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Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site-specific conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG-catalyzed bioconjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPIP ). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2-targeting antibody trastuzumab variants. Moreover, an antibody-drug conjugate assembled from trastuzumab that was C-terminally tagged with the novel recognition sequence revealed a higher payload-antibody ratio than the reference antibody.

Functional papers are the subject of extensive research efforts and have already become an irreplaceable part of our modern society. Among other issues, they enable fast and inexpensive detection of a plethora of analytes and simplify laboratory work, for example in medical tests. This article focuses on the molecular and structural fundamentals of paper and the possibilities of functionalization, commercially available assays and their production, as well as on current and future challenges in research in this field. Graphic abstract

Antibody‐drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody‐drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug‐to‐antibody ratios, remarkable hydrophilicity, and high toxicity in vitro.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point-of-care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo-triggered cycloaddition followed by site-specific sortase A-catalyzed transamidation. This site-directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper-based diagnostics.

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.

The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid‐phase peptide synthesis strategy that is based on a water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real‐time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non‐natural Smoc‐protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions., Peptide synthesis goes green: A water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group has been developed that enables solid‐phase peptide synthesis under aqueous conditions as well as efficient postsynthetic purification of the peptides. This protecting group is fluorescent both when attached to an Nα atom and when cleaved and so also allows real‐time monitoring of building block coupling.

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC50 =2-20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50 =2-7 nm), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

Wir prasentieren eine umfassende Studie uber neuartige, hocheffiziente und bioabbaubare molekulare Transporter. Hierzu entwarfen wir verschiedene kubisch-oktamere Silsesquioxane (“cube-octameric silsesquioxane”, COSS) und untersuchten deren Zellaufnahme mit Konfokalmikroskopie und Duchflusszytometrie. Fur ein mit kompakt angeordneten Guanidinogruppen funktionalisiertes COSS wurde bereits bei nanomolaren Konzentrationen eine schnelle Aufnahme in lebende HeLa-Zellen beobachtet. Diese effiziente Aufnahme wurde ebenfalls in Bakterien, Hefen und Archaeen festgestellt. Der COSS-basierte Transporter war deutlich effizienter als zellpenetrierende Peptide und zeigte geringe Toxizitat. Mit seiner Hilfe wurde der kovalent gekuppelte zytotoxische Wirkstoff Doxorubicin in lebende Tumorzellen transportiert. Da die Zellaufnahme eines fluoreszenzmarkierten Transporters in Gegenwart von Serum erhalten blieb, konnte das System auch als Wirkstofftransporter fur In-vivo-Anwendungen interessant sein.

Basierend auf der Kristallstruktur eines naturlichen Proteinsubstrats von mikrobieller Transglutaminase, einem Enzym, das die Vernetzung von Proteinen katalysiert, wurde eine Erkennungssequenz fur ortsspezifische Konjugation rational entworfen. Dieses Strukturmimetikum einer nativen Konjugationsstelle wurde konformativ uber eine intramolekulare Disulfidbrucke fixiert und ermoglichte dadurch die effiziente Konjugation eines Reportermolekuls an den therapeutischen monoklonalen Antikorper Cetuximab, ohne dessen Bindungseigenschaften zu beeintrachtigen.

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.

A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.

Neuartige, bioaktive Derivate des Sunflower Trypsin Inhibitors-1 (SFTI-1), die fur die Hyperpolarisation mittels Parawasserstoff-induzierter Kernspinpolarisation (PHIP) geeignet sind, wurden entwickelt. Die PHIP-Aktivitat dieser Derivate wurde durch den Einbau der Marker L-Propargylglycin, O-Propargyl-L-tyrosin oder 4-Pentinsaure erreicht. In wassrigen Losungen konnten 1H-NMR-Signalverstarkungen um bis zu 70 erzielt werden. Eine isolierte raumliche Lage der Dreifachbindung und die Zuganglichkeit des Markers fur den Katalysator sind essenziell fur den Grad der erreichbaren Signalverstarkung.

Die Immobilisierung von bioaktiven Molek len auf Nanocellulose liefert Konjugate, die die biologischen Eigenschaften der gekuppelten Verbindungen mit Vorteilen der Cellulose wie Biokompatibilit t, geringe Zytotoxizit t und Nanodimension vereinen. Nachteile der klassischen Methoden zur Proteinkonjugation sind h ufig ein Mangel an Selektivit t, lange Reaktionszeiten, nichtphysiologische pH-Bereiche oder der Einsatz organischer Lcsungsmittel sowie die Notwendigkeit, eine masgeschneiderte Synthesestrategie f r jeden Einzelfall zu entwickeln. Diese Einschr nkungen umgeht das hier vorgestellte generische Zweistufenverfahren, das die Vorteile einer hoch effizienten Oximligation mit enzymatischer Proteinkonjugation auf der Oberfl che peptidmodifizierter kristalliner Nanocellulose kombiniert. Es basiert auf effizienten und orthogonalen Transformationen, erfordert keine organischen Lcsungsmittel und akzeptiert physiologische Bedingungen. Durch die Ortsund Regiospezifit t kcnnte es f r die Immobilisierung einer Vielzahl funktioneller Proteine einge

Sunflower trypsin inhibitor-1 (SFTI-1), a bicyclic tetradecapeptide, has become a versatile tool as a scaffold for the development of the inhibitors of therapeutically relevant serine proteases, among them matriptase and kallikreins. Herein, we report the rational design of potent monocyclic and bicyclic inhibitors of human matriptase-1. We found that the presence of positive charge and lack of bulky residues at the peptide N-terminus is required for the maintenance of inhibitory activity. Replacement of the N-terminal glycine residue by lysine allowed for the chemical conjugation with a fluorophor via the e-amino group without significant loss of inhibitory activity. Head-to-tail and side-chain-to-tail cyclization resulted in potent inhibitors with comparable activities against matriptase-1. The most potent synthetic bicyclic inhibitor found in this study (Ki=2.6nM at pH 7.6) is a truncated version of SFTI-1 (cyclo-KRCTKSIPPRCH) lacking a C-terminal proline and aspartate residue. It combines an internal disulfide bond with a peptide macrocycle that is formed through side-chain-to-tail cyclization of the e-amino group of an N-terminal lysine and a C-terminal proline. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site.

The architecture of protein macromolecules fundamentallydepends on the sequential arrangement of peptide backbonebonds in defined conformations. Among the three torsionangles(f,y,andw)presentateachaminoacid,itistheamidebond (w) which is intrinsically hindered as a result of itspartial double-bond character and it is thus more or lessrestricted to either a trans or a cis conformation (Figure 1).

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.

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.

Sunflower trypsin inhibitor-1 (SFTI-1), a bicyclic tetradecapeptide, has become a versatile tool as a scaffold for the development of the inhibitors of therapeutically relevant serine proteases, among them matriptase and kallikreins. Herein, we report the rational design of potent monocyclic and bicyclic inhibitors of human matriptase-1. We found that the presence of positive charge and lack of bulky residues at the peptide N-terminus is required for the maintenance of inhibitory activity. Replacement of the N-terminal glycine residue by lysine allowed for the chemical conjugation with a fluorophor via the ε-amino group without significant loss of inhibitory activity. Head-to-tail and side-chain-to-tail cyclization resulted in potent inhibitors with comparable activities against matriptase-1. The most potent synthetic bicyclic inhibitor found in this study (Ki = 2.6 nM at pH 7.6) is a truncated version of SFTI-1 (cyclo-KRCTKSIPPRCH) lacking a C-terminal proline and aspartate residue. It combines an internal disulfide bond with a peptide macrocycle that is formed through side-chain-to-tail cyclization of the ε-amino group of an N-terminal lysine and a C-terminal proline.

The unsaturated side chain of l-propargylglycine (Pra) was used to study parahydrogen-induced polarization (PHIP) in synthetic oligopeptides. For the first time PHIP-induced NMR signal enhancement was demonstrated using model peptides bearing various functional side chains.

Here we describe the facile generation of tetravalent peptide conjugates via a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) using a cyclic peptide template as a versatile conjugation scaffold. This stable and rigid framework is a conformationally constrained cyclic beta-sheet decorated with spatially defined alkyne moieties that serve as selectively addressable coupling sites. The proposed method allows for the effective coupling of unprotected peptide monomers in water at room temperature within comparatively short reaction times. The resulting conjugates display the ligands in an oriented manner, thus allowing for multivalent interactions with given target molecules, which may contribute to enhanced affinity and specificity. In addition, the selected scaffold offers an orthogonal coupling site for the incorporation of fluorescent labels or radioligands.

Background Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack. Results In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding. Conclusion In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.