The design, synthesis and evaluation of Nrf2-Keap1 PPI inhibitors: a modular, virtual screening-led approach

Nrf2 (nuclear factor erythroid 2-related factor 2) is a cap’n’collar bZIP transcription factor and is the main activator of the transcription of over 100 genes that play roles in responses to oxidative stress and detoxifying xenobiotics. The main control of Nrf2 levels is exercised by Keap1 (Kelchlike ECH-associated protein 1) which facilitates the ubiquitination of Nrf2 and therefore its degradation. Keap1 is oxidation-sensitive and upon exposure to oxidants, it changes its conformation and binds Nrf2 tightly. Consequently, de novo-synthesised Nrf2 can accumulate. Following its discovery, Nrf2 received most attention in relation to cancer. Over the time, however, its implication in other pathologies have been more and more acknowledged, namely in inflammation and most importantly in neurodegenerative diseases. Especially Parkinson’s disease (PD), which is the second most common neurodegenerative disease, caused by the progressive loss of dopaminergic neurons in the substantia nigra, has been linked to oxidative stress. The role Nrf2 plays has been demonstrated in animal models of α-synuclein aggregation or chemically induced parkinsonism, where an increase in Nrf2 expression provided neuroprotection and a slowing of disease progression. Therefore, the inhibition of Keap1- mediated Nrf2 degradation presents a promising strategy for the mechanistic study and the therapy of PD. Several structures showing high potency towards Keap1 inhibition have been described, with activities in the nanomolar range. However, these compounds are large, or hydrophilic and charged. In order to develop new scaffolds, extensive virtual screening assays have been conducted which resulted in hits with promising molecular scaffolds. At the same time, chemical modifications on a known triazole structure have been performed in order to elucidate structure-activity relationships. In this thesis, the molecular modelling lead, as well the synthetic approach to both project components are described. Finally, the results of a competitive fluorescence polarisation (FP) assay for the second set of compounds are presented

Development of Novel Treatment Strategies in Complement-mediated Thromboinflammatory and Autoimmune Diseases: Tethering of Host Immune Modulators by Modified Peptide Conjugates

Host-defense pathways are essential to protect the organism against pathogens and maintain tissue homeostasis. The complement system is an integral part of innate immunity and recognizes a broad set of danger signals, upon which it initiates a swift and powerful effector response, including immune cell activation, enhanced phagocytosis and direct tissue damage. Furthermore, complement activation provides a platform for cross-talk to other host-defense pathways such as adaptive immunity and coagulation. While complement activation is beneficial under most circumstances, undesired activation on surfaces recognized as non-self (e.g. biomaterials and transplants) or in autoimmune diseases can have deleterious effects. The complement system is tightly controlled under physiologic conditions due to the broad and severe reactions it may entrain on host cells. One major regulator of complement activation is the plasma protein factor H (FH), which inhibits the central amplification loop where all three complement initiation pathways converge. The recruitment of FH to foreign surfaces is not only employed by pathogens as evasion strategy but is also considered an attractive therapeutic option. We could previously show that the 14 amino acid-long cyclic peptide 5C6 binds potently to FH and can, when conjugated to appropriate surface tethers, inhibit complement activation on biomedically relevant surfaces. Although initial proof-of-concept studies of 5C6 have been conducted previously, little was known about the interaction determinants within FH and 5C6 nor about the peptide’s specificity, stability or activity in clinically relevant models. We addressed all these questions here and could demonstrate that 5C6 is highly selective for FH and binds murine and monkey FH in addition to the human regulator, thereby facilitating future translational studies. Additionally, we could demonstrate that 5C6’s minimal binding region in FH consist of domains 10-14, suggesting a conformational binding epitope, and that 5C6 binding to FH is highly selective. By performing in-depth structure activity relationship studies on 5C6, we not only elucidated activity determinants but also identified a next-generation 5C6 analog with improved affinity, activity and stability. In addition to evaluating 5C6 in models representing complement activation by nanoparticles and liposomal drug formulations, we also explored other peptide-based strategies to interfere in complement-related immune disorders. In the autoimmune disease IgA nephropathy (IgAN), for example, immune complexes can activate the complement system. Although the role of complement in IgAN has not been fully elucidated, the presence of the immune complexes is thought to drive the condition. The removal of autoantibodies causal to the immune complexes from circulation is therefore considered a promising therapeutic approach. One strategy to achieve this goal is to sequester the autoantibodies by immobilizing the respective epitope, an erroneously glycosylated polypeptide stretch in IgA1, to a polymer. We developed an improved synthesis protocol for producing the challenging glycopeptide epitope, a 20-mer peptide containing five glycosylated residues, at sufficient quantities to enable proof-of-concept studies. Our finding that the polymer-immobilized synthetic epitope is able to bind patient-derived autoantibodies serves as important validation of this approach and may pave the way for novel therapeutic strategies in IgAN. Finally, based on the critical role of complement-coagulation cross-talk in many thromboinflammatory conditions, we investigated whether the coagulation cascade regulator FXIII-B, which is structurally similar to FH, also exerts complement-regulatory functions in analogy to FH. We could demonstrate that neither the ligand binding nor functional profile of FXIII-B corresponds to that of FH, thereby untangling a part of the complex interaction network between different host-defense pathways. The described projects underline the potential impact that peptidic modalities may have in the treatment of complement-mediated diseases. Future optimization and translational efforts will reveal their full potential and, hopefully, bring these promising approaches closer to the bedside