Virtual screening is a fast, low cost method to identify potential small molecule therapeutics from large chemical databases for the vast amount of target proteins emerging from the life sciences and bioinformatics. In this work, we applied several conventional and newly developed virtual screening approaches to identify novel non-steroidal ligands for the human and zebrafish sex hormone binding globulin (SHBG). The ‘benchmark set of steroids’ is a set of steroids with known affinities for human SHBG that has been widely used for validation in the development of different virtual screening methods. We have updated this data set by including additional steroidal SHBG ligands and by modifying the predicted binding orientations of several benchmark steroids in the SHBG binding site based on the use of an improved docking protocol and information from recent crystallographic data. The new steroid binding orientations and the expanded version of the benchmark set was then used to create new in silico models which were applied in virtual screening to identify high-affinity non-steroidal human SHBG ligands from a large chemical database. Anthropogenic compounds with the capacity to interact with the steroid-binding site of SHBG pose health risks to humans and other vertebrates including fish. We constructed a homology model of SHBG from zebrafish and applied virtual screening to identify ligands for zebrafish SHBG from a set of 80 000 existing commercial substances, many of which can be exposed to the aquatic environment. Six hits from this in silico screen were tested experimentally for zebrafish SHBG binding and three of them, hexestrol, 4-tert-octylcatechol, dihydrobenzo(a)pyren-7(8H)-one demonstrated micromolar binding affinity for the zebrafish SHBG. These findings demonstrate the feasibility of using virtual screening to identify anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Studies applying this new computational toxicology method could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.

Caused by the organism Mycobacterium tuberculosis (Mtu), the globally distributed disease tuberculosis was responsible for the deaths of 1.4 million people in 2011. Anthranilate phosphoribosyltransferase (AnPRT) is an enzyme that catalyses the second committed step of the tryptophan biosynthetic pathway within Mtu, and is a promising target for antibiotics. This research aimed to further understand the mechanics of the AnPRT active site, in order to provide useful information towards AnPRT drug design. AnPRT inhibition and alternate substrates were investigated as well as variant AnPRT proteins, the results of which aided in unravelling a complex active site mechanism and illuminating several decisive inhibition strategies.

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

<p>Leonotis leonorus and Mentha longifolia are two herbs commonly used in South Africa, mostly in oral liquid dosage forms. Several disadvantages are associated with these traditional dosage forms which can perhaps be remedied by using an appropriate oral solid dosage form, provided the actual plant material in the latter still resemble, as closely as possible, the traditionally used material and provide products of suitable pharmaceutical quality. The objectives of this study were to prepare and evaluate the pharmaceutical suitability of the freeze-dried aqueous extracts of Leonotis Leonorus and Mentha Longifolia as plant raw material for the capsule dosage of these two therapies and to formulate and manufacture capsules of Leonotis Leonorus and Mentha Longifolia aqueous extract that would contain amounts of the plant materials equivalent to that found in their traditional liquid dosage forms, and have immediate release characteristics and suitability stability.</p>

Virtual screening is a fast, low cost method to identify potential small molecule therapeutics from large chemical databases for the vast amount of target proteins emerging from the life sciences and bioinformatics. In this work, we applied several conventional and newly developed virtual screening approaches to identify novel non-steroidal ligands for the human and zebrafish sex hormone binding globulin (SHBG). The ‘benchmark set of steroids’ is a set of steroids with known affinities for human SHBG that has been widely used for validation in the development of different virtual screening methods. We have updated this data set by including additional steroidal SHBG ligands and by modifying the predicted binding orientations of several benchmark steroids in the SHBG binding site based on the use of an improved docking protocol and information from recent crystallographic data. The new steroid binding orientations and the expanded version of the benchmark set was then used to create new in silico models which were applied in virtual screening to identify high-affinity non-steroidal human SHBG ligands from a large chemical database. Anthropogenic compounds with the capacity to interact with the steroid-binding site of SHBG pose health risks to humans and other vertebrates including fish. We constructed a homology model of SHBG from zebrafish and applied virtual screening to identify ligands for zebrafish SHBG from a set of 80 000 existing commercial substances, many of which can be exposed to the aquatic environment. Six hits from this in silico screen were tested experimentally for zebrafish SHBG binding and three of them, hexestrol, 4-tert-octylcatechol, dihydrobenzo(a)pyren-7(8H)-one demonstrated micromolar binding affinity for the zebrafish SHBG. These findings demonstrate the feasibility of using virtual screening to identify anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Studies applying this new computational toxicology method could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.

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.

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.