@phdthesis{Wu2019,
  author    = {Wu, Fang},
  title     = {Adding new functions to insulin-like growth factor-I (IGF-I) via genetic codon expansion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175330},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Insulin-like growth factor-I (IGF-I) is a 70-amino acid polypeptide with a molecular weight of approximately 7.6 kDa acting as an anabolic effector. It is essential for tissue growth and remodeling. Clinically, it is used for the treatment of growth disorders and has been proposed for various other applications including musculoskeletal diseases. Unlike insulin, IGF-I is complexed to at least six high-affinity binding proteins (IGFBPs) exerting homeostatic effects by modulating IGF-I availability to its receptor (IGF-IR) on most cells in the body as well as changing the distribution of the growth factor within the organism.1-3 Short half-lived IGF-I have been the driving forces for the design of localized IGF-I depot systems or protein modification with enhanced pharmacokinetic properties. In this thesis, we endeavor to present a versatile biologic into which galenical properties were engineered through chemical synthesis, e.g., by site-specific coupling of biomaterials or complex composites to IGF-I. For that, we redesigned the therapeutic via genetic codon expansion resulting in an alkyne introduced IGF-I, thereby becoming a substrate for biorthogonal click chemistries yielding a site-specific decoration. In this approach, an orthogonal pyrrolysine tRNA synthetase (PylRS)/tRNAPyl CUA pair was employed to direct the co-translational incorporation of an unnatural amino acid—¬propargyl-L-lysine (plk)—bearing a clickable alkyne functional handle into IGF-I in response to the amber stop codon (UAG) introduced into the defined position in the gene of interest. We summarized the systematic optimization of upstream and downstream process alike with the ultimate goal to increase the yield of plk modified IGF-I therapeutic, from the construction of gene fusions resulting in (i) Trx-plk-IGF-I fusion variants, (ii) naturally occurring pro-IGF-I protein (IGF-I + Ea peptide) (plk-IGF-I Ea), over the subsequent bacterial cultivation and protein extraction to the final chromatographic purification. The opportunities and hurdles of all of the above strategies were discussed. Evidence was provided that the wild-type IGF-I yields were pure by exploiting the advantages of the pHisTrx expression vector system in concert with a thrombin enzyme with its highly specific proteolytic digestion site and multiple-chromatography steps. The alkyne functionality was successfully introduced into IGF-I by amber codon suppression. The proper folding of plk-IGF-I Ea was assessed by WST-1 proliferation assay and the detection of phosphorylated AKT in MG-63 cell lysate. The purity of plk-IGF-I Ea was monitored with RP-HPLC and SDS-PAGE analysis. This work also showed site-specific coupling an alkyne in plk-IGF-I Ea by copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) with potent activities in vitro. The site-specific immobilization of plk-IGF-I Ea to the model carrier (i.e., agarose beads) resulted in enhanced cell proliferation and adhesion surrounding the IGF-I-presenting particles. Cell proliferation and differentiation were enhanced in the accessibility of IGF-I decorated beads, reflecting the multivalence on cellular performance. Next, we aimed at effectively showing the disease environment by co-delivery of fibroblast growth factor 2 (FGF2) and IGF-I, deploying localized matrix metalloproteinases (MMPs) upregulation as a surrogate marker driving the response of the drug delivery system. For this purpose, we genetically engineered FGF2 variant containing an (S)-2-amino-6-(((2-azidoethoxy)carbonyl)amino)hexanoic acid incorporated at its N-terminus, followed by an MMPs-cleavable linker (PCL) and FGF2 sequence, thereby allowing site-directed, specific decoration of the resultant azide-PCL-FGF2 with the previously mentioned plk-IGF-I Ea to generate defined protein-protein conjugates with a PCL in between. The click reaction between plk-IGF-I Ea and azide-PCL-FGF2 was systematically optimized to increase the yield of IGF-FGF conjugates, including reaction temperature, incubation duration, the addition of anionic detergent, and different ratios of the participating biopharmaceutics. The challenge here was that CuAAC reaction components or conditions might oxidize free cysteines of azide-PCL-FGF2 and future work needs to present the extent of activity retention after conjugation. Furthermore, our study provides potential options for dual-labeling of IGF-I either by the introduction of unnatural amino acids within two distinct positions of the protein of interest for parallel "double-click" labeling of the resultant plk-IGF-I Ea-plk or by using a combination of enzymatic-catalyzed and CuAAC bioorthogonal coupling strategies for sequentially dual-labeling of plk-IGF-I Ea. In conclusion, genetic code expansion in combination with click-chemistry provides the fundament for novel IGF-I analogs allowing unprecedented site specificity for decoration. Considerable progress towards IGF-I based therapies with enhanced pharmacological properties was made by demonstrating the feasibility of the expression of plk incorporated IGF-I using E. coli and retained activity of unconjugated and conjugated IGF-I variant. Dual-labeling of IGF-I provides further insights into the functional requirements of IGF-I. Still, further investigation warrants to develop precise IGF-I therapy through unmatched temporal and spatial regulation of the pleiotropic IGF-I.},
  subject      = {Insulin-like Growth Factor I},
  language  = {en}
}
@phdthesis{Gador2018,
  author    = {Gador, Eva},
  title     = {Strategies to improve the biological performance of protein therapeutics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161798},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {199},
  year      = {2018},
  abstract  = {During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance. Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found. The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects. Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold. The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold. For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use. Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling. We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability. Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields. Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS. The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds. The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3's bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3's bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area. With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds.},
  subject      = {biologics},
  language  = {en}
}
@phdthesis{Braun2018,
  author    = {Braun, Alexandra Carolin},
  title     = {Bioresponsive delivery of anticatabolic and anabolic agents for muscle regeneration using bioinspired strategies},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169047},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Progressive loss of skeletal muscle mass, strength and function poses a major threat to independence and quality of life, particularly in the elderly. To date, sarcopenia therapy consists of resistance exercise training in combination with protein supplementation due to the limited efficacy of available pharmacological options in counteracting the effects of muscle wasting. Therapeutic intervention with growth factors including insulin-like growth factor I (IGF-I) or inhibitors of myostatin  a potent suppressor of myogenesis  hold potential to rebalance the altered activity of anabolic and catabolic cytokines. However, dosing limitations due to acute side effects and disruptions of the homeostasis have so far precluded clinical application. Intending to provide a therapy with a superior safety and efficacy profile by directing drug release to inflamed tissue and minimizing off-target activity, we designed bioresponsive delivery systems for an anti-catabolic peptide and anabolic IGF-I responding to local flares of muscle wasting. In Chapter I, current concepts for bioorthogonal conjugation methods are discussed and evaluated based on various drug delivery applications. With a focus on protein delivery, challenges and potential pitfalls of each chemical and enzymatic conjugation strategy are analyzed and opportunities regarding their use for coupling of biomolecules are given. Based on various studies conjugating proteins to polymers, particles and biomaterials using different site-directed approaches, the chapter summarizes available strategies and highlights certain aspects requiring particular consideration when applied to biomolecules. Finally, a decision process for selection of an optimum conjugation strategy is exemplarily presented. Three of these bioorthogonal coupling reactions are applied in Chapter II detailing the potential of site-directed conjugation in the development of novel, homogenous drug delivery systems. The chapter describes the design of a delivery system of a myostatin inhibitor (MI) for controlled and local release counteracting myositis flares. MI release from the carrier is driven by increased matrix metalloproteinase (MMP) levels in compromised muscle tissues cleaving the interposed linker, thereby releasing the peptide inhibitor from the particulate carrier. Release experiments were performed to assess the response towards various MMP isoforms (MMP-1, -8, -9 and -13) - as upregulated during skeletal muscle myopathies - and the release pattern of the MI in case of disease progression was analyzed. By selection of the protease-sensitive linker (PSL) showing variable susceptibilities to proteases, release rates of the MI can be controlled and adapted. Immobilized MI as well as released MI as response to MMP upregulation was able to antagonize the effects of myostatin on cell signalling and myoblast differentiation. The approach of designing bioresponsive protein delivery systems was also applied to the anabolic growth factor IGF-I, as described in Chapter III. Numerous studies of PEGylated proteins or peptides reveal, that successful therapy is challenged by safety and efficacy issues, as polymer attachment considerably alters the properties of the biologic, thereby jeopardizing clinical efficacy. To this end, a novel promising approach is presented, intending to exploit beneficial effects of PEGylation on pharmacokinetics, but addressing the pharmacodynamic challenges by releasing the protein upon entering the target tissue. This was realized by integration of a PSL between the PEG moiety and the protein. The soluble polymer conjugate was produced by site-directed, enzymatic conjugation of IGF-I to the PSL, followed by attachment of a 30 kDa-PEG using Strain-promoted azide-alkyne cycloaddition (SPAAC). This strategy illustrates the potential of bioorthogonal conjugation (as described in Chapter I) for generation of homogenous protein-polymer conjugates with reproducible outcome, but also emphasizes the altered protein properties resulting from permanent polymer conjugation. As compared to wild type IGF-I, the PEGylated protein showed considerable changes in pharmacologic effects - such as impaired insulin-like growth factor binding protein (IGFBPs) interactions, submaximal proliferative activity and altered endocytosis patterns. In contrast, IGF-I characteristics were fully restored upon local disintegration of the conjugate triggered by MMP upregulation and release of the natural growth factor. For successful formulation development for the proteins and conjugates, the careful selection of suitable excipients is crucial for a safe and reliable therapy. Chapter IV addresses one aspect by highlighting the chemical heterogeneity of excipients and associated differences in performance. Polysorbate 80 (PS80) is a surfactant frequently used in protein formulations to prevent aggregation and surface adsorption. Despite being widely deployed as a standard excipient, heterogeneous composition and performance entails the risk of eliciting degradation and adverse effects on protein stability. Based on a comprehensive study using different batches of various suppliers, the PS80 products were characterized regarding chemical composition and physicochemical properties, facilitating the assessment of excipient performance in a formulation. Noticeable deviations were recorded between different suppliers as well as between batches of the same suppliers. Correlation of all parameters revealed, that functionality related characteristics (FRCs) could be reliably predicted based on chemical composition alone or by a combination of chemical and physicochemical properties, respectively. In summary, this thesis describes and evaluates novel strategies for the targeted delivery and controlled release of biologics intended to counteract the imbalance of anabolic and catabolic proteins observed during aging and musculoskeletal diseases. Two delivery platforms were developed and characterized in vitro - (i) using anti-catabolic peptides immobilized on a carrier for local delivery and (ii) using soluble IGF-I polymer conjugates for systemic application. Both approaches were implemented by bioorthogonal coupling strategies, which were carefully selected in consideration of limitations, side reactions and efficiency aspects. Bioresponsive release of the active biomolecules following increased protease activity could be successfully realized. The therapeutic potential of these approaches was demonstrated using various cell-based potency assays. The systems allow targeted and controlled release of the growth factor IGF-I and anti-catabolic peptides thereby overcoming safety concerns of current growth factor therapy and thus positively impacting the benefit-risk profile of potent therapeutics. Taking potential heterogeneity and by-product concerns into account, comprehensive excipient characterization was performed and a predictive algorithm for FRCs developed, in order to facilitate formulation design and guarantee a safe and efficient therapy from start to finish.},
  subject      = {Muskelatrophie},
  language  = {en}
}
@phdthesis{Gunesch2021,
  author    = {Gunesch, Sandra},
  title     = {Molecular Mode of Action of Flavonoids: From Neuroprotective Hybrids to Molecular Probes for Chemical Proteomics},
  doi       = {10.25972/OPUS-23936},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-239360},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Alzheimer's disease (AD) is the most common form of dementia, and currently, there is no treatment to cure or halt disease progression. Because the one-target strategy focusing on amyloid-β has failed to generate successful pharmaceutical treatment, this work studies natural products with pleiotropic effects focusing on oxidative stress and neuroinflammation as key drivers of disease progression. The central part of this work focused on flavonoids as neuroprotectants. 7-O-Esters of taxifolin and cinnamic or ferulic acid were synthesized and investigated towards their neuroprotective potential addressing aging and disease. 7-O-Feruloyl- and 7-O-cinnamoyltaxifolin showed overadditive effects in oxidative stress-induced assays in the mouse neuronal cell line HT22 and proved to be protective against neuroinflammation in microglial BV-2 cells. The overadditive effect translated to animals using an Aβ25-35-induced memory-impaired AD mouse model where the compounds were able to ameliorate short-term memory defects. While the disease-modifying effects in vivo were observed, the detailed mechanisms of action and intracellular targets of the compounds remained unclear. Hence, a chemical probe of the neuroprotective flavonoid ester 7-O-cinnamoyltaxifolin was developed and applied in an activity-based protein profiling approach. SERCA and ANT-1 were identified as potential targets. Further, chemical modifications on the flavonoids taxifolin, quercetin, and fisetin were performed. The achievements of this work are an important contribution to the use of secondary plant metabolites as neuroprotectants. Chemical modifications increased the neuroprotective effect of the natural products, and distinct intracellular pathways involved in the neuroprotective mechanisms were identified. The results of this work support the use of secondary plant metabolites as potential therapeutics and hint towards new pharmacological targets for the treatment of neurodegenerative disorders.},
  subject      = {Alzheimerkrankheit},
  language  = {en}
}