We have initiated the development of a screening platform to design a library of small molecules on the same solid support surface. This solid support surface, and the chemistry involved, can be utilized as a means of developing lead target molecules, namely ligands and catalysts. Evidence shows the successful assembly of both simple amino acids, as well as successful employment of our synthetic compounds. We support our efforts by showing compatibility for binding studies with larger macromolecules. Thus, intrigue remains by the prospects of this project. Challenges within our efforts are highlighted and emphasis is placed on presenting solutions to current issues, in order to attain further development. Notwithstanding difficulty, the desire to establish efficient processes for the discovery of lead target molecules and to ascertain the utility of our synthesized compounds, can be captured within this body of work. Lastly, the framework for continued efforts has been set to enable future progression. / Chemistry

Peptide drugs are used to treat a broad spectrum of diseases such as cancer and HIV and have many more promising applications, such as new vaccines against SARS-CoV-2. The most popular manufacturing method for peptides is solid-phase peptide synthesis (SPPS). The main drawback of SPPS is that it is a costly and wasteful process.  SpinChem is a company that provides technology solutions for chemical processes. Recently, SpinChem has started investigating if their Rotating Bed Reactor (RBR) is suitable for peptide synthesis. The goal of this project is to investigate how the RBR can make processes like SPPS more resource-efficient. The idea is that the RBR-system can maximize the solid-phase to liquid ratio (STL). The STL is the ratio of the volume of solid-phase material and the volume of liquid. By maximizing the STL, it is possible to manufacture peptides using less solvents and chemicals. The main quest of the project is formulated into a single question:  How does a high STL affect the efficiency of the RBR-system?  To answer the question, Minitab's statistical software and design of experiments (DOE) will be used to plan and perform experiments in both lab- and industrial scales. DOE factorial experiments are used to gain as much information as possible about the new RBR-system. The results are analyzed and summarized to make a solid foundation for the continued work on the new RBR application.  Peptide synthesis efficiency in the RBR-system is measured using ionic adsorption. The ionic adsorption rate is measured in both lab-scale and industrial-scale experiments. In the lab-scale experiments, the decrease of ions was on average 86,5% after just 15 s with an average STL of 0,936. The industrial-scale experiments showed a similar result where the average decrease in ions was 92,9% after 20 s with an average STL of 0,947. It was concluded that the RBR-system can reduce the consumption of washing-solvent in SPPS by up to 82%.

Cystinosis is a metabolic disorder characterised by the abnormal accumulation of the amino acid cystine in cells leading to a slow destruction of all major organs. If patients diagnosed with cystinosis are untreated, death due to kidney failure ensues in the second decade of life. A number of studies have shown the ability of the drug cysteamine (Cystagon®) to lower cystine accumulation within cells resulting in reduced organ and tissue damage. Cysteamine therapy however, is associated with a number of side effects involving the gastrointestinal tract and the central nervous system. Most of these arise due to the large amount of cysteamine present in the stomach and gut following administration. In addition, cysteamine possesses an unpleasant taste and smell, resulting in poor patient compliance. In an attempt to overcome these problems, a number of pro-drug derivatives of cysteamine and cystamine, the disulfide analogue of cysteamine, have been synthesised and evaluated. Pro-drugs were synthesised using a route established in our laboratories. Briefly, cystamine dihydrochloride was basified and allowed to react with a number of cyclic anhydrides under basic conditions. The resulting di-acids were reacted with carbonyldiimidazole and monoBoc-cystamine to yield the desired pro-drugs. Removal of the tBoc-protecting group was achieved in a facile manner by use of trifluoroacetic acid to yield product. The efficacy of the synthesised pro-drugs was determined by incubation of 50μM compound in a suspension of cultured cystinotic fibroblasts, with 50μM cysteamine as control. Cell growth was measured at 72 h and the level of thiol determined. All except one of the pro-drugs tested were significantly more effective than the control at lowering the cystine burden of the cells. Further work will concentrate on repeating these studies and evaluating a more robust Structure Activity Relationship for these compounds. The overall aim of all this work remains the production of an odourless, tasteless and orally active treatment for cystinosis and, if possible, improve on the current dosing regimen of every 6h. By using pro-drugs, cysteamine will be chemically camouflaged and hence, the side effects associated with its administration will be minimised or even entirely abolished.

The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels. A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides’ biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds. Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation.

Câncer é a denominação para um grupo de doenças devastadoras caracterizadas pelo crescimento e multiplicação descontrolados de células anormais que são capazes de invadir estruturas próximas e se espalhar por diversas regiões do organismo.Trata-se de um grande problema de saúde pública mundial, fazendo milhares de novas vítimas a cada ano. De acordo com o Instituto Nacional do Câncer (INCA), são estimados cerca de 580 mil casos novos da doença no Brasil para 2014. A presente tese de doutorado teve como foco principal a identificação e o desenvolvimento de novas moléculas com atividade anticâncer, através de triagens biológicas de compostos de origem natural e sintética, e do estudo das relações entre a estrutura e atividade (SAR). Triagens in vitro de derivados sintéticos do ácido gálico, ácido protocatecuico e guanidínicos, além de uma variedade de produtos naturais, possibilitaram a identificação de agentes inibidores da migração e proliferação de células tumorais metastáticas. Uma série de derivados sintéticos indólicos e espirocicloexadienonas, com potente efeito inibitório da migração celular, teve caracterizada a sua ação frente à proteína tubulina, alvo molecular de compostos importantes como o taxol, a vimblastina e a colchicina. Ensaios de imunofluorescência revelaram a ação dos compostos associada a alterações do citoesqueleto celular. Também foram realizados o planejamento e a síntese de três cadeias peptídicas por meio da metodologia de síntese peptídica em fase sólida. Os resultados da avaliação biológica dos peptídeos indicaram o efeito de inibição na migração e proliferação de células tumorais de mama. Finalmente, estudos de metabolômica de duas linhagens tumorais de mama foram conduzidos através de análises de RMN do material celular cultivado. / Cancer is a group of devastating diseases characterized by the abnormal growth of defective cells which invade adjacent tissues and eventually disseminate to several locations of the body. It is a major public health problem, affecting thousands of people each year. According to the brazilian National Institute of Cancer (INCA), approximately 580,000 new cases of cancer are predicted for the year of 2014 in Brazil. The main goal of this PhD thesis was the identification and development of new molecules possessing anticancer activity, through biological screenings of compounds from natural and synthetic sources, as well as the investigation of structure-activity relationships (SAR). In vitro screening of synthetic derivatives of gallic acid, protocatechuic acid and guanidines, along with a diverse set of natural products, allowed the identification of inhibitors of cellular migration and metastatic cell proliferation. A series of synthetic indolic derivatives and cyclohexanediones, having potent inhibitory activity in cellular migration, was characterized upon tubulin, an important macromolecular target for compounds such as taxol, vinblastine and colchicine. Immunofluorescence assays revealed that the compounds act by altering the cellular cytoskeleton. The design and synthesis of three polypeptides were also performed through solid phase synthesis. The biological evaluation of the peptides demonstrated their inhibition effects on the migration and proliferation of breast cancer cells. Finally, metabolomic studies of two strains of breast cancer cells were conducted by NMR analyses of cellular cultures.

Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective  and  flexible  radiolabeling  and  enhancing  the  molecular  imaging  in  terms  of  imaging contrast and resolution. In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino  acids,  thereby  allowing  for  both  synthetic  and  recombinant  production.  By  grafting  the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with  99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with  111 In, while the peptide sequence was able to chelate  99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds. In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with  111 In via chelation by  DOTA  and  directly  compared  in  terms  of  biodistribution  and  targeting  properties.  Results showed  that  the  smaller  variant  can  provide  higher  contrast  images,  at  the  cost  of  lower  tumor uptake,  in  high-expressing  HER2-tumors.  However,  neither  the  tumor  uptake  nor  the  contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2. In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with  18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum  18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the  18 F-NOTA-approach is a very promising method of labeling Affibody molecules with  18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work  18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation. / <p>QC 20130203</p>

Effective detection and treatment of tumor malignancies depends upon identifying targets – molecular markers that differentiate cancer cells from healthy cells. Current cancer therapies involve targeting overexpressed specific gene products. An alternative approach is proposed here: to specifically target combinations of cell-surface receptors using heteromultivalent ligands (htMVLs). There are about 2500 genes encoding for cellsurface proteins in the human genome that can potentially be targeted. Taken as sets, there can be ~ 10⁶ two-receptor combinations and ~ 10⁹ three-receptor combinations available. Our group envisions that using cell-surface protein combinations that are expressed on a cancer cell but not on a normal cell, multivalent constructs displaying complementary ligands of weak affinities can be assembled. These multivalent ligands should bind with high avidity to cancer populations in vivo, and provide a degree of specificity not seen with current approaches. As a proof-of-concept, a series of multivalent ligands were designed and synthesized for a model system consisting of the human Melanocortin subtype 4 receptor (hMC4R) and the Cholecystokinin subtype 2 receptor (CCK-2R). Modeling studies on GPCR dimers predicted that a minimum linker span of 20 - 50 Å would be required to non-covalently crosslink these two receptors. The multivalent ligands were assembled using a modular parallel synthesis approach and using solidphase chemistries. A variety of linkers were explored ranging from highly rigid to highly flexible, and using natural and/or synthetic building blocks. Ligand binding affinities were evaluated using a lanthanide based competitive binding assay in cells that expressed both receptors (bivalent binding) vs those that expressed only one of the receptors (monovalent binding), and were demonstrated to have enhanced binding affinities of up to nearly two orders of magnitude. The promising ligands were further explored by synthesizing fluorescently labeled and/or lanthanide chelate labeled monovalent and heterobivalent ligands designed for in vitro and in vivo studies. More explorative work using these labeled constructs is in progress. To the best of our knowledge, the author believes this is the first such demonstration of a 'synthetic htMVL' directed recruitment and crosslinking of two heterologous cell-surface receptors. This receptor combination approach opens up new possibilities for single cell imaging, cancer detection and therapeutic intervention, and can provide a revolutionary new platform technology with which to direct therapeutics to defined cell populations.

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering. / <p>QC 20140207</p>

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>

Η Heparin affin regulatory peptide (HARP) είναι ένας αυξητικός παράγοντας με μοριακό βάρος 18 kDa, που έχει μεγάλη συγγένεια με την ηπαρίνη. Είναι συντηρημένη μεταξύ διαφόρων ειδών και παρουσιάζει 50% ομολογία με τη Midkine και την RI-HBP. Οι πρωτεΐνες αυτές συγκροτούν μια σχετικά νέα οικογένεια αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Η HARP απομονώθηκε για πρώτη φορά από τον εγκέφαλο νεογέννητου βοός ως ένα μόριο που μπορεί να επάγει την προέκταση των νευρικών κυττάρων. Επίσης, εκφράζεται στη μήτρα, στους χόνδρους και στα οστά. Αρκετές αναφορές αποδεικνύουν ότι υπάρχει μεγάλη συσχέτιση μεταξύ της έκφρασης της HARP και της ανάπτυξης καρκινικού όγκου και της αγγειογένεσης. Η HARP αποτελεί μιτογόνο παράγοντα για διάφορους τύπους ενδοθηλιακών κυττάρων, ενώ μπορεί να επάγει την αγγειογένεση in vivo και in vitro. Ασκεί τη βιολογική της δράση μετά από αλληλεπίδραση με πρωτεογλυκάνες της επιφάνειας του κυττάρου, όπως η N-συνδεκάνη, ή μετά από δέσμευση σε πιο ειδικούς υποδοχείς. Η RPTPβ/ζ, η εκκρινόμενη μορφή της (φωσφακάνη), αλλά και η κινάση ALK, έχει αναφερθεί ότι μπορούν να δεσμεύουν τη HARP και να συμμετέχουν στη μεταγωγή του σήματός της. Ο κύριος σκοπός αυτής της διατριβής είναι η μελέτη της δομής και της δράσης της HARP, χρησιμοποιώντας μικρότερα τμήματα της HARP τα οποία τα έχουμε βιοτινυλιώσει για να διερευνήσουμε τις περαιτέρω δράσεις της, τους διάφορους μηχανισμούς και με ποιους υποδοχείς αλληλεπιδρά. Τα βιοτινυλιωμένα-σεσημασμένα πεπτίδια αποτελούν χρήσιμα εργαλεία για την βιοτεχνολογία, σε διάφορες εφαρμογές στερεής φάσης ανοσολογικών δοκιμών καθώς και τον εντοπισμό των υποδοχέων. Τα συνθετικά πεπτίδια της HARP συντέθηκαν με μεθοδολογία Fmoc/t-Bu σε στερεά φάση χρησιμοποιώντας ως στερεό υπόστρωμα τη 2-χλωροτρίτυλ-ρητίνη για την παραλαβή μιας ελεύθερης αμινοομάδας (ΝΗ2) και C-τελικού καρβοξυλικού οξέος αντίστοιχα. Για την παραγωγή βιοτινυλιωμένων πεπτιδίων, περιλαμβάνει τον σχηματισμό ενός δεσμού: της ελεύθερη αμινοομάδα με την βιοτίνη στην διάρκεια της πεπτιδικής σύνθεσης στερεής φάσης. Επειδή η βιοτίνη έχει μικρή διαλυτότητα στα διαλύματα της πεπτιδικής σύνθεσης χρησιμοποιούμε τους προσχηματισμένους ενεργοποιημένους εστέρες όπως την βιοτίνη- ONp (biotin p-nitrophenyl ester ). Τα συνθετικά πεπτίδια P(13-39) και Ρ (65-97), που αντιστοιχούσαν στην Ν-CTR-I και C-TSR-I περιοχή της HARP μελετήθηκαν ως προς την επίδρασή τους στον πολλαπλασιασμό και στην μετανάστευση των καρκινικών κυττάρων του προστάτη (PC3). / Heparin affin regulatory peptide (HARP) is an 18 kDa growth factor that has a high affinity for heparin. HARP is highly conserved among species and shares 50% homology with Midkine and RI-HBP. The above proteins constitute a relatively new family of growth factors with high affinity for heparin. HARP has been originally purified from perinatal rat and bovine brain as a molecule that induces neurite outgrowth. HARP is also expressed in uterus, cartilage and bone extracts. Several reports have established a strong correlation between HARP expression and tumour growth and angiogenesis. HARP has been reported to be mitogenic for different types of endothelial cells and angiogenic in vivo and in vitro. HARP exerts its biological activity through interactions with cell surface proteoglycans, such as N-syndecan, or binding to more specific cell surface receptors. Receptor-type protein tyrosine-phosphatase β/ζ (RPTPβ/ζ) and its secreted variant phosphacan, as well as ALK are implicated in HARP signalling. The main target of this study is to investigate the structure and the biological activities of HARP, using smaller fragments of HARP which are biotinylated in order to study its further activities, the mechanisms and the interactions with the receptor. Biotin-labelled peptides are extremely useful tools for biochemistry, with applications in solid-phase immunoassays, affinity purification and receptor localization. The synthetic peptides of HARP were synthesized by Fmoc/t-Bu solid phase methodology utilizing a 2-chlorotrityl-chloride resin to provide a free NH2- amino-group and carboxyl acid, respectively. The simplest approach for the production of biotin-labelled peptides involves capping of a resin-bound free amino group with biotin, prior to cleavage of the peptide from the resin and side-chain deprotection. Because of the poor solubility of biotin in peptide synthesis solvents, biotin is most frequently introduced using a pre-formed active ester, such as biotin p-nitrophenyl ester. The synthetic peptides, P(13-39) and P(65-97) with amino acids sequence corresponding to the N- and C-TSR-I domains of HARP, were studied for their impact to the proliferation and chemotactic (migration) on prostate cancer cells (PC3).