The use of peptide drugs has gained popularity recently. Peptides are attractive drug targets due to their high specificity and potency towards their biological targets. A drawback for peptide drugs is a lack of stability for oral delivery. Two classes of disulfide-rich peptides, conotoxins and cyclotides, have been shown to have higher stability than linear peptides thanks to their disulfide connectivity. Conotoxins are present in the venom of cone snails, a carnivorous marine mollusk that preys upon fish, worms, or other mollusks. Conotoxins are promising drugs leads with great prospects in the treatment of diseases and disorders such as chronic pain, multiple sclerosis and Parkinson’s and Alzheimer’s diseases. Cyclotides, which are cyclic cysteine knot containing peptides, isolated from the Violaceae (violet), Rubiaceae (coffee), and Cucurbitaceae (cucurbit) families and they have a wide range of biological activities, such as anti-HIV, uterotonic, and antimicrobial. P-superfamily framework IX conotoxins (C-C- C-CXC- C) contain the same cysteine framework, homologous sequences, and similar 3D structures to cyclotides. The knot containing conotoxins have been identified in several Conus species, but this work focuses on those from Conus brunneus, Conus purpurascens, and Conus gloriamaris. The cysteine knot motif of cyclotides and P-superfamily conotoxins is characterized by a cyclic backbone and six-conserved cysteine residues that form the three-disulfide bridges of the “knot”. This motif provides cyclotides and conotoxins with superior stability against thermal, chemical, and enzymatic degradation; marking them as potential frameworks for peptide drug delivery. Presented are details on the isolation of conotoxins and cyclotides, from Viola tricolor, and the characterization of their activity in the well-characterized Drosophila melanogaster giant fiber system (GFS) neuronal circuit, which contains GAP, acetylcholine, and glutamate synapses. The transcriptomes of two Conus brunneus specimens were assembled and mined for P-superfamily framework IX conotoxins. Eleven mature P-superfamily framework IX conotoxins were identified in the crude venom. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 6 uppsatser.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

The broad aim of this thesis is to generate fundamental knowledge about the structure and function of cyclotides, which are a topologically unique family of proteins. A long-term goal is to use the fundamental knowledge to assist in the development of drugs based on the stable cyclotide framework. Cyclotides are small proteins that are characterised by a cyclic cystine knot (CCK) motif, which is defined as a circular backbone combined with a cystine knot core. So far cyclotides have been found in plants of the Violaceae (violet) and Rubiaceae (coffee) plant families, and are believed to have a defence-related function. From an application perspective, the CCK framework has potential as a drug scaffold, being an ultra-stable alternative to linear peptide models. The reasons why cyclotides show promise as a drug template are three-fold – they have naturally high sequence diversity, suggesting that their framework can accommodate a range of epitopes; they are remarkably stable under various chemical, enzymatic and thermal conditions, which means that they have increased bioavailability; and they have a diverse range of bioactivities, supporting the notion that they can be used in a number of therapeutic applications. These three reasons are intimately linked to three core knowledge domains of cyclotide research, namely cyclotide sequences, structures and interactions. Thus, fundamental research into these three domains, as investigated in this thesis, is important as it may assist in the development of drugs based on the CCK scaffold. Chapter 1 of this thesis provides the background information to define the molecules studied and to highlight their importance. Chapter 2 describes the main experimental techniques that were used in this thesis, including nuclear magnetic resonance spectroscopy and mass spectrometry. The development of the CCK technology may benefit from a thorough understanding of the natural diversity of cyclotide sequences and the significance of this diversity on activity. Chapter 3 reports on the discovery of cyclotides in Viola yedoensis, a Chinese violet that is interesting because it is widely used in Traditional Chinese Medicine to treat a number of illnesses including swelling and hepatitis. In this study, a total of eight cyclotides was characterised, including five novel sequences. Based on anti-HIV and haemolytic assays, a strong relationship between surface hydrophobicity and activity was established. The stability of cyclotides, which underpins their potential as a drug scaffold, is examined at a structural level in Chapter 4. The solution structure of varv F, a cyclotide from the European field pansy, Viola arvensis, was solved and compared to the crystal structure of the same peptide, confirming the core structural features of cyclotides responsible for their stability, including the topology of the cystine knot, which has previously attracted some debate. From a comparison of biophysical measurements of a representative group of five cyclotides, a conserved network of hydrogen bonds, which also stabilises the cyclotide framework, was defined. A subset of hydrogen bonds involving the highly conserved Glu in loop 1 of cyclotides was examined in more detail by solving the structure of kalata B12, the only naturally occurring cyclotide with an Asp instead of a Glu in loop 1. By comparison with the prototypical cyclotide kalata B1 and an Ala mutant E7A-kalata B1, it was shown that the highly conserved Glu is important for both stability and activity. Chapter 5 reports on studies that add to our understanding of the mechanism of action of cyclotides, which is believed to involve membrane interactions. Spin-label experiments were performed for two cyclotides, kalata B2 and cycloviolacin O2, which are representative cyclotides from the two cyclotide sub-families, Möbius and bracelet, respectively. This study showed that different cyclotides have different but very specific binding modes at the membrane surface. Currently, it is believed that for Möbius cyclotides at least (e.g. kalata B1 and kalata B2), self-association may lead to the formation of membrane pores. Oligomerisation of cyclotides was also studied in this chapter using NMR relaxation. A computer program, NMRdyn, was developed to extract microdynamic and self-association parameters from NMR relaxation data. This program was used to analyse 13C relaxation data on kalata B1, providing clues about the tetramer structure of kalata B1. Although the three areas of cyclotide research examined in this thesis – sequence, structure and interactions – are reported in separate sections, the areas are not independent of each other. For example, the mechanism of action of cyclotides, which is reported in Chapter 3, requires an understanding of cyclotide structures, which is reported in Chapter 4. Chapter 6 describes a database, CyBase, which integrates sequence/structure/activity data on cyclotides so that relationships between the three areas can be examined. The database also provides tools to assist in discovery and engineering of cyclic proteins. In summary, several key areas that are fundamental to our understanding of cyclotides have been investigated in this thesis, ranging from cyclotide sequence diversity to their mechanism of action. The work described in this thesis represents a significant advance in our current understanding of cyclotides by providing, for example, explanations to their observed structural stability and how they work through interactions with other biomolecules. The information presented in this thesis is potentially useful in facilitating the long-term goal of developing peptide therapeutics based on the stable cyclotide framework.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

Peptides are considered as drugs of the future because of their advantageous features of high specificity and low toxicity. However, the complete therapeutic potential of peptides has not yet been realized because of the in vivo instability displayed by most potential peptides. In this thesis, two naturally derived cyclic peptides, cyclotides and sunflower trypsin inhibitor 1 (SFTI-1), were utilized to impart stability to linear bioactive epitopes and enhance their therapeutic potential in a biological environment. Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclised backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Sunflower typsin inhibitor 1 (SFTI-1) is a stable cyclic peptide containing a single disulfide bond. Taking advantage of these stable cyclic peptide frameworks, novel drug leads to inhibit/stimulate angiogenesis were developed by using the approach of ‘epitope grafting’ in which linear epitopes were grafted onto the cyclic peptide frameworks. Angiogenesis is a physiological condition that is unregulated in the progression of many diseases, including cancers and cardiovascular diseases. Thus the drug leads designed in the current project have potential therapeutic applications to combat cancers and cardiovascular diseases. To fully exploit cyclotides as drug scaffolds, it is imperative to understand their folding. Two main subfamilies, referred to as the Möbius and bracelet cyclotides have been identified and interestingly, they require dramatically different in vitro folding conditions to achieve formation of the conserved cyclic cystine knot motif. To determine the underlying structural elements that influence cyclotide folding, the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1 was examined in this thesis. The pathways of folding of the two cyclotide subfamilies were found to be different and primarily dictated by specific residues harboured within inter-cysteine loops 2 and 6. Two changes in these loops, an amino acid substitution in loop 2 and an amino acid addition in loop 6 enabled the folding of cycloviolacin O1 under conditions where folding does not occur in vitro for the native peptide. Thus, the study identified key residues that are not in close proximity in the primary sequence or three-dimensional structure which assist folding in cyclotides. A key intermediate species in the folding pathway was isolated and characterised, and found to contain a native-like hairpin structure that appears to be a nucleation locus early in the folding process. The intermediate does not have native disulfide connectivities, but disulfide shuffling processes ultimately lead to a rearrangement to the native form. Overall these mechanistic findings on the folding of cyclotides are potentially valuable for protein engineering applications that utilize cystine-rich peptides as scaffolds in the design of new drug leads. The current study has also enabled the extention of the grafting studies to the bracelet cyclotide subfamily, which was intractable to grafting prior to this work. Cyclotides are gene encoded macrocyclic proteins and another way to exploit their potential as drug scaffolds, would be to develop combinatorial cyclotide libraries. The most efficient way to generate engineered cyclotides would be via recombinant expression, which currently remains unsuccessful, partly due to lack of understanding of the mechanism of cyclotide backbone cyclization. Understanding how the cyclotide precursor folds may provide clues to how cyliclization occurs. A conserved region known as the N-terminal repeat (NTR) region in the cyclotide precursor has been speculated to play an important role in precursor folding. In this thesis, the function of the NTR in the folding of the cyclotide precursor in vitro was examined via the design of a series of constructs for the precursor protein for the prototypic kalata B1 cyclotide, with incremental additions of the NTR region. Analysis of the constructs by NMR spectroscopy for evidence of secondary structure revealed that the NTR does not assist folding of the cyclotide precursor in vitro. Using diffusion NMR, the unstructured nature of the constructs was localized to the NTR region. In a complementary study, structural analysis of the full length cyclotide precursor was carried out by expressing the precursor gene for kalata B1 in a bacterial expression system. The full-length precursor was found to be unstructured in solution despite approximately half of the precursor comprising the mature domain and NTR, both of which are structured in isolation. The unstructured nature of the cyclotide precursor suggested that a different environment, or indeed interaction of the NTR with a particular enzyme involved in processing, is necessary for it to adopt a well-defined conformation and allow processing to produce the mature circular protein. The information that NTR alone may not assist folding of the cyclotide precursors has provided new impetus to examine the role of other potential folding auxiliaries such as protein disulfide isomerase in cyclotide folding and has indirectly advanced the production of cyclotides via transgenic means. In summary, this thesis has provided a fundamental insight into the folding of cyclotides, both when expressed as part of a precursor protein and in isolation via solid phase chemical synthesis, and has exploited the potential of cyclic peptide scaffolds in drug design applications.

The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.

Orientador: Vanderlan da Silva Bolzani / Co-orientador: Alberto José Cavalheiro / Banca: Humberto Márcio Santos Milagre / Banca: Alessandra Regina Pepe Ambrozin / Resumo: Os ciclotídeos são produtos natura is de estrutura polipeptídica, contendo de 2 8 a 37 resíduos de aminoácidos, sendo seis deles resíduos de cisteína, altamente conservados e formados por ligações dissulfeto as quais exib em ciclização tipo "cabeça - cauda". Ess e arranjo estrutura l característico co nfere aos ciclotí deos uma estabilidade excepcional e resistência à proteólise . Devido a estas peculiaridades moleculares os ciclotídeos são substâncias com importante s funç ões biológica s, entre elas, de stacam - se as de defesa e adaptação dos organismos que as acumulam . São de gran de interesse para agricultura, agindo como inseticida s e, no uso medicinal p or apresentarem atividades anti - HIV, anti - helmíntic a, antimicrobian a e efeitos uterotônicos . A presente dis sertação trata d o estudo de ciclotídeos isolados d a planta Noisettia orchidiflora (Rudge) Gingins, pertencente à família Violaceae, um táxon de ocorrência frequente n a Mata Atlântica . Já existem dados na literatura sobre o estudo de ciclotídeos das raíze s e o presente estudo é importante na medida que registra a ocorrência desta classe de substâncias e a sua caracterização nas folhas e galhos desta espécie, levando a informações mais completas sobre a ocorrência de ciclotídeos em Violaceae . A extração de c iclotídeos do material vegetal ( galhos e folhas secos e moído s ) foi feita mediante maceração hidrometanólica até esgotamento do material vegetal, seguid a de partição líquido - líquido com diclorometano. A fase polar foi concentrada e submetida à crom atografia em coluna, empregando octadecilsilano (C 18 ) como fase estacioná ria, obtendo - se frações ricas em peptídeos. O processo de purificação foi realizado p or HPLC preparativ o e/ou analític o com eluição gradiente, empregando - se acetonitrila e ág ua como fase móv e l, o que permitiu a obt... / Abstract: C yclotides are pol ypeptide str uctures, c omprise 2 8 - 37 amino acid residues, six of the m, are cysteine residues, highly conserved and formed by disulfide bonds, which exhibit "head - tail" cyclization type. This structural feature gives to cyclotides remarkable stability and resistance to proteolysis. Due to these singularities, such compound s have disclosed important biological functions . A mong the m ; the literature has highlighted important ecological properties as defense and adaptation of the organisms, which accumulate these natural products. Accordingly, they are of great interest to agri culture, acting as insecticide, and to medicine, since some compounds have displayed anti - HIV activity, anthelmintic, antimicrobial and uterotonic properties. This research deals with the study of cyclotides isolated Noisettia orchidiflora (Rudge) Gingins, belonging to the taxon Violaceae, which frequently occurs in the Atlantic Forest. There are several studies in the literature on the occurr ence of this class of compounds and their characterization in the leaves and branches of Violaceae plant species, le ading to more complete information on the occurrence of cyclotides, and their biological significance for these plants. The cyclotides extraction of plant material (dried and ground branches and leaves) was carried out by maceration with methanol/water, fo llowed by a liquid - liquid partition with dichloromethane. The polar phase was concentrated and subjected to chromatographic column using octadecylsilane (C 18 ) as stationary phase, achieving several fractions rich in peptides. The purification process was p erformed by analytical and/or preparative HPLC, in gradient of acetonitrile and water as mobile phase, which led to the isolation of the cyclotides. The amino acid sequence (primary structure) of the isolated compounds was established by MALDI - TOF /TOF by r eduction and alkylation re... / Mestre