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Anna Spanopoulou Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Fachgebiet für Peptidbiochemie Synthesis and study of conformationally constrained peptides as inhibitors of amyloid self-assembly Anna Spanopoulou Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan, für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr.rer.nat.) genehmigten Dissertation. Vorsitzende: Prof. Dr. Iris Antes Prüfer der Dissertation: 1. Prof. Dr. Aphrodite Kapurniotu 2. Prof. Dr. Dr.h.c. Horst Kessler Die Dissertation wurde am 18.01.2019 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am.07.06.2019 angenommen. Abstract Abstract Protein aggregation is linked to many cell degenerative diseases such as Alzheimer’s disease (AD) and type 2 diabetes (T2D). AD is associated with the self-assembly of the 40-42 residue β-amyloid peptide (Aβ) whereas T2D is linked to the aggregation of the 37-residue long islet amyloid polypeptide (IAPP). Both polypeptides Aβ and IAPP share in addition to their amyloidogenicity 50% of sequence similarity. It has been previously shown that non-fibrillar and non-toxic Aβ and IAPP species bind to each other with low nanomolar affinity and that Aβ-IAPP hetero-association suppresses cytotoxic self-association and amyloidogenesis by both peptides. In addition, clinical and pathophysiological studies suggest that the two diseases AD and T2D might be linked to each other. Earlier work has identified IAPP segments IAPP(8-18) and IAPP(22-28) as “hot segments” in both IAPP self- and its cross-interaction with A. More recently, these segments were linked to each via different linkers and a series of linear IAPP-derived peptides were synthesized. These peptides, termed cross-amyloid interaction surface mimics (ISMs), have been shown to be potent inhibitors of amyloid self-assembly of Aβ, IAPP, or both polypeptides. Notably, the nature of the linker has been shown to determine ISM structure and inhibitory function, including both potency and target (A versus IAPP) selectivity. In the first part of the thesis, three cyclic analogues of R3-GI (1), one of the above mentioned ISMs which is a potent inhibitor of amyloid self-assembly of A, were synthesized and studied. These peptides comprised the Cys-bridged cc-R3-GI (1a) and the two “Cys-stapled" (via aromatic linker moieties) peptides cc-L1-R3-GI and cc-L2-R3-GI. The aim was to find out whether cyclization would be (a) compatible with inhibitor function and (b) confer stability toward proteolytic degradation in human plasma. In fact, all three peptides were found to be nanomolar inhibitors of amyloid self- assembly of both Aβ and IAPP. Thus, cyclization via Cys-bridges and “Cys-stapling" likely stabilized a peptide conformation which is important for amyloid inhibitor function. However, only slight improvements were observed regarding the resistance of all three cyclic analogues toward proteolytic degradation in human plasma as compared to the linear precursor. In the second part of the thesis, cyclization via flanking cysteines of N-terminal truncated analogues of the three ISMs L3-GI, R3-GI and G3-GI was first performed; its effects on their structures and inhibitory effects were studied. The cyclic 17-residue long cc-R3-GIsh (2a) containing three Arg residues in the linker tripeptide was thus identified. 2a was a nanomolar inhibitor of amyloid self- assembly of both Aβ and IAPP; by contrast, its linear precursor was unable to do so. The cyclic cc-R3-GIsh (2a) was then used as a template for the development of a next generation of cyclic peptides. Their design was based on recent findings which suggested that IAPP residues F15, L16, F23 and I26 are key residues of IAPP self-assembly and its interaction with Aβ. The question was whether these four residues together with the linker tripeptide RRR and the cyclic structure via the two flanking cysteines would be sufficient for inhibitory activity. To investigate this hypothesis, all other amino acids of 2a were substituted with Gly resulting in cc-ALLG-R3-GIsh (2b). In fact, 2b turned out to be a nanomolar inhibitor of amyloid self-assembly of both Aβ and IAPP. In addition, six “2b-linker” analogues were also synthesized and studied; the results suggest that all three arginines of the linker tripeptide are required for inhibitory function. Furthermore, detailed studies on the effects of systematic substitutions of L-amino acid residues in 2b with D-ones were performed. Toward this aim, the three cyclic 2b analogues cc-ALLG-r3-GI (2c), cc- ALLG-(f15,l16,f23)-r3-GIsh (2d), and cc-ALLD-ALLG-r3-GIsh (2e) were synthesized and studied. Here, the focus was on both inhibitory function and proteolytic stability. Analogue 2e was identified as a selective nanomolar inhibitor of A amyloid self-assembly. Most importantly, 2e was found to exhibit a highly improved proteolytic stability in human plasma in comparison to other analogues being >30- 1 Abstract fold more resistant than R3-GI (1) and >15-fold more resistant than its L-precursor 2b. 2e is thus a promising candidate for further development as an anti-amyloid drug in AD. In the third part of my thesis, I studied the effects of the previously developed ISMs on insulin amyloid self-assembly. These studies were done based on the facts that IAPP is able to bind insulin and inhibit its amyloidogenesis and that IAPP-GI, an earlier designed soluble IAPP analogue, is a potent inhibitor of amyloid self-assembly of insulin. My results show that all ISMs containing hydrophobic linkers can inhibit insulin oligomerization and amyloid self-assembly whereas all but two of the ISMS containing polar amino acids in the linker were unable to do so. Together with previous results, my studies on 14 different ISMs revealed the following amyloid inhibitor selectivities: L3-GI, Nle3-GI and Cha3-GI are able to inhibit all three polypeptides A, IAPP and insulin; V3-GI, I3-GI and F3-GI inhibit A and insulin but not IAPP, 2-Aoc3-GI inhibits IAPP and insulin but not A, and KAc3-GI and SpG-GI inhibit only insulin while R3-GI inhibits only A. Based on their selectivity profiles, some of these ISMs could be suitable leads for the design of anti-amyloid drugs in AD and/or T2D likely also in combination with insulin-based treatments. Taken together, the results of my studies should contribute in both understanding how cross-interactions between different amyloidogenic polypeptides may affect amyloid self-assembly and the development of novel highly potent inhibitors of amyloid self-assembly of Aβ, IAPP and/or insulin as promising leads for anti-amyloid drugs in AD and/or T2D. 2 Zusammenfassung Zusammenfassung Die Proteinaggregation wird mit zelldegenerativen Erkrankungen wie der Alzheimer-Krankheit (AD) und dem Typ-2-Diabetes (T2D) in Verbindung gebracht. AD ist mit der Selbstassoziation des β- Amyloid-Peptids (Aβ) assoziiert, während die Aggregation des Islet-Amyloid-Polypeptids (IAPP) T2D begünstigt. Die beiden Polypeptide Aβ und IAPP weisen zusätzlich zu ihrer ähnlichen Amyloidogenität 50% Sequenzähnlichkeit auf. Es wurde bereits gezeigt, dass nicht-fibrilläre und nicht-toxische Aβ- und IAPP-Spezies mit nanomolarer Affinität aneinanderbinden; dadurch wird die zytotoxische Selbstassoziation und die Amyloidogenese beider Peptide unterdrückt. Darüber hinaus deuten klinische und pathophysiologische Studien darauf hin, dass die beiden Erkrankungen AD und T2D im Zusammenhang stehen könnten. Frühere Studien haben die IAPP-Segmente IAPP(8-18) und IAPP(22-28) als "Hot-Segmente", sowohl in der IAPP-Selbst-, als auch in seiner Kreuz-Interaktion mit A, identifiziert. Vor kurzem wurden diese Segmente über verschiedene Linker miteinander verknüpft und eine Reihe von linearen, von IAPP abgeleiteten, Peptiden wurde entworfen. Es zeigte sich, dass diese Peptide, die auch als Cross-Amyloid- Interaktionsoberflächen-Mimetika (ISMs) bezeichnet werden, hochpotente Inhibitoren der Amyloid- Selbstassemblierung von Aβ, IAPP oder beiden Polypeptiden sind. Dabei bestimmt die Zusammensetzung des Linkers die ISM-Struktur, ihre inhibitorische Funktion und ihre Zielselektivität (Aβ versus IAPP). Im ersten Teil der Arbeit wurden drei zyklische Analoga von R3-GI (1), einem der oben genannten ISMs, der ein potenter Inhibitor der amyloiden Selbstassoziation von Aβ ist, synthetisiert und untersucht. Diese Peptide bestehen aus dem Cys-verbrückten cc-R3-GI (1a) und den zwei „Cys- gestapelten“ Analoga cc-L1-R3-GI und cc-L2- R3-GI. Letztere sind über aromatische Linker-Einheiten verbrückt. Ziel war es, herauszufinden, ob die Zyklisierung (a) mit der inhibitorischen Funktion kompatibel wäre, und (b) Stabilität gegenüber der Proteolyseanfälligkeit im humanen Plasma verleihen würde. Es stellte sich heraus, dass alle drei Peptide Inhibitoren im nanomolaren Konzentrationsbereich der amyloiden Selbstassoziation von sowohl Aβ als auch IAPP sind. Diese Ergebnisse deuten darauf hin, dass die Zyklisierung über eine Cys-Brücke und das "Cys-Stapeln" eine Peptidkonformation, stabilisiert hat, die für die Inhibitor-Funktion wichtig ist. Allerdings war die Proteolysestabilität der drei Analoga nicht stark verbessert im Vergleich zum linearen Vorläuferpeptid. Im zweiten Teil der Arbeit wurden zunächst N-terminal verkürzte Analoga der drei ISMs L3-GI, R3- GI und G3-GI über flankierende Cysteine zyklisiert; die Analoga wurden dann im Bezug auf ihre
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