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1.
Nat Commun ; 11(1): 295, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31941901

ABSTRACT

The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.


Subject(s)
Cystine-Knot Miniproteins/metabolism , Fibronectins/metabolism , Glioblastoma/blood supply , Recombinant Proteins/metabolism , Amino Acid Motifs , Animals , Binding Sites , Cell Line, Tumor , Cystine-Knot Miniproteins/chemistry , Cystine-Knot Miniproteins/genetics , Cystine-Knot Miniproteins/therapeutic use , Fibronectins/genetics , Fluorescent Dyes/chemistry , Fluorescent Dyes/therapeutic use , Glioblastoma/diagnostic imaging , Glioblastoma/metabolism , Humans , Mice , Mice, Nude , Optical Imaging , Peptide Library , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Surface Plasmon Resonance , Xenograft Model Antitumor Assays
2.
J Pept Sci ; 21(8): 651-60, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25964162

ABSTRACT

Cystine-knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine-knot peptide MCoTI-II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine-knot trypsin inhibitor into a CTLA-4 binder by screening a library of variants using yeast surface display. A set of cystine-knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400-fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy.


Subject(s)
CTLA-4 Antigen/metabolism , Cyclotides/genetics , Cyclotides/pharmacology , Avidin/metabolism , CTLA-4 Antigen/chemistry , Cyclotides/chemistry , Humans , Models, Molecular , Peptide Library , Protein Binding , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
3.
Biotechnol J ; 9(4): 545-54, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24497417

ABSTRACT

As membrane proteins play an important role in a variety of life-threatening diseases, the development of therapeutic monoclonal antibodies against membrane proteins is of significant interest. Among many other requirements, the process of antibody drug development requires a set of tailor-made assays for the characterization of the antibodies and for monitoring their activity. Designing assays to characterize antibodies directed to membrane proteins is challenging, because the natural targets are often not available in a format that is compatible with a biochemical assay setup. Thus, alternatives that mimic the targeted membrane proteins are needed. In this study, we developed optimal peptidic mimotopes for the ELISA-based detection of the novel therapeutic antibody IMAB362 in biological samples. Initial hits were identified using phage display and these hits were optimized with the help of structure-activity relationship analysis on peptide microarrays. The optimized peptides showed binding constants in the low nanomolar to picomolar range, an improvement by a factor of up to 30 compared to the initial hits. The best mimotope (apparent KD = 0.15 nM) was successfully used for the ELISA-based quantification of IMAB362 in samples from a mouse pharmacokinetic study. The process described allows the rapid discovery of mimotopes for target proteins that are difficult to produce or handle, which can then be used in pre-clinical and clinical assays or for the purification of biological products.


Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/pharmacokinetics , Peptides/chemistry , Peptides/metabolism , Protein Array Analysis/methods , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Mice, Inbred BALB C , Peptide Library , Protein Binding , Structure-Activity Relationship
4.
J Mol Biol ; 395(1): 167-75, 2010 Jan 08.
Article in English | MEDLINE | ID: mdl-19852971

ABSTRACT

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.


Subject(s)
Cyclotides/chemistry , Cyclotides/pharmacology , Cystine Knot Motifs , Protein Engineering , Tryptases/antagonists & inhibitors , Amino Acid Sequence , Animals , Cattle , Humans , Models, Molecular , Molecular Sequence Data , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Structural Homology, Protein , Tryptases/chemistry
5.
Methods Mol Biol ; 535: 361-72, 2009.
Article in English | MEDLINE | ID: mdl-19377985

ABSTRACT

Microbodies are novel pharmacophoric entities which are derived from naturally occurring cystine-knot microproteins. They provide extremely stable scaffolds that can be engineered to high-affinity binding proteins. A peptide-grafting approach yielded specific ligands for human thrombopoietin receptor (TPO-R). Thrombopoietin (TPO) is the primary regulator of platelet production and acts by dimerization of its cognate receptor. Chemical cross linking of two anti TPO-R Microbodies resulted in highly potent TPO mimetics which are promising candidates for the treatment of TPO deficiencies. The approach demonstrates the high potential of dimeric Microbodies as synthetic receptor agonists.


Subject(s)
Peptides/genetics , Peptides/metabolism , Protein Engineering/methods , Receptors, Thrombopoietin/agonists , Receptors, Thrombopoietin/metabolism , Amino Acid Sequence , Biomimetics , Cell Line , Cell Proliferation , Cysteine/chemistry , Humans , Ligands , Molecular Sequence Data , Peptides/chemistry , Plasmids , Protein Binding , Protein Multimerization
7.
FEBS J ; 274(1): 86-95, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17147697

ABSTRACT

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high-affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin-mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c-Mpl receptor agonists with EC(50) values in the low nanomolar or picomolar range. One selected miniprotein-derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand-induced dimerization.


Subject(s)
Cystine Knot Motifs , Peptides/chemistry , Peptides/pharmacology , Thrombopoietin/agonists , Thrombopoietin/antagonists & inhibitors , Animals , Bone Marrow Cells/metabolism , Cells, Cultured , Dimerization , Humans , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice , Receptors, Thrombopoietin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
8.
J Mol Biol ; 356(1): 1-8, 2006 Feb 10.
Article in English | MEDLINE | ID: mdl-16337652

ABSTRACT

We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.


Subject(s)
Disulfides/chemistry , Momordica/chemistry , Plant Proteins/chemistry , Plant Proteins/metabolism , Ribonucleases/metabolism , Amino Acid Sequence , Bacterial Proteins , Crystallography, X-Ray , Disulfides/metabolism , Histidine/genetics , Histidine/metabolism , Models, Molecular , Molecular Sequence Data , Momordica/genetics , Nuclear Magnetic Resonance, Biomolecular , Plant Proteins/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleases/genetics , Sequence Alignment
9.
Biol Chem ; 386(12): 1301-6, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16336125

ABSTRACT

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.


Subject(s)
Cucurbitaceae/enzymology , Cyclotides/chemical synthesis , Trypsin/metabolism , Alanine/chemistry , Amino Acid Sequence , Arginine/chemistry , Chromogranins/chemistry , Cyclization , Cyclotides/pharmacology , Lysine/chemistry , Molecular Mimicry , Molecular Sequence Data , Protein Conformation
10.
Protein Expr Purif ; 39(1): 82-9, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15596363

ABSTRACT

The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple beta-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.


Subject(s)
Cloning, Molecular/methods , Cystine Knot Motifs , Peptides/genetics , Recombinant Fusion Proteins/genetics , Ribonucleases/genetics , Amino Acid Sequence , Bacterial Proteins , Base Sequence , Cystine/genetics , Cystine/metabolism , Molecular Sequence Data , Peptides/metabolism , Recombinant Fusion Proteins/biosynthesis , Ribonucleases/biosynthesis
11.
Curr Opin Biotechnol ; 15(4): 323-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15296929

ABSTRACT

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.


Subject(s)
Bacteria/enzymology , Enzymes/analysis , Enzymes/metabolism , Flow Cytometry/methods , Fluorescence Resonance Energy Transfer/methods , Biological Assay/methods , Catalysis , Enzyme Activation , Membrane Proteins/analysis , Membrane Proteins/metabolism , Peptide Library , Protein Engineering/methods , Protein Interaction Mapping/methods
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