The cationic tetradecapeptide mastoparan as a privileged structure for drug discovery: Enhanced antimicrobial properties of mitoparan analogues modified at position-14

Mastoparan (MP) peptides, distributed in insect venoms, induce a local inflammatory response post envenomation. Most endogenous MPs share common structural elements within a tetradecapeptide sequence that adopts an amphipathic helix whilst traversing biological membranes and when bound to an intracellular protein target. Rational modifications to increase cationic charge density and amphipathic helicity engineered mitoparan (MitP), a mitochondriotoxic bioportide and potent secretagogue. Following intracellular translocation, MitP is accreted by mitochondria thus indicating additional utility as an antimicrobial agent. Hence, the objectives of this study were to compare the antimicrobial activities of a structurally diverse set of cationic cell penetrating peptides, including both MP and MitP sequences, and to chemically engineer analogues of MitP for potential therapeutic applications. Herein, we confirm that, like MP, MitP is a privileged structure for the development of antimicrobial peptides active against both prokaryotic and eukaryotic pathogens. Collectively, MitP and target-selective chimeric analogues are broad spectrum antibiotics, with the Gram-negative A. baumannii demonstrating particular susceptibility. Modifications of MitP by amino acid substitution at position-14 produced peptides, Δ14MitP analogues, with unique pharmacodynamic properties. One example, [Ser14]MitP, lacks both cytotoxicity against human cell lines and mast cell secretory activity yet retains selective activity against the encapsulated yeast C. neoformans

Bike peptides: a ride through the membrane

Several natural peptides have a biaryl or biaryl ether motif in their biologically active structures. A model bicyclic pentapeptide containing a biaryl bridge has been synthesized by solid-phase peptide synthesis combining on-resin Suzuki and Miyaura cross-coupling reactions. Its biological properties in terms of permeability, stability and cytotoxicity have been studied, demon-strating the positive contribution of the biaryl bridge, excellent membrane penetration and serum stabilit

Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.

C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs

Design of Novel Pini Inhibitors Incorporating a p-Phospho-Glutamate Analogue

Pin1 is an enzyme essential to cell cycle regulation and has a key role in cancer proliferation. This thesis reports ongoing efforts to obtain a Pin1 inhibitor exhibiting an inhibition constant in the nanomolar range. It was previously found that Pin 1 activity could be inhibited using a short proline containing peptide sequence which also contains a stereospecific (3- substituted a-amino acid. Several proline analogues were tested for greater inhibition against Pin1 than previously synthesized Cbz-/_-Glu((3-phos)-Pro-NH2. It was found that using full length Pin1 in the chymotrypsin-coupled photometric assay rendered different values than using only the catalytic PPIase domain for Cbz-Z_-Glu((3-phos)-Pro-NH2, Kj = 54.1+1.8 pM and 20 ± 0.9 pM respectively. The potential effectiveness of the synthesized inhibitors against Pin1 was assessed using the same convenient chymotrypsin-coupled photometric assay against full length Pin1. This thesis describes the synthesis of two novel compounds which exhibited an increase in inhibition relative to Cbz-/_-Glu((3-phos)-Pro-NH2(Kj = 54.1+1.8 pM) with results in the micromolar range; Cbz-Z_-Glu(f3-phos)-Pip-NH2 (Kj = 45.4+1.1 pM) and Cbz-/_-Glu(p-phos)-Methylpiperazine (Kj = 22.7+1.4 pM

Design of Orexin Based Imaging Agents

Orexin A (33 amino acids) and orexin B (28 amino acids) are two naturally occurring ligands of the G-protein coupled receptor: orexin receptor 1 (OXiR) and orexin receptor 2 (OX2R). Based on the expression of OXiR in colon cancer cells, gallium-68 labelled orexin A (15-33) and orexin B (6-28) were developed as potential PET imaging agents targeting colon cancer cells. Orexin A (15-33) and orexin B (6-28) were synthesized by Fmoc solid phase peptide synthesis (SPPS). A bifunctional chelator 1,4,7,10-tetra azacyclo dodecane- N,N\u27,N”,N”\u27-tetraacetic acid (DOTA) was used to attach gallium-68 to orexin A (15-33) and orexin B (6-28). A linker aminohexanoic acid (Ahx) was incorporated between the targeting peptide sequence and 68Ga-DOTA complex to reduce the bulky effect of the imaging label on the binding affinities. It was determined that the IC50 values of Ga-DOTA-Ahx-orexin A (15-33) and Ga-DOTA-Ahx-orexin B (6-28) were 629 nM and 168 nM respectively. In order to evaluate the binding affinities in cells, orexin A (13-33) and orexin B (6-28) were also labeled with fluorescein isothiocyanate (FITC) allowing for fluorescence microscopy studies. Besides orexin A and orexin B analogues, a potential orexin receptor antagonist was developed as a potential imaging agent as well and preliminary synthetic steps were undertaken. This antagonist was proposed to be labelled with fluorine-18 to form the compound [l8F](4-fluoro-phenyl)-(4-quinozolin- 2-yl-[l,4]diazepan-l-yl)-methanone as an imaging agent targeting colon cancer cell

Design and characterisation of dendritic molecules.

Dendritic molecules with highly versatile and synthetically controllable structures have been examined closely in the fields of drug delivery and targeting. This thesis focuses on the design and synthesis of a series of poly(lysine) dendritic molecules, and how their structural characteristics affect their physical and pharmaceutical properties. All molecules studied in this thesis were prepared using solid phase peptide synthesis. Amphipathic dendrons having three lipidic (C14) chains coupled to dendritic lysine head groups with 8, 16 or 32 free terminal amino groups were synthesised. Their interactions with charged and neutral liposomes were studied, and the interaction efficiency for all three dendrons was greater than 88%. The dendrimers produced cationic liposomes regardless of initial liposome charge and showed evidence of hydrophobic interactions with the liposome membranes. Membrane disruption of the liposomes was also seen with the highly cationic dendrons. The interaction of the series of dendrons with albumin, was studied in the presence of NaC1, using dynamic dialysis and molecular modelling. Diffusion and membrane permeability studies (using cellulose membranes) were also performed. The stoichiometry of dendron: albumin interactions was found to be 1:1.5, 1:4 and 1:5 for the dendrons with 8, 16 and 32 amino groups, respectively. Calculation of shape factors showed that the dendrons had a more spherical confirmation with each generation of growth. The synthetic approaches used to synthesise other dendrons as well as a novel didendron is detailed. Viscosity studies showed that a didendron with 128 terminal amino groups (2 x 64) had a dynamic viscosity greater than that of a single dendron with 128 terminal amino groups. A dendron with acetylated terminal groups was found to form unusual aggregates on the surfaces of glass slides

Design, synthesis and biological evaluation of hepcidin analogues

Hepcidin is a peptide hormone involved in the control of iron homeostasis. It has 25 amino acids with an antiparallel beta-sheet structure stabilized by four disulfide bonds. Hepcidin binds to the sole known iron exporter, ferroportin, leading to its internalization and degradation by a mechanism not fully understood. Hepcidin has an important role in iron metabolism disorders, such as hemochromatosis and anaemia. However, hepcidin analogues are currently not available for clinical use. The aims of this project are twofold: - to design and synthesise a fluorescent hepcidin analogue that can be used as a biological tool to further investigate hepcidin-ferroportin interactions; - to design and synthesise small peptide-like hepcidin analogues which can bind and internalize ferroportin. At the end of the thesis there is a pullout which summarises the structures of the peptides synthesised in this project. Chapter 2 describes the synthesis of eleven linear peptides, synthesised to facilitate a structure-activity relationship study on the N-terminus of hepcidin, which is the most active part of the peptide. Chapter 3 describes the synthesis of two hepcidin analogues (15 and 19) containing intramolecular disulfides. Peptide 15 represents the N-terminus of hepcidin constrained by one intramolecular disulfide. Peptide 19 sequence contains amino acids from the N-terminus of hepcidin and from the C-terminus, which we considered to be also relevant for the binding to ferroportin. The cysteines in peptide 19 were oxidised to disulfides. Chapter 4 focuses on the synthesis of two hepcidin analogues (27 and 35) with intermolecular disulfides: peptide 27 containing one intermolecular disulfide and peptide 35 containing two. Intermolecular disulfide formation is more challenging to achieve, as oxidation of the cysteines needs to be selective. A successful approach was developed by carefully selecting the protecting groups for the thiol group of the cysteines, which were removed stepwise in order to achieve selectivity. Chapter 5 discusses the synthesis and folding of [Lys21] 6-carboxy tetramethylrhodamine (TMR) labelled hepcidin and N13 6-carboxy uorescein (CF) labelled hepcidin 20. Hepcidin sequence presents a methionine in position 21, near the C-terminus, which was replaced, in the synthesis of [Lys21] TMR hepcidin, with a lysine protected at the N with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde). This approach provides a site where TMR can be selectively attached. In N13 CF hepcidin 20 the last ve amino acids of hepcidin were not included in the synthesis and the peptide was labelled at the lysine in position 13. This peptide is not active and was synthesised as a negative control for biological evaluation purposes. Chapter 6 is divided in two sections. The first section describes the structural analysis by circular dichroism of the analogues synthesised in this project. The second section illustrates the biological evaluation results. Biological assays were performed at Vifor Pharma laboratories in Zurich. The [Lys21] TMR hepcidin was found to possess appreciable biological activity, being able to bind and internalize ferroportin with a potency only 4 fold lower than that of synthetic hepcidin. The structure-activity relationship study, conducted with peptides mimicking the N-terminus of hepcidin, suggests that a disul de exchange may be involved in the binding between the N-terminus and ferroportin. Furthermore, between these analogues, peptides 5 and 8 were found to be able to bind ferroportin without leading to its internalisation, suggesting an interesting antagonist activity. Peptides 19 and 27 show some activity, being 189 and 13 fold less active than hepcidin 25, respectively

Development of novel molecular tools for the characterisation of zebrafish renin

The renin angiotensin system (RAS) is highly conserved across vertebrates. However, until recently it has not been extensively explored in zebrafish, an important model organism suitable for developmental studies, high-resolution in vivo imaging, and genetic and chemical screens. In common with mammals, the adult zebrafish kidney contains specialised renin-expressing mural cells that contain granules indicative of the processing and regulated secretion of active renin. Due to the translucent nature of zebrafish larvae, the functional pronephros is easily accessible for real-time imaging and in vivo studies of the RAS. The primary aim of this project was to develop novel tools to help elucidate the function of renin and the role of the RAS in zebrafish. I designed a zebrafish renin-luciferase reporter transgene using a previously published promoter sequence to investigate the transcriptional regulation of renin in real-time. The expression vector was injected into fertilised WIK zebrafish eggs and successful integration of the plasmid into the zebrafish genome was demonstrated. An in vivo assay was designed to select for highest luciferase expressers, but bioluminescent imaging revealed that the high signal stemmed from the yolk sac of zebrafish. Yolk sac expression appeared to originate from episomal transcription of the injected plasmid and, in the F0 fish, masked potential bona fide expression from the renin-expressing cells of the pronephros. A new transgene was generated using two reporters, driven by the same promoter sequence and the two reporters were separated by a ’2A’ sequence which codes for a self-cleaving peptide. This zebrafish line was generated to permit selection of tg(ren:LUC-2A-mCherry) fish for the strongest mCherry signal originating from renin expressing cells. However, no expression was observed at the anterior mesenteric artery (AMA). Further investigation revealed that when generating novel transgenic zebrafish, the reporter proteins are transiently expressed in the yolk sac of F0 founder fish and the ectopic expression can influence studies relying on quantitative reporters. Current studies largely rely on the transcriptional activity of RAS components and are restricted to timepoint-specific observations. The lack of antibodies prevents the measurement of RAS proteins in zebrafish. The amino acid sequence for zebrafish angiotensinogen is known and by comparison to mammalian sequences, I identified the amino acid sequences for angiotensin (Ang) I and II. The production of zebrafish Ang I and II peptides by solid phase peptide synthesis provided standards for the development of assays for zebrafish angiotensins. I used these assays to demonstrate that Captopril, which in mammals prevents the conversion of Ang I to Ang II by inhibiting the Angiotensin Converting Enzyme (ACE), is active in zebrafish, and that its administration leads to a dramatic decrease in Ang II. In parallel I designed and synthesised a fluorescent resonant energy transfer (FRET) probe to enable zebrafish renin activity to be measured. Using various zebrafish models I was able to demonstrate dynamic changes in renin activity and measure these using the first renin zebrafish FRET probe. I characterised a renin knockout zebrafish, generated by CrispRCas9 gene editing. Knockouts were identified by DNA sequencing which identified an 8bp deletion in exon 2 of the renin gene, causing a frameshift mutation and early termination of renin translation. Renin knockout fish proved to be viable and were screened for a phenotype using the high throughput automated screening system (VAST) which permits the precise imaging of a large number of live fish. Imaging revealed delayed development of the swim bladder and reduced fish length compared to age-matched controls. This was indicative of an overall developmental delay. The knockout fish were intercrossed with existing renal reporter lines to investigate phenotypes at a cellular level. Ren−/− tg(wt1b:EGFP) fish showed a delay in glomerular fusion of the pronephric kidney and a cross of the ren−/− with tg(ren:RFP-LifeAct) fish indicated a dramatic increase in renin-expressing cell along the renal mesonephric vasculature. Moreover, morphological examination revealed vacuolation of proximal tubules in the mesonephric kidney. The intercrossing of the ren−/− zebrafish to the tg(ren:RFP-LifeAct) and tg(acta2:EGFP) fluorescent reporters lead to the optimisation of a FAC sorting protocol and resulted in the first zebrafish ren- and acta2-expressing cells to be cultured and imaged using high-resolution microscopy imaging. In summary, my work has led to the first successful measurement of zebrafish AngI and AngII in mesonephric zebrafish tissue to demonstrate the effectiveness of Captopril in zebrafish as well as the first development of a zebrafish renin FRET probe to allow the measurement and quantification of renin activity in zebrafish, enabling more accurate studies of the zebrafish RAS. Lastly, successful phenotypic characterisation of the ren−/− fish will further our understanding of the role of renin and the RAS in zebrafish