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  1. Article ; Online: Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix Metalloproteinase Inhibitor

    Bonadio, Alessandro / Wenig, Bernhard L. / Hockla, Alexandra / Radisky, Evette S. / Shifman, Julia M.

    Journal of Molecular Biology. 2023 Apr. 15, p.168095-

    2023  , Page(s) 168095–

    Abstract: Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of selective probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The ... ...

    Abstract Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of selective probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.
    Keywords ligands ; metalloproteinases ; molecular biology ; mutagenesis ; sequence alignment ; yeasts ; protein loop engineering ; protein engineering ; protein design ; protein–protein interaction ; binding specificity ; protein structure ; molecular modelling ; matrix metalloproteinase, MMP ; tissue inhibitor of metalloproteinases, TIMP
    Language English
    Dates of publication 2023-0415
    Publishing place Elsevier Ltd
    Document type Article ; Online
    Note Pre-press version
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2023.168095
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  2. Article ; Online: Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix MetalloproteinaseInhibitor.

    Bonadio, Alessandro / Wenig, Bernhard L / Hockla, Alexandra / Radisky, Evette S / Shifman, Julia M

    Journal of molecular biology

    2023  Volume 435, Issue 13, Page(s) 168095

    Abstract: Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N- ... ...

    Abstract Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.
    MeSH term(s) Matrix Metalloproteinase 14/genetics ; Matrix Metalloproteinase 14/chemistry ; Matrix Metalloproteinase 14/metabolism ; Matrix Metalloproteinase 3 ; Matrix Metalloproteinase Inhibitors/chemistry ; Matrix Metalloproteinase Inhibitors/pharmacology ; Mutagenesis ; Protein Engineering
    Chemical Substances Matrix Metalloproteinase 14 (EC 3.4.24.80) ; Matrix Metalloproteinase 3 (EC 3.4.24.17) ; Matrix Metalloproteinase Inhibitors
    Language English
    Publishing date 2023-04-15
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80229-3
    ISSN 1089-8638 ; 0022-2836
    ISSN (online) 1089-8638
    ISSN 0022-2836
    DOI 10.1016/j.jmb.2023.168095
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    Zs.A 867: Show issues Location:
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  3. Article ; Online: Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10.

    Raeeszadeh-Sarmazdeh, Maryam / Coban, Mathew / Mahajan, Shivansh / Hockla, Alexandra / Sankaran, Banumathi / Downey, Gregory P / Radisky, Derek C / Radisky, Evette S

    The Journal of biological chemistry

    2022  Volume 298, Issue 3, Page(s) 101654

    Abstract: Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive ... ...

    Abstract Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.
    MeSH term(s) Humans ; Matrix Metalloproteinase 10/chemistry ; Matrix Metalloproteinase 10/genetics ; Matrix Metalloproteinase 10/metabolism ; Matrix Metalloproteinase 3/chemistry ; Matrix Metalloproteinase 3/genetics ; Matrix Metalloproteinase 3/metabolism ; Protein Engineering ; Tissue Inhibitor of Metalloproteinase-1/chemistry ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Tissue Inhibitor of Metalloproteinase-1/metabolism
    Chemical Substances TIMP1 protein, human ; Tissue Inhibitor of Metalloproteinase-1 ; MMP3 protein, human (EC 3.4.24.17) ; Matrix Metalloproteinase 3 (EC 3.4.24.17) ; MMP10 protein, human (EC 3.4.24.22) ; Matrix Metalloproteinase 10 (EC 3.4.24.22)
    Language English
    Publishing date 2022-01-29
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1016/j.jbc.2022.101654
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    Uc I Zs.108: Show issues Location:
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  4. Article ; Online: Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma.

    Mehner, Christine / Hockla, Alexandra / Coban, Mathew / Madden, Benjamin / Estrada, Rosendo / Radisky, Derek C / Radisky, Evette S

    The Journal of biological chemistry

    2022  Volume 298, Issue 8, Page(s) 102146

    Abstract: Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among ... ...

    Abstract Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.
    MeSH term(s) Adenocarcinoma, Clear Cell/enzymology ; Adenocarcinoma, Clear Cell/pathology ; Female ; Humans ; Ovarian Neoplasms/enzymology ; Ovarian Neoplasms/pathology ; Serine Proteases/metabolism ; Tissue Plasminogen Activator/metabolism ; Trypsin ; Urokinase-Type Plasminogen Activator/metabolism
    Chemical Substances Serine Proteases (EC 3.4.-) ; Trypsin (EC 3.4.21.4) ; Tissue Plasminogen Activator (EC 3.4.21.68) ; Urokinase-Type Plasminogen Activator (EC 3.4.21.73)
    Language English
    Publishing date 2022-06-16
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1016/j.jbc.2022.102146
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  5. Article ; Online: Targeting an autocrine IL-6-SPINK1 signaling axis to suppress metastatic spread in ovarian clear cell carcinoma.

    Mehner, Christine / Miller, Erin / Hockla, Alexandra / Coban, Mathew / Weroha, S John / Radisky, Derek C / Radisky, Evette S

    Oncogene

    2020  Volume 39, Issue 42, Page(s) 6606–6618

    Abstract: A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near- ... ...

    Abstract A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.
    MeSH term(s) Adenocarcinoma, Clear Cell/mortality ; Adenocarcinoma, Clear Cell/prevention & control ; Adenocarcinoma, Clear Cell/secondary ; Animals ; Anoikis/drug effects ; Antibodies, Monoclonal, Humanized/pharmacology ; Antibodies, Monoclonal, Humanized/therapeutic use ; Autocrine Communication/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Interleukin-6/antagonists & inhibitors ; Interleukin-6/genetics ; Interleukin-6/metabolism ; Mice ; Ovarian Neoplasms/drug therapy ; Ovarian Neoplasms/mortality ; Ovarian Neoplasms/pathology ; Ovary/pathology ; Peritoneal Neoplasms/prevention & control ; Peritoneal Neoplasms/secondary ; Prognosis ; Signal Transduction/drug effects ; Trypsin Inhibitor, Kazal Pancreatic/genetics ; Trypsin Inhibitor, Kazal Pancreatic/metabolism ; Xenograft Model Antitumor Assays
    Chemical Substances Antibodies, Monoclonal, Humanized ; IL6 protein, human ; Interleukin-6 ; SPINK1 protein, human ; Trypsin Inhibitor, Kazal Pancreatic (50936-63-5) ; tocilizumab (I031V2H011)
    Language English
    Publishing date 2020-09-14
    Publishing country England
    Document type Journal Article
    ZDB-ID 639046-8
    ISSN 1476-5594 ; 0950-9232
    ISSN (online) 1476-5594
    ISSN 0950-9232
    DOI 10.1038/s41388-020-01451-4
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    Zs.A 2218: Show issues Location:
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  6. Article ; Online: Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

    Cohen, Itay / Naftaly, Si / Ben-Zeev, Efrat / Hockla, Alexandra / Radisky, Evette S / Papo, Niv

    The Biochemical journal

    2018  Volume 475, Issue 7, Page(s) 1335–1352

    Abstract: High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases ... ...

    Abstract High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI
    MeSH term(s) Amyloid beta-Protein Precursor/chemistry ; Amyloid beta-Protein Precursor/genetics ; Amyloid beta-Protein Precursor/metabolism ; Binding, Competitive ; Humans ; Models, Molecular ; Molecular Docking Simulation ; Peptide Library ; Protease Inhibitors/chemistry ; Protease Inhibitors/metabolism ; Substrate Specificity ; Trypsin/genetics ; Trypsin/metabolism
    Chemical Substances APP protein, human ; Amyloid beta-Protein Precursor ; Peptide Library ; Protease Inhibitors ; Trypsin (EC 3.4.21.4)
    Language English
    Publishing date 2018-04-16
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2969-5
    ISSN 1470-8728 ; 0006-2936 ; 0306-3275 ; 0264-6021
    ISSN (online) 1470-8728
    ISSN 0006-2936 ; 0306-3275 ; 0264-6021
    DOI 10.1042/BCJ20180070
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    Uc I Zs.110: Show issues Location:
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  7. Article ; Online: Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries.

    Naftaly, Si / Cohen, Itay / Shahar, Anat / Hockla, Alexandra / Radisky, Evette S / Papo, Niv

    Nature communications

    2018  Volume 9, Issue 1, Page(s) 3935

    Abstract: Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot ... ...

    Abstract Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a novel and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to each of the four human serine proteases (kallikrein-6, mesotrypsin, and anionic and cationic trypsins). We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the four proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein-protein interactions.
    MeSH term(s) Combinatorial Chemistry Techniques ; Protein Interaction Maps ; Serine Proteases/metabolism ; Serine Proteinase Inhibitors/genetics ; Yeasts
    Chemical Substances Serine Proteinase Inhibitors ; Serine Proteases (EC 3.4.-)
    Language English
    Publishing date 2018-09-26
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Validation Studies
    ISSN 2041-1723
    ISSN (online) 2041-1723
    DOI 10.1038/s41467-018-06403-x
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  8. Article ; Online: PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma.

    Ma, Honghai / Hockla, Alexandra / Mehner, Christine / Coban, Matt / Papo, Niv / Radisky, Derek C / Radisky, Evette S

    Scientific reports

    2019  Volume 9, Issue 1, Page(s) 1844

    Abstract: Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated ...

    Abstract Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.
    MeSH term(s) Adenocarcinoma of Lung/metabolism ; Adenocarcinoma of Lung/mortality ; Adenocarcinoma of Lung/pathology ; Carcinogenesis ; Cell Growth Processes ; Cell Line, Tumor ; Cell Movement ; Gene Expression Regulation, Neoplastic ; Humans ; Kallikreins/genetics ; Kallikreins/metabolism ; Lung Neoplasms/metabolism ; Lung Neoplasms/mortality ; Lung Neoplasms/pathology ; Microarray Analysis ; Neoplasm Invasiveness ; Prognosis ; RNA, Small Interfering/genetics ; Trypsin/genetics ; Trypsin/metabolism
    Chemical Substances RNA, Small Interfering ; KLK5 protein, human (EC 3.4.21.-) ; Kallikreins (EC 3.4.21.-) ; PRSS3 protein, human (EC 3.4.21.4) ; Trypsin (EC 3.4.21.4)
    Language English
    Publishing date 2019-02-12
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-018-38362-0
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  9. Article ; Online: Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin.

    Cohen, Itay / Coban, Matt / Shahar, Anat / Sankaran, Banumathi / Hockla, Alexandra / Lacham, Shiran / Caulfield, Thomas R / Radisky, Evette S / Papo, Niv

    The Journal of biological chemistry

    2019  Volume 294, Issue 13, Page(s) 5105–5120

    Abstract: Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop ... ...

    Abstract Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (
    MeSH term(s) Amyloid beta-Protein Precursor/chemistry ; Amyloid beta-Protein Precursor/genetics ; Amyloid beta-Protein Precursor/metabolism ; Animals ; Aprotinin/chemistry ; Aprotinin/genetics ; Aprotinin/metabolism ; Crystallography, X-Ray ; Disulfides/chemistry ; Disulfides/metabolism ; Humans ; Models, Molecular ; Protein Conformation ; Protein Domains ; Protein Engineering ; Proteolysis ; Trypsin/chemistry ; Trypsin/metabolism
    Chemical Substances APP protein, human ; Amyloid beta-Protein Precursor ; Disulfides ; Aprotinin (9087-70-1) ; PRSS3 protein, human (EC 3.4.21.4) ; Trypsin (EC 3.4.21.4)
    Language English
    Publishing date 2019-01-30
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.RA118.007292
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.

    Sananes, Amiram / Cohen, Itay / Shahar, Anat / Hockla, Alexandra / De Vita, Elena / Miller, Aubry K / Radisky, Evette S / Papo, Niv

    The Journal of biological chemistry

    2018  Volume 293, Issue 33, Page(s) 12663–12680

    Abstract: Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity ...

    Abstract Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPI
    MeSH term(s) Amyloid beta-Protein Precursor/pharmacology ; Breast Neoplasms/drug therapy ; Breast Neoplasms/metabolism ; Breast Neoplasms/pathology ; Cell Movement ; Cell Proliferation ; Female ; Genetic Engineering ; High-Throughput Screening Assays ; Humans ; Kallikreins/antagonists & inhibitors ; Kallikreins/chemistry ; Models, Molecular ; Protease Inhibitors/pharmacology ; Protein Binding ; Protein Conformation ; Proteolysis ; Tumor Cells, Cultured
    Chemical Substances APP protein, human ; Amyloid beta-Protein Precursor ; Protease Inhibitors ; KLK6 protein, human (EC 3.4.21.-) ; Kallikreins (EC 3.4.21.-)
    Language English
    Publishing date 2018-06-22
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.RA117.000871
    Database MEDical Literature Analysis and Retrieval System OnLINE

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