Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes' plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency.

Investigating the Role of Sulfate Groups for the Binding of Gd3+ Ions to Glycosaminoglycans with NMR Relaxometry.

Glycosaminoglycans (GAGs) are highly negatively charged macromolecules with a large cation binding capacity, but their interaction potential with exogeneous Gd3+ ions is under-investigated. These might be released from chelates used as Gadolinium-based contrast agents (GBCAs) for clinical MR imaging due to transmetallation with endogenous cations like Zn2+ . Recent studies have quantified how an endogenous GAG sequesters released Gd3+ ions and impacts the thermodynamic and kinetic stability of some GBCAs. In this study, we investigate and compare the chelation ability of two important GAGs (heparin and chondroitin sulfate), as well as the homopolysaccharides dextran and dextran sulfate that are used as models for alternative macromolecular chelators. Our combined approach of MRI-based relaxometry and isothermal titration calorimetry shows that the chelation process of Gd3+ into GAGs is not just a long-range electrostatic interaction as proposed for the Manning model, but presumably a site-specific binding. Furthermore, our results highlight the crucial role of sulfate groups in this process and indicate that the potential of a specific GAG to engage in this mechanism increases with its degree of sulfation. The transchelation of Gd3+ ions from GBCAs to sulfated GAGs should thus be considered as one possible explanation for the observed long-term deposition of Gd3+ in vivo and related observations of long-term signal enhancements on T1 -weighted MR images.

Aptamer BC 007's Affinity to Specific and Less-Specific Anti-SARS-CoV-2 Neutralizing Antibodies.

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0-21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.

Apolipoprotein E peptide-modified colloidal carriers: the design determines the mechanism of uptake in vascular endothelial cells.

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.

Functionalized Lipopeptide Micelles as Highly Efficient NMR Depolarization Seed Points for Targeted Cell Labelling in Xenon MRI.

Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16 000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics.

Aptamer BC 007 - Efficient binder of spreading-crucial SARS-CoV-2 proteins.

Corona virus disease 2019 (COVID-19) is a respiratory disease caused by a new coronavirus (SARS-CoV-2) which causes significant morbidity and mortality. The emergence of this novel and highly pathogenic SARS-CoV-2 and its rapid international spread poses a serious global public health emergency. To date 32,174,627 cases, of which 962,613 (2.99%) have died, have been reported (https://www.who.int/westernpacific/health-topics/coronavirus, accessed 23 Sep 2020). The outbreak was declared a Public Health Emergency of International Concern on 30 January 2020. There are still not many SARS-CoV-2-specific and effective treatments or vaccines available. A second round of infection is obviously unavoidable. Aptamers had already been at the centre of interest in the fight against viruses before now. The selection and development of a new aptamer is, however, a time-consuming process. We therefore checked whether a clinically developed aptamer, BC 007, which is currently in phase 2 of clinical testing for a different indication, would also be able to efficiently bind DNA-susceptible peptide structures from SARS-CoV-2-spreading crucial proteins, such as the receptor binding domain (RBD) of the spike protein and the RNA dependent RNA polymerase of SARS-CoV-2 (re-purposing). Indeed, several such sequence-sections have been identified. In particular for two of these sequences, BC 007 showed specific binding in a therapy-relevant concentration range, as shown in Nuclear magnetic resonance (NMR)- and Circular dicroism (CD)-spectroscopy and isothermal titration calorimetry (ITC). The excellent clinical toxicity and tolerability profile of this substance opens up an opportunity for rapid clinical testing of its COVID-19 effectiveness.

Bacterial Aggregation Triggered by Fibril Forming Tryptophan-Rich Sequences: Effects of Peptide Side Chain and Membrane Phospholipids.

The influence of side chain residue and phospholipid characteristics of the cytoplasmic membrane upon the fibrillation and bacterial aggregation of arginine (Arg) and tryptophan (Trp) rich antimicrobial peptides (AMPs) has not been well described to date. Here, we utilized the structural advantages of HHC-10 and 4HarHHC-10 (Har, l-homoarginine) that are highly active Trp-rich AMPs and investigated their fibril formation and activity behavior against bacteria. The peptides revealed time-dependent self-assembly of polyproline II (PPII) α-helices, but by comparison, 4HarHHC-10 tended to form higher ordered fibrils due to relatively strong cation-π stacking of Trp with Har residue. Both peptides rapidly killed S. aureus and E. coli at their MICs and caused aggregation of bacteria at higher concentrations. This bacterial aggregation was accompanied by the formation of morphologically distinct electron-dense nanostructures, likely including but not limited to peptides alone. Both HHC-10-derived peptides caused blebs and buds in the E. coli membrane that are rich in POPE phospholipid that promotes negative curvature. However, the main population of S. aureus cells retained their cocci structure upon treatment with HHC peptides even at concentration higher than the MICs. In contrast, the cell aggregation was not induced by HHC fibrils that were most likely stabilized through intra-/intermolecular cation-π stacking. It is proposed that masking of these interactions might have resulted in diminished membrane association/insertion of the HHC nanostructures. The peptides caused aggregation of POPC/POPG (1/3) and POPE/POPG (3/1) liposomes. Nonetheless, disaggregation of the former vesicles was observed at ratios of lipid to peptide of greater than 6 and 24 for HHC-10 and 4HarHHC-10, respectively. Collectively, our results revealed dose-dependent bacterial aggregation mediated by Trp-rich AMPs that was profoundly influenced by the degree of peptide's self-association and the composition and intrinsic curvature of the cytoplasmic membrane lipids.

Insight into the role of HSPG in the cellular uptake of apolipoprotein E-derived peptide micelles and liposomes.

Liposomes and micellar carriers equipped with targeting and cellular uptake mediating peptides have attracted attention for numerous applications. The optimization of the carrier requires an understanding of how their properties influence target cell recognition and uptake. We developed a dipalmitoylated apolipoprotein E-derived peptide, named P2A2 as promising vector to mediate cellular uptake of potential micellar and liposomal carriers. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) were used to get insight into the internalization mediated by carboxyfluoresceine-labeled P2fA2 and the all-D amino acid analogue P2fa2 into brain capillary endothelial cells. Both peptide micelles and liposomes entered cells via endocytosis. Cell surface heparan sulfate proteoglycans (HSPGs) were involved in the internalization process of peptide-bearing liposomes characterized by a diameter of 100 nm, a low surface density of 100 peptide molecules per vesicle and a helical conformation of the vector. In contrast, peptide micelles characterized by a diameter of about 10 nm, a high peptide density caused by 19 associated molecules and a high conformational flexibility of the vector sequence did not address HSPG. Unspecific interactions between the carriers and membrane constituents predominate the two uptake processes but stereospecific components seem to be involved. Both routes differ with respect to transport efficiency. The results provide a prospective basis to optimize liposomes and micelles as drug delivery systems.

Characterization of Aptamer BC 007 Substance and Product Using Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy.

Possible unwanted folding of biopharmaceuticals during manufacturing and storage has resulted in analysis schemes compared to small molecules that include bioanalytical characterization besides chemical characterization. Whether bioanalytical characterization is required for nucleotide-based drugs, may be decided on a case-by-case basis. Nucleotide-based pharmaceuticals, if chemically synthesized, occupy an intermediate position between small-molecule drugs and biologics. Here, we tested whether a physicochemical characterization of a nucleotide-based drug substance, BC 007, was adequate, using circular dichroism (CD) spectroscopy. Nuclear magnetic resonance confirmed CD data in one experimental setup. BC 007 forms a quadruplex structure under specific external conditions, which was characterized for its stability and structural appearance also after denaturation using CD and nuclear magnetic resonance. The amount of the free energy (ΔG0) involved in quadruplex formation of BC 007 was estimated at +8.7 kJ/mol when dissolved in water and +1.4 kJ/mol in 154 mM NaCl, indicating structural instability under these conditions. However, dissolution of the substance in 5 mM of KCl reduced the ΔG0 to -5.6 kJ/mol due to the stabilizing effect of cations. These results show that positive ΔG0 of quadruplex structure formation in water and aqueous NaCl prevents BC 007 from preforming stable 3-dimensional structures, which could potentially affect drug function.

Dipalmitoylation of a cellular uptake-mediating apolipoprotein E-derived peptide as a promising modification for stable anchorage in liposomal drug carriers.

Liposomes equipped with cellular uptake-mediating peptidic vector compounds have attracted much attention as target-specific drug delivery systems. Aside from the development of the target recognition motif itself, vector coupling to liposomes while conserving the active conformation constitutes an important element in carrier development. To elucidate the most efficient way for adsorptive peptide binding to liposomes, we synthesized and characterized two-domain peptides comprising a cationic sequence derived from the binding domain of apolipoprotein E (apoE) for the low-density lipoprotein receptor and different lipid-binding motifs, that is, an amphipathic helix, a transmembrane helix, single fatty acids or two palmitoyl chains. Peptide properties considered relevant for peptide-liposome complexes to initiate an endocytotic cellular uptake such as lipid binding, helicity, stability of anchorage, bilayer-disturbing activity, and toxicity showed that the dipalmitoyl derivative was the most suitable to associate the apoE peptide to the surface of liposomes. The peptide showed pronounced lipid affinity and was stably anchored within the lipid bilayer on a time scale of at least 30 min. The helicity of about 40% in the lipid-bound state and the location of the amphipathic helix on the liposomal surface provided the prerequisites for interaction of the complex with the cell surface-located receptor. The concentration of the dipalmitoylated peptide to permeabilize neutral lipid bilayers (lipid concentration 25 microM) was 0.06 microM and a 2 microM concentration reduced cell viability to about 80%. Efficient internalization of liposomes bearing about 180 peptide derivatives on the surface into brain capillary endothelial cells was monitored by confocal laser scanning microscopy. The concept of complexation using dipalmitoylated peptides may offer an efficient substitute to covalent vector coupling and a prospective way to optimize the capacity of liposomes as drug delivery systems also for different targets.

Lipopeptide-based micellar and liposomal carriers: Influence of surface charge and particle size on cellular uptake into blood brain barrier cells.

Lipopeptide-based micelles and liposomes were found to differ in cell recognition and uptake mode into blood brain barrier (BBB) endothelial cells. Here we analyse the role of size and surface charge of micelles and liposomes composed of different lipopeptide sequences with respect to uptake into human brain capillary (HBMEC) and aortic (HAoEC) endothelial cells. Comparable to the dipalmitoylated apolipoprotein E-derived P2A2, lipopeptides of cationic poly-arginine (P2Rn), poly-lysine (P2Kn) and an anionic glutamic-acid sequence (P2En) self assemble into micelles (12-14nm in diameter) with high surface charge density, and bind to small (SUVs, about 24nm in diameter) and large (LUV, about 100nm in diameter) liposomes at variable lipid to peptide ratios. The interaction pattern of the resulting particles with endothelial cells is highly variable as revealed by confocal laser scanning microscopic (CLSM) and fluorescence assisted cell sorting (FACS) studies. Micelles and SUVs with high P2A2 density are efficiently and selectively internalized into HBMEC. P2Kn micelles strongly accumulate in both the cytosol and at the cell membrane, while the interaction of liposomes tagged with a low amount of P2A2 and P2Kn with the cells was reduced. Anionic micelles seem to dissociate in the presence of cells and P2En molecules incorporate into the cellular membrane whereas the negatively charged liposomes hardly interact with cells. Surprisingly, all poly-R-based particles show high selectivity for HBMEC compared to HAoEC, independent of particle size and peptide surface density. The P2Rn-mediated internalization is highly efficient and partially clathrin-dependent. The oligo-R lipopeptide is considered to be most promising to selectively transport different drug carriers into the blood brain barrier.

Evidence for a novel mechanism of antimicrobial action of a cyclic R-,W-rich hexapeptide.

The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research.

What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides.

Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Cyclization increases the antimicrobial activity and selectivity of arginine- and tryptophan-containing hexapeptides.

Arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with various secondary structures. We synthesized a set of linear hexapeptides derived from the sequence AcRRWWRF-NH(2) by substitution of tryptophan (W) by tyrosine (Y) or naphthylalanine (Nal) and by replacement of arginine (R) by lysine (K) to investigate the role of cationic charge and aromatic residues in membrane activity and selectivity. A second set of corresponding head-to-tail cyclic analogues was prepared to analyze the role of conformational constraints. The biological activity of the linear peptides followed the order Nal- >> W- > Y-containing compounds and slightly decreased upon R-K substitution. A pronounced activity-improving and bacterial selectivity-enhancing effect was found upon cyclization of the R- and W-bearing parent peptide, whereas the activity-modifying effect of cyclization of Y- and Nal-containing peptides was low. The analysis of the driving forces of peptide interaction with model membranes showed that the activities correlated with the partition coefficients and the depths of peptide insertion into neutral and negatively charged lipid bilayers. Spectroscopic studies, RP-HPLC, and titration calorimetry implied that the combination of cationic and aromatic amino acid composition and conformational rigidity afforded a membrane-active, amphipathic structure with a highly charged face opposed by a cluster of aromatic side chains. However, threshold values of low and high hydrophobicity seemed to exist beyond which the activity-enhancing effect of cyclization was negligible. The results suggest that cyclization of small peptides of an appropriate amino acid composition may serve as a promising strategy in the design of antimicrobial peptides.