Macrocyclic peptides have been identified as key backbones in several biologically active compounds. They have been considered as great inspiration in the development of novel cyclic scaffolds in medicinal chemistry, notably in the introduction of α-helically constricted stapled peptides with the ability to mimic biologically relevant α-helices. DNA-binding transcription factors often bind their DNA promoter through an α-helix, making a parallel with stapled peptides as inhibitors. Despite this relevant feature, DNA-binding stapled peptides are highly unrepresented in the literature, as will be discussed here in a review. We also further expand this area of research with a study of DNA binding stapled peptide ana-logues with the goal of optimizing and investigating the DNA binding and antivirulence of an RpoN-based stapled peptide. Cyclic depsipeptides are highly biologically active natural product molecules however their synthesis can be challenging with the presence of a macrolactone. Due to this complexity, solid phase peptide synthesis strategies have been utilized to access peptide intermediates that can be synthetically macrocyclized using solution phase or on-resin approaches via macrolactam or macrolactone formation. A representative number of total syntheses in the literature is reviewed. Furthermore, we describe here the chemical total synthesis and chemoenzymatic synthesis of seongsanamide E cyclic depesipeptide via thioesterase medi-ated macrolactonization. Cyclic depsipeptides also play major roles in their producing organisms, notably siderophores capable of chelating and transporting iron. The biosynthesis of fungal siderophores is poorly explored, such as the iterative mechanism of oligomeric compound fusarinine C. We explore the synthesis of the previously never synthesized fusarinine C monomer to be utilized as a di-domain inhibitor of the adenylating-thiolation domains of the non-ribosomal peptide synthetase (NRPS) SidD.

Photodynamic therapy (PDT) is an emerging therapy for the treatment of cancer and various other human disorders. 5-Aminolaevulinic acid (ALA) is a simple natural product that is of great interest for PDT because it can be converted within cells via the haem biosynthetic pathway to the photosensitiser, protoporphyrin IX (PpIX). ALA-PDT has become a first line clinical approach for the treatment of cancerous and precancerous skin lesions (e.g Bowen’s disease, basal skin carcinomas, and actinic keratosis) that would otherwise require significant conventional surgery. However, ALA being a zwitterion suffers from poor lipid solubility and at the same time has stability issues at physiological or alkaline pH. The work herein describes some novel strategies to enhance the delivery of ALA to specific cell types using targeted ALA dendrimeric prodrugs. Specifically, it describes the synthesis of molecules consisting of branched units with 3 or more copies of ALA attached to a central core structure (e.g. gallic acid) using copper-catalysed azide-alkyne click chemistry (CuAAC). Selective delivery of the dendrimeric ALA cargo was achieved by attachment of a homing peptide to an independently addressable functional group on the prodrug core. As proof of concept of this approach, systems were prepared containing a peptide that allows selective targeting of the epidermal growth factor receptor (EGFR) which is overexpressed in a variety of tumours. Targeted ALA delivery and PpIX production was studied with these prodrugs in EGFR-expressing breast adenocarcinoma cells (MDA-MB-231 cells) and a peptide-targeted derivative with 9 ALA units was found to have enhanced PDT efficacy compared to an equimolar dose of ALA. Other targeting units that have been attached to these dendrimeric ALA prodrugs include biomolecules such as vitamin E, thymidine (a nucleoside) and a glucose derivative. Additionally, strain-promoted azide-alkyne cycloadditions (SPAAC) of the same EGFR-targeting peptide with some classical photosensitisers were also investigated and biological studies in EGFR-overexpressing cell lines were carried out. Lastly, a group of cell penetrating peptide-ALA conjugates have been synthesised via CuAAC as a novel approach for targeted ALA delivery.

Design of self-assembling ABC-type collagen triple helical heterotrimers is challenging due to the number of competing species that can be formed in ternary mixture of peptides with a high propensity to fold into triple helices and the fact that well understood rules for pair-wise amino acid stabilization of the canonical collagen triple helix have remained elusive. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population towards a desired target but the presence of competing states in mixtures has remained an outstanding problem. In this work we use high-resolution structural biology techniques to do a detailed study of stabilizing pair-wise interactions between positively and negatively charged amino acids in triple helices. Two types of contacts with distinct sequence requirements depending on the relative stagger of the interacting chains are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. Using this knowledge we developed distinct design strategies to improve the specificity of our designed systems towards the desired ABC heterotrimeric target state. We validate our strategies through the synthesis and characterization of the designed sequences and show that they self-assemble into a highly stable ABC triple helices with control over composition in the case of the rational approach and with control over both composition and register in the case of the computational approach.

Οι Κωνοτοξίνες είναι μικρά πεπτίδια πλούσια σε δισουλφιδικούς δεσμούς, τα οποία προέρχονται από τα δηλητήρια των θαλάσσιων σαλιγκαριών. Οι κωνοτοξίνες αυτές στοχεύουν διαφορετικούς υποδοχείς στο νευρικό σύστημα με υψηλή εκλεκτικότητα και ισχύ, αποτελώντας χρήσιμους φαρμακευτικούς δείκτες ή και φάρμακα. Μια ομάδα κωνοτοξινών είναι οι μ-κωνοτοξίνες, οι οποίες έχουν παρουσιάσει αναλγητική δράση. Οι μ-κωνοτοξίνες έχουν συγκεκριμένα δομικά χαρακτηριστικά πολύ σημαντικά για την βιολογική τους δραστικότητα. Περιέχουν τρείς δισουλφιδικούς δεσμούς μεταξύ των Cys1-Cys4, Cys2-Cys5 και Cys3-Cys6, σχηματίζοντας τρείς θηλιές (loop) στον σκελετό του πεπτιδίου. Οι μ-κωνοτοξίνες ερευνήθηκαν σαν πιθανά φαρμακευτικά εργαλεία εξαιτίας της ικανότητας τους να αναστέλλουν διαφορετικούς νευρικούς υποτύπους διαύλων Νατρίου και συνεπώς να στοχεύουν όχι μόνο κατά του χρόνιου πόνου αλλά και κατά άλλων ασθενειών. Στόχος της παρούσας διατριβής είναι η σύνθεση νέων μ-κωνοπεπτιδίων με πιθανή αναλγητική δράση, τα οποία στοχεύουν κυρίως σε πρωτεΐνες –κανάλια ιόντων Νατρίου, προκαλώντας αναστολή της μετάδοσης σήματος στα νευρικά κύτταρα. Η σύνθεση των αναλόγων πραγματοποιήθηκε με την Fmoc/But μεθοδολογία επί στερεάς φάσεως, χρησιμοποιώντας ως στερεό υπόστρωμα την Sieber Amide ρητίνη. Ο σχηματισμός των τριών δισουλφιδικών γεφυρών επιτεύχθηκε σε ένα στάδιο στην υγρή φάση με την χρήση ρυθμιστικών αναγωγικών μέσων. Στην παρούσα φάση, τα συντιθέμενα πεπτίδια βρίσκονται σε στάδιο βιολογικής αξιολόγησης μέσω ηλεκτροφυσιολογικών ανταγωνιστικών πειραμάτων. / Conotoxins are small disulfide rich peptides derived from the venom of Conus snails. They target different receptors in the nervous system with high selectivity and potency making them valuable as drug leads or drug themselves. One group of conotoxins, μ-conotoxins have been shown to have potential as analgesic treatment. μ-conotoxins have concrete conformational features, which are very crucial to their biological activity. They contain three disulfide bonds among Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6, forming three loops in their backbone. μ-conotoxins studied as potent pharmacological tools due to their ability to inhibit different neuronal subtypes of sodium channels and likewise conotoxins target not only the chronic pain treatment but also target against other disease’s treatment. The goal of this present thesis is to synthesize new μ-conopeptides, with a potent analgesic activity, which target mostly proteins-sodium channels, inhibiting the nerve impulse transmission at the neuromuscular injection. These analogues were synthesized using the Fmoc/But methodology by SPPS, using Sieber Amide resin as solid support. The formation of three disulfide bonds was accomplished in one step in the solution phase using the redox buffer strategy. At present, the synthesized cono-peptides are under biological evaluation through electrophysiological competitive experiments.

Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.

<p><b>Part I: Design of a Photolabile Backbone Amide Linker for the Synthesis of C-terminally Modified Peptides</b></p> <p>A new photolabile backbone amide linker has been developed for the on-resin synthesis of cyclic and C-terminally modified peptides. The linker (Hcnb) is stable to strongly acidic conditions and instead releases the completed peptide through photolytic cleavage at 365 nm. Hcnb possesses four degrees of orthogonality and is amenable to the preparation of cyclic peptides, C-terminally modified peptides, and fully protected peptides due to its photolabile backbone amide linkage. The Hcnb precursor can be conveniently synthesized in 4 steps from commercially available 4-methyl-3,5-dinitrobenzoic acid. The C-terminal amino acid residue is loaded via reductive amination of the precursor followed by an O→N transacylation for the addition of the second residue in quantitative yields, even when employing sterically bulky residues. Standard Fmoc- or Boc-based synthesis can then be utilized to complete the desired peptide. Hcnb has been used to demonstrate the linear synthesis and subsequent on-resin cyclization of various cyclic peptides of interest, as well as synthesis of C-terminal thioesters on-resin. </p> <p><b>Part II: Development of II-HMG CoA Reductase Inhibitors for use as Gram-Positive Selective Antimicrobials. </b></p> <p>Bacterial resistance to antibiotic drugs is an issue that humans have faced since the first use of sulfa drugs in the 1930s. In recent years, the rate of production of new antimicrobial drugs has diminished, as they are no longer financially beneficial to pharmaceutical companies due to short term use and rapid resistance development. This places the burden of the development of new antimicrobial drug on the academic research field. In the work presented here, progress has been made toward the development of a novel class of antimicrobial compounds. These small molecule inhibitors target II-HMG CoA Reductase, a key enzyme involved in cell wall synthesis in gram-positive bacteria. Based on analysis of co-crystal structures obtained from first- and second- generation inhibitors, structural alterations were made to design a new generation of compounds. Efforts have also been made toward identification of a potential secondary target of these inhibitors. </p>

Cyclization reactions play an important role in synthesizing a significant number of small molecule scaffolds for various purposes, including drug discovery. However, the application of cyclization reactions in the modifications of biomolecules in a single step is still limited. This dissertation reports a stereoselective thiomorpholine ring formation reaction to site-selectively modify N-terminal cysteines of unprotected peptides or proteins in a single step. We showed that α-fluoroketone molecules afford the cyclization reaction with the beta amino thiol group of N-terminal cysteine. Both chemo and stereo-selectivity of this reaction have been studied using 2D NMR analyses. Cysteine located at the Nterminal of a short protein (VHP protein) has been modified site-selectively with a fluorescein isothiocyanate (FITC) containing an α-fluoroketone linker to demonstrate the applicability of this reaction in modifying biomolecules. This chemistry has the potential to generate homogeneous and stable antibody-drug conjugates (ADCs) for the treatment of cancer. This dissertation also demonstrates a fluorine-thiol displacement reaction (FTDR) to synthesize various macrocyclic and stapled peptides, which renders medicinally privileged peptides with improved biological properties such as binding affinities and cell membrane permeability. The cyclization of fluoroacetamide containing peptides with benzenedimethane thiol linkers enhances peptides' alpha helicity. These FTDR stapled peptides exhibit better cellular uptake compared to the classic ring-closing metathesis (RCM) stapled peptides. Compared to the proteoglycan-aided cell penetration by peptides stapled with RCM, the preliminary mechanistic studies of our FTDR-stapled peptides revealed that our thiolated linkers allowed peptides to enter cells in multiple pathways. Taken together, our FTDR-based stapling approach may provide a novel class of cell-permeable peptides that might open a new window to probe intracellular targets. This dissertation reports another cyclization reaction between hydrazine and carbamate, synthesized from 7-hdyroxy coumarin derivatives. We demonstrate that the secondary nitrogen of hydrazine is much more reactive than the primary nitrogen for intramolecular cyclization reactions. Hydrazine affords urea bonds upon the substitution of a coumarin moiety of carbamates to generate 6 or 7 membered cyclic scaffolds. The exocyclic free amine might allow facile generation of a library of compounds. In addition, further optimization of this reaction might allow using these hydrazine containing molecules in monitoring the real-time activity of histone deacetylases (HDACs). / Chemistry

Using three complementary approaches, this work sought to tackle the widespread problem of antibiotic resistance. To circumvent the resistance mechanisms developed by bacteria, it is necessary to establish drug candidates that act on novel therapeutic targets, such as the ion channels used by bacteria to modulate homeostasis. Examples include the potassium efflux channel, Kef, and the mechanosensitive channel of small conductance, MscS, which are not found in humans. How these targets function must be well understood before drug candidates can be developed, as such, their identification and investigation is often accompanied by the evolution of the analytical techniques used to study them. Membrane protein mass spectrometry is one technique showing potential in the study of ion channels. However, spectra can be clouded by the detergents used to solubilise ion channels from their native membranes. Undertaken herein was the synthesis of some fluorescent glycolipid detergents, which it was hypothesised could be encouraged to dissociate from ion channels via laser-induced excitation within the gas phase of a mass spectrometer, thereby improving the clarity with which spectra can be obtained. For Kef, an unconfirmed mechanism of action had previously been proposed. To explore the suggestion that sterically-demanding central residues are important for channel activation, solid phase peptide synthesis was used to isolate three tripeptide analogues of N-ethylsuccinimido glutathione, a known activator with a high affinity for Kef. A competition fluorescence assay suggested these tripeptides bound to Kef with an affinity lower than predicted, allowing the conclusion that a more detailed assessment of the steric bulk required for activation was necessary before a mechanism of action could be confirmed. Lysophosphatidylcholine has been shown to activate MscS, although it is not known how. Affinity chromatography between MscS and lysophosphatidylcholine was proposed as a means by which specific binding interactions could be investigated. For this technique an amino-derivative of lysophosphatidylcholine was necessary and its challenging synthesis is also detailed herein.

Editorial on the Research Topic. Antimicrobial and Anticancer Peptides.

Correct functioning of the brain relies upon the precise connectivity between the billions of neurons that make up this crucial organ. Aberrations in the formation of these elaborate neural networks lead to neurodegenerative and neuropsychiatric disorders. A synapse-spanning molecular triad, involving members of the Neurexin, Cbln and ionotropic glutamate receptor delta families of proteins, is crucial for the accurate formation and proper function of synapses in the cerebellum. This trans-synaptic complex has been implicated in the molecular mechanisms behind motor control and motor learning, and furthermore individual members have been linked to diseases such as Alzheimer’s, autism spectrum disorders and schizophrenia. The major findings presented in this thesis include: crystal structures of the amino-terminal domains (ATD) of the two members of the ionotropic glutamate receptor delta (iGluR-Delta) family, functional characterisation of the effects of disrupting the ATD interface in one member of the iGluR-Delta family, a crystal structure of the C1q domain of Cbln1, biophysical analysis of the molecular interactions within the Neurexin-Cbln1-GluD2 trans-synaptic complex, as well as evidence for the domain arrangement of the ecto-domain of the iGluR-Delta proteins. Together, these data enhance our knowledge of the molecular details of this macro-molecular complex and provide evidence to support models for the mechanisms of their involvement in synapse formation and function, thereby making a contribution to the vast and medically relevant field of molecular neurobiology.