The protein cross‐linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG‐binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper‐like cross‐linking mechanism of self‐assembled substrate proteins by MTG.
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.
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
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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.
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