10.1016/j.jbc.2022.101654

Cited by 18 publications

(15 citation statements)

References 80 publications

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“…In similar approaches, N-TIMP2 has been engineered for enhanced specificity toward other MMP targets such as MMP-1 (28), MMP-3 (56), and MMP-9 (30) by randomizing several N-TIMP2 positions and using competitive directed evolution approaches. In all these studies, the introduction of several mutations resulted in increased specificity but slightly decreased binding affinity to the target MMP.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Human N‐TIMP‐1 (N‐terminal domain of TIMP‐1) and the minimal TIMP library variant genes were inserted at the N‐terminus of Aga2p, yeast surface protein was inserted in pCHA yeast display vector between BsrGI and BamHI restriction enzymes, and displayed on the surface of the yeast as previously described (Raeeszadeh‐Sarmazdeh et al, 2022; Raeeszadeh‐Sarmazdeh, Greene, et al, 2019). The yeast strain was Saccharomyces cerevisiae , strain EBY100 ( MATa AGA1::GAL1‐AGA1::URA3 ura3‐52 trp1 leu2‐Δ200 his3‐Δ200 pep4::HIS3 prb11.6R can1 GAL ).…”

Section: Methodsmentioning

confidence: 99%

“…Binding between bMMP‐3 and N‐TIMP‐1 or minimal TIMP variants was performed on the yeast surface similar to protocols done previously (Raeeszadeh‐Sarmazdeh et al, 2022; Toumaian & Raeeszadeh‐Sarmazdeh, 2022). The yeast cells displaying N‐TIMP‐1 or minimal TIMP variants were grown and induced as previously described in SD‐CAA (pH 6) media at 30°C shaker and induced in SG‐CAA media at 30°C for 20 h. The induced yeast cells were pelleted at OD 600 of 0.4 by centrifuge at 5000 Xg for 4 min at 4°C and washed three times with PBSA buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Full‐length TIMP‐1, or N‐terminal TIMPs (Arkadash et al, 2017; Shirian et al, 2018) have been previously engineered to target MMPs with higher affinity (Raeeszadeh‐Sarmazdeh, Greene, et al, 2019) and selectivity (Raeeszadeh‐Sarmazdeh et al, 2022), however, it is important to understand the key motifs in the human TIMP family that derive binding to specific MMPs. Furthermore, minimal TIMP variants offer higher tissue penetration and ease of modulation and synthesis.…”

Section: Introductionmentioning

confidence: 99%

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Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Hosseini,

Kumar,

Hedin

et al. 2023

Protein Science

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Overexpression of specific matrix metalloproteinases (MMPs) has a key role in development of several diseases, such as cancer, neurological disorders, and cardiovascular diseases due to their critical role in degradation and remodeling of the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs), a family of four in human, are endogenous inhibitors of MMPs. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible in inhibition of MMPs for developing efficient therapeutics targeting specific MMPs. We used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library to identify the dominant minimal MMP inhibitory regions in yeast. The minimal TIMP variants screened toward MMP‐3 and MMP‐9 using fluorescent‐activated cell sorting (FACS). Interestingly, several minimal TIMP variants selected after screening toward MMP‐3cd or MMP‐9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP‐3 and MMP‐9. The TIMP‐MMP binding dissociation constant (KD), in the nM range, and MMP inhibition constants (Ki), in the pM range, of these minimal TIMP variants were similar to the N‐terminal domain of TIMP‐1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular protein docking of the minimal TIMP variants in complex with MMP‐3cd to understand the binding and inhibition mechanism of these variants.This article is protected by copyright. All rights reserved.

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“…Substantial efforts to tailor the specificities of naturally occurring, protein-based inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), have utilized competitive binding assays in display format to distinguish between on-and off-target binding 15,16 . This competition strategy has also been utilized to evaluate whether inhibitory antibodies compete for binding with TIMPs on the yeast surface [17][18][19][20] . Selections of inhibitory antibody fragments from naïve antibody libraries have been described utilizing phage display 21 and selections in the periplasm of E. coli 17 using competitions with small-molecule inhibitors and a selection marker for protease cleavage, respectively.…”

Section: Introductionmentioning

confidence: 99%

Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface

Rezhdo

Lessard

Islam

et al. 2022

Preprint

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Display technologies are powerful tools for discovering antibodies and other binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of such targets because the phenotypic readout during display screens is binding. The goal of this work is to investigate approaches for discovering inhibitory antibodies in yeast display format using a well-defined series of constructs and the target matrix metalloproteinase-9 (MMP-9). Three previously reported antibodies (DX-2802, M0076 and FAPB2.3.6) were used to create model libraries that are representative of protein libraries consisting of inhibitory binders, non-inhibitory binders, and non-binding constructs. Conditions that preferentially enrich for inhibitory clones were identified for both magnetic bead-based enrichments and fluorescence-activated cell sorting (FACS). Finally, we used direct titration of yeast to estimate inhibitor IC50 values with yeast-displayed and soluble constructs and found that the IC50 obtained for DX-2802 in yeast display format (20.01 ± 9.01 nM) falls within the confidence interval of IC50 the soluble scFv-Fc form of DX-2802 (17.56 ± 6.16 nM). Thus, it is possible to obtain IC50 values on the yeast surface, which greatly streamlines initial characterizations of inhibitory properties. Overall, we used these well-defined constructs to identify strategies for the discovery and characterization of inhibitory clones directly in surface display format.

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

Cited by 18 publications

References 80 publications

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

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

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display

Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface

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