Novel amino-Li resin for water-based solid-phase peptide synthesis.

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

Toward Fabrication of Bioactive Papers: Covalent Immobilization of Peptides and Proteins.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

Multivalent dextran hybrids for efficient cytosolic delivery of biomolecular cargoes.

The development of novel biotherapeutics based on peptides and proteins is often limited to extracellular targets, because these molecules are not able to reach the cytosol. In recent years, several approaches were proposed to overcome this limitation. A plethora of cell-penetrating peptides (CPPs) was developed for cytoplasmic delivery of cell-impermeable cargo molecules. For many CPPs, multimerization or multicopy arrangement on a scaffold resulted in improved delivery but also higher cytotoxicity. Recently, we introduced dextran as multivalent, hydrophilic polysaccharide scaffold for multimerization of cell-targeting cargoes. Here, we investigated covalent conjugation of a CPP to dextran in multiple copies and assessed the ability of resulted molecular hybrid to enter the cytoplasm of mammalian cells without largely compromising cell viability. As a CPP, we used a novel, low-toxic cationic amphiphilic peptide L17E derived from M-lycotoxin. Here, we show that cell-penetrating properties of L17E are retained upon multivalent covalent linkage to dextran. Dextran-L17E efficiently mediated cytoplasmic translocation of an attached functional peptide and a peptide nucleic acid (PNA). Moreover, a synthetic route was established to mask the lysine side chains of L17E with a photolabile protecting group thus opening avenues for light-triggered activation of cellular uptake.

Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are hybrid molecules intended to overcome the drawbacks of conventional small molecule chemotherapy and therapeutic antibodies by merging beneficial characteristics of both molecule classes to develop more efficient and patient-friendly options for cancer treatment. During the last decades a versatile bioconjugation toolbox that comprises numerous chemical and enzymatic technologies have been developed to covalently attach a cytotoxic cargo to a tumor-targeting antibody. Microbial transglutaminase (mTG) that catalyzes isopeptide bond formation between proteinaceous or peptidic glutamines and lysines, provides many favorable properties that are beneficial for the manufacturing of these conjugates. However, to efficiently utilize the enzyme for the constructions of ADCs, different drawbacks had to be overcome that originate from the enzyme's insufficiently understood substrate specificity. Within this review, pioneering methodologies, recent achievements and remaining limitations of mTG-assisted assembly of ADCs will be highlighted.

Sustainable Peptide Synthesis Enabled by a Transient Protecting Group.

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.

Efficient Site-Specific Antibody-Drug Conjugation by Engineering a Nature-Derived Recognition Tag for Microbial Transglutaminase.

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.

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering.

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.

Microbial transglutaminase for biotechnological and biomedical engineering.

Research on bacterial transglutaminase dates back to 1989, when the enzyme has been isolated from Streptomyces mobaraensis. Initially discovered during an extensive screening campaign to reduce costs in food manufacturing, it quickly appeared as a robust and versatile tool for biotechnological and pharmaceutical applications due to its excellent activity and simple handling. While pioneering attempts to make use of its extraordinary cross-linking ability resulted in heterogeneous polymers, currently it is applied to site-specifically ligate diverse biomolecules yielding precisely modified hybrid constructs comprising two or more components. This review covers the extensive and rapidly growing field of microbial transglutaminase-mediated bioconjugation with the focus on pharmaceutical research. In addition, engineering of the enzyme by directed evolution and rational design is highlighted. Moreover, cumbersome drawbacks of this technique mainly caused by the enzyme's substrate indiscrimination are discussed as well as the ways to bypass these limitations.

Light-Controlled Chemoenzymatic Immobilization of Proteins towards Engineering of Bioactive Papers.

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.

Ultrafast Single-Scan 2D†NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

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.

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

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.

Synthetic Cystine-Knot Miniproteins - Valuable Scaffolds for Polypeptide Engineering.

Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.

An Apoptosis-Inducing Peptidic Heptad That Efficiently Clusters Death Receptor†5.

Multivalent ligands of death receptors hold particular promise as tumor cell-specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptor 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP-derived peptides in different spatial orientations using a set of enzyme-promoted conjugations or recombinant production. Heptameric constructs based on a short (60-75 residues) scaffold of a C-terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC50=3 nm) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter-ligand distance, relative spatial orientation and copy number of receptor-binding modules are key prerequisites for receptor activation and cell killing.

Nanoscale Biodegradable Organic-Inorganic Hybrids for Efficient Cell Penetration and Drug Delivery.

We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the in vivo delivery of therapeutics.

Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors.

We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI-1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin-like serine protease inhibitor into a subtilisin-like one by substitutions in the canonical and, particularly, in the substrate-binding loop. Although the substrate sequence for furin is Arg-X-Arg/Lys-Arg↓, the most potent inhibitor had a lysine at position P1. C-terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by molecular modeling. With an inhibition constant of 0.49 nm, the engineered peptide H-KRCKKSIPPICF-NH2 is a promising compound for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.

Cystine-knot peptides targeting cancer-relevant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

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.

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation.

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Potent inhibitors of human matriptase-1 based on the scaffold of sunflower trypsin inhibitor.

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.

Effective PHIP labeling of bioactive peptides boosts the intensity of the NMR signal.

A series of novel bioactive derivatives of the sunflower trypsin inhibitor-1 (SFTI-1) suitable for hyperpolarization by parahydrogen-induced polarization (PHIP) was developed. The PHIP activity was achieved by labeling with L-propargylglycine, O-propargyl-L-tyrosine, or 4-pentynoic acid. (1) H NMR signal enhancements (SE) of up to a factor of 70 were achieved in aqueous solution. We found that an isolated spatial location of the triple bond within the respective label and its accessibility for the hydrogenation catalyst are essential factors for the degree of signal enhancement.