Design and characterization of EGFR-specific peptides and re-targeted rAAVs for tumor therapy

Feiner R. Design and characterization of EGFR-specific peptides and re-targeted rAAVs for tumor therapy. Bielefeld: Universität Bielefeld; 2019

Size-Dependent Cellular Uptake of RGD Peptides

Kemker I, Feiner R, Müller K, Sewald N. Size-Dependent Cellular Uptake of RGD Peptides. ChemBioChem. 2020;21(4):496-499.Monomeric RGD peptides show unspecific fluid-phase uptake in cells, whereas multimeric RGD peptides are thought to be internalized by integrin-mediated endocytosis. However, a potential correlation between uptake mechanism and molecular mass has been neglected so far. A dual derivatization of peptide c(RGDw(7Br)K) was performed to investigate this. A fluorescent probe was installed by chemoselective Suzuki-Miyaura cross-coupling of the 7-bromotryptophan and a poly(ethylene glycol) (PEG) linker was attached to the lysine residue. Flow cytometry and live cell imaging confirmed unspecific uptake of the small, non-PEGylated peptide, whereas the PEG(5000) peptide conjugate unveiled a selective internalization by M21 cells overexpressing alpha(v)beta(3) and no uptake in alpha(v)-deficient M21L cells

Peptide stapling by late-stage Suzuki-Miyaura cross-coupling

The development of peptide stapling techniques to stabilise alpha-helical secondary structure motifs of peptides led to the design of modulators of protein-protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki-Miyaura cross-coupling of bromotryptophan-containing peptides of the catenin-binding domain of axin. Optimisation of the linker length in order to find a compromise between both sufficient linker rigidity and flexibility resulted in a peptide with an increased alpha-helicity and enhanced binding affinity to its native binding partner beta-catenin. An increased proteolytic stability against proteinase K has been demonstrated

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

Feiner R, Teschner J, Teschner K, et al. rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations. International Journal of Molecular Sciences. 2019;20(22): 5702.Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.</jats:p

The U.S. Inland Creel and Angler Survey Catalog (CreelCat): Development, Applications, and Opportunities

Inland recreational fishing, defined as primarily leisure-driven fishing in freshwaters, is a popular pastime in the USA. State natural resource agencies endeavor to provide high-quality and sustainable fishing opportunities for anglers. Managers often use creel and other angler survey data to inform state- and waterbody-level management efforts. Despite the broad implementation of angler surveys and their importance to fisheries management at state scales, regional and national coordination among these activities is minimal, limiting data applicability for larger-scale management practices and research. Here, we introduce the U.S. Inland Creel and Angler Survey Catalog (CreelCat), a first-of-its-kind, publicly available national database of angler survey data that establishes a baseline of national inland recreational fishing metrics. We highlight research and management applications to help support sustainable inland recreational fishing practices, consider cautions, and make recommendations for implementation

Atmospheric fates of Criegee intermediates in the ozonolysis of isoprene

We use a large laboratory, modeling, and field dataset to investigate the isoprene + O_3 reaction, with the goal of better understanding the fates of the C_1 and C_4 Criegee intermediates in the atmosphere. Although ozonolysis can produce several distinct Criegee intermediates, the C_1 stabilized Criegee (CH_2OO, 61 ± 9%) is the only one observed to react bimolecularly. We suggest that the C_4 Criegees have a low stabilization fraction and propose pathways for their decomposition. Both prompt and non-prompt reactions are important in the production of OH (28% ± 5%) and formaldehyde (81% ± 16%). The yields of unimolecular products (OH, formaldehyde, methacrolein (42 ± 6%) and methyl vinyl ketone (18 ± 6%)) are fairly insensitive to water, i.e., changes in yields in response to water vapor (≤4% absolute) are within the error of the analysis. We propose a comprehensive reaction mechanism that can be incorporated into atmospheric models, which reproduces laboratory data over a wide range of relative humidities. The mechanism proposes that CH_2OO + H_2O (k_((H_2O)) ∼ 1 × 10^(−15) cm^3 molec^(−1) s^(−1)) yields 73% hydroxymethyl hydroperoxide (HMHP), 6% formaldehyde + H_2O_2, and 21% formic acid + H_2O; and CH_2OO + (H_2O)_2 (k_((H_2O)_2) ∼ 1 × 10^(−12) cm^3 molec^(−1) s^(−1)) yields 40% HMHP, 6% formaldehyde + H_2O_2, and 54% formic acid + H_2O. Competitive rate determinations (k_(SO_2/k(H_2O)n=1,2) ∼ 2.2 (±0.3) × 10^4) and field observations suggest that water vapor is a sink for greater than 98% of CH2OO in a Southeastern US forest, even during pollution episodes ([SO_2] ∼ 10 ppb). The importance of the CH_2OO + (H_2O)n reaction is demonstrated by high HMHP mixing ratios observed over the forest canopy. We find that CH_2OO does not substantially affect the lifetime of SO_2 or HCOOH in the Southeast US, e.g., CH_2OO + SO_2 reaction is a minor contribution (<6%) to sulfate formation. Extrapolating, these results imply that sulfate production by stabilized Criegees is likely unimportant in regions dominated by the reactivity of ozone with isoprene. In contrast, hydroperoxide, organic acid, and formaldehyde formation from isoprene ozonolysis in those areas may be significant

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme &beta;-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with &beta;-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology

Recent progress in protein-protein interaction study for EGFR-targeted therapeutics

Feiner R, Müller K. Recent progress in protein-protein interaction study for EGFR-targeted therapeutics. Expert Review of Proteomics. 2016;13(9):817-832.Introduction: Epidermal growth factor receptor (EGFR) expression is upregulated in many tumors and its aberrant signaling drives progression of many cancer types. Consequently, EGFR has become a clinically validated target as extracellular tumor marker for antibodies as well as for tyrosine kinase inhibitors. Within the last years, new mechanistic insights were uncovered and, based on clinical experience as well as progress in protein engineering, novel bio-therapeutic approaches were developed and tested.Areas covered: The potential therapeutic targeting arsenal in the fight against cancer now encompasses bispecific or biparatopic antibodies, DARPins, Adnectins, Affibodies, peptides and combinations of these binding molecules with viral- and nano-particles. We review past and recent binding proteins from the literature and include a brief description of the various targeting approaches. Special attention is given to the binding modes with the EGFR.Expert commentary: Clinical data from the three approved anti EGFR antibodies indicate that there is room for improved therapeutic efficacy. Having choices in size, affinity, avidity and the mode of EGFR binding as well as the possibility to combine various effector functions opens the possibility to rationally design more effective therapeutics

EGF-mCherry Fusion Protein Expressed in E. coli Shows Product Heterogeneity but a High Biological Activity.

Feiner R, Pennè I, Müller B, Müller K. EGF-mCherry Fusion Protein Expressed in E. coli Shows Product Heterogeneity but a High Biological Activity. Biochemistry. 2019;58(8):1043-1047.The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes, and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with a high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure. Biolayer interferometry data of the protein-protein interaction of EGF-mCherry with the soluble EGFR are comparable to that of unmodified EGF. Cell culture experiments demonstrated that this fusion replicates all important features of the natural ligand. Finally, fluorescent assays based on EGF-mCherry provided a simple and convenient method to compare EGFR levels on cells and to determine competition of EGFR-binding molecules. These assays will help to rank competitive properties of EGFR inhibitors