| 1. |
- Hakobyan, Shoghik, 1981-
(författare)
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Antivirulent and antibiofilm salicylidene acylhydrazide complexes in solution and at interfaces
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The growing bacterial resistance against antibiotics creates a limitation for using traditional antibiotics and requests development of new approaches for treatment of bacterial infections. Among the bacterial infections that are most difficult to treat, biofilm-associated infections are one of the most hazardous. Consequently, the prevention of biofilm formation is a very important issue. One of the techniques that are widely investigated nowadays for this purpose is surface modification by polymer brushes that allows generating antifouling antibacterial surfaces. Previously, it was reported that salicylidene acylhydrazides (hydrazones) are good candidates as antivirulence drugs targeting the type three secretion system (T3SS). This secretion system is used by several Gramnegative pathogens, including Pseudomonas aeruginosa, to deliver toxins into a host cell. Furthermore, the chemical structure of these substances allows formation of complexes with metal ions, such as Fe3+ and Ga3+. The antibacterial activity of Ga3+ is well known and attributed to its similarity to the Fe3+ ion. It has also been shown that Ga3+ ions are able to suppress biofilm formation and growth in bacteria. In this thesis the chemistry of antibacterial and antivirulence Ga3+-Hydrazone complexes in solution was studied. First, to get insights in the solution chemistry, the protonation and the stability constants as well as the speciation of the Ga3+-Hydrazone complexes were determined. Additionally, a procedure for anchoring one of the hydrazone substances to antifouling polymer brushes was optimized, and the resulting surfaces were characterized. Results showed that the complexation with Ga3+ ions stabilizes the ligand and increases its solubility. Ga3+ ion binds to the hydrazone molecule forming a strong chelate that should be stable at physiological conditions. The different biological assays, such as Ga3+ uptake, antivirulence and antibiofilm effects, indicated very complex interaction of these complexes with the bacterial cell. Negatively charged and zwitterionic surfaces strongly reduced protein adsorption as well as biofilm formation. Therefore, the antifouling zwitterionic poly-[2-(methacryloyloxy)ethyl]dimethyl-3- sulfopropyl)-ammonium hydroxide (pMEDSAH) brushes were post-modified and successfully functionalized with bioactive substances via a block-copolymerization strategy. However, in order to maintain the availability of the bioactive substance after functionalization, the hydrophobic polyglycidylmethacrylate (pGMA) top block is probably better to functionalize with a lipophilic molecules to reduce diblock copolymer brush rearrangement.
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| 2. |
- Johansson, Emil, 1985-
(författare)
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Tailored conjugates of N-acetylneuraminic acid and small molecules that block virus cell attachment and entry
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Viruses are obligate intracellular parasites, unable to replicate without exploiting machinery andmaterials from host cells. Pandemics of viral diseases have had large impacts on human socieities and are continued threats to global health. The most efficient means of controlling viral diseases are preventive measures such as immunization of the population, social distancing, and basic hygiene routines. Another mean is development of antiviral drugs that could be used as preventive measures and in treatment of infected individuals. Coxsackievirus A24 variant (CVA24v) is a highly contagious pathogen that cause large outbreaks and pandemics of the eye infection acute hemorrhagic conjunctivitis. Human adenovirus D species type 37 (HAdV-D37) causes epidemics of the severe eye infection epidemic keratoconjunctivitis, that can become life-threatening in immunocompromised individuals. Currently, no specific treatments (vaccine or antivirals) are available to combat the diseases caused by these two pathogens.CVA24v and HAdV-D37 bind to N-acetylneuraminic acid (Neu5Ac) glycans on host cells facilitating attachment and subsequent infection. In this thesis, we explored inhibition of this common recognition motif by development of pentavalent Neu5Ac containing molecules with radial topology to act as decoy receptors. This allowed us to study the potential of development of a general inhibitor targeting both these viruses. The developed compounds inhibited attachment of CVA24v and HAdVD37 to cells.Furthermore, we developed divalent Neu5Ac tools to validate if targeting the Neu5Acmediated attachment of CVA24v to cells were a potential target for antiviral drug discovery and development. The results from these studies indicate that development of a Neu5Ac-based antiviral targeting CVA24v looks bleak as the primary receptor utilized by this virus is ICAM-1. The work with developing Neu5Ac tools led to a side project with synthesis of 4-O-alkyl Neu5Ac analogs. In this project we provided a method to synthesize 4-O-alkyl analogs of Neu5Ac and gave insights into the scope of the reaction. This work could have have value in drug discovery.Targeting enterovirus uncoating is a well explored strategy for the inhibition of enterovirus infection. In this thesis, we synthezied novel branched probes of pleconaril (a well-known pocket binding molecule) to study if targeting the unique branched pocket of CVA24v could have potential as a target for antiviral drug discovery. Further experiments are needed to draw conclusions in regards to the future prospects of targeting this unique feature.At last, two novel classes of trivalent Neu5Ac conjugates were develop using a structure-based approach targeting HAdV-D37, -D36, and -D26. This led to a more potent compound towards HAdVD37 further validating that targeting the attachment of this virus to cells is a reasonable strategy for antiviral drug development. Towards HAdV-D26 the inhibitory effect was saturated at 50%, likely due to engagement of other receptors. Evaluation towards HAdV-D36 is currently ongoing. Structural biology studies, indicates the compounds bind to the viruses via chelation of their trimeric binding sites. Taken together, these compounds have potential to be used as chemical tools to study the biology of human adenoviruses and perhaps other Neu5Ac binding proteins.
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| 3. |
- Johansson, Leif B.G.
(författare)
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Asymmetric Oligothiophenes : Chemical Evolution of Multimodal Amyloid Ligands
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Luminescent conjugated polymers (LCPs) and luminescent conjugated oligothiophenes (LCOs) can be used as molecular probes to study diseases associated with protein aggregation. The conventionally used dyes to study and detect protein aggregates, denoted amyloid, have been Congo red (CR) and Thioflavin T (ThT). In contrast to these amyloid ligands, LCOs offer the possibility to detect aggregated proteinaceous species occurring at earlier stages of amyloid formation as well as to distinguish different morphotypes of protein aggregates. The interaction between the LCOs and the protein deposits can be studied by fluorescence spectroscopy and microscopy both in vitro and ex vivo. In this thesis we report the development of multimodal asymmetric LCOs that can be utilized with two novel techniques, Surface Plasmon Resonance (SPR) and Positron Emission Tomography (PET), to study the interaction between LCO and amyloid fibrils in real time. With SPR, we have been able to determine binding affinities between LCO and amyloid, and with PET we have shown that radiolabelled LCOs can be used as a non-invasive method to study amyloid deposits in vivo. In addition, by alteration of the backbone (change of thiophene units), and of adding different side chains functionalities, we have shown that the properties of the amyloid ligands have a huge impact of the binding to different stages or forms of protein aggregates. By making asymmetrical LCOs, which can be attached to a surface, we also foresee a methodology that will offer the possibility to create a sensitive and selective detection method, and maybe lead to a lab-on-a-chip-application.
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| 4. |
- Strand, Mårten, 1982-
(författare)
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The discovery of antiviral compounds targeting adenovirus and herpes simplex virus : assessment of synthetic compounds and natural products
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease.Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types.To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity.This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.
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| 5. |
- Ur-Rehman, Tofeeq, 1971-
(författare)
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Controlled release gel formulations and preclinical screening of drug candidates
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications. In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel. All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.
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| 6. |
- Behren, Sandra, 1990-
(författare)
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Development and Evaluation of Tools to Explore Posttranslational HexNAc-Tyrosine and Mucin-Type O-Glycosylation
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glycosylation is the most abundant form of post-translational modification (PTM). Recently, O-glycosylation attracted much attention in the glycoproteomic field due to its association with various diseases, such as pathogenic infections and cancer. However, glycoproteomic analysis of O-linked glycosylation is highly challenging due its structural diversity and complexity. New and efficient methods need to be developed to obtain a better understanding of the biological functions of O-glycans. In the presented thesis, glycopeptide microarrays were used as tools to explore the role of mucin type O-glycosylation in cancer, bacterial adhesion processes and galectin recognition on a molecular level, and to get insights into a new group of tyrosine O-glycosylation. A better understanding of these carbohydrate-protein interactions on a molecular level could facilitate the development of glycomimetic inhibitors to fight bacterial infections or block glycan binding proteins involved in cancer progression, or improve the design of novel carbohydrate-based cancer vaccines.In the first part of this work, tools were developed to elucidate the role of a novel group of PTMs, where N-acetylhexosamine (HexNAc = α-GalNAc, α- or β-GlcNAc) was found to modify the hydroxyl group of tyrosine. Synthetic glycopeptides carrying this new modification, as well as glycopeptide microarray libraries were prepared to evaluate the abilities of plant lectins (carbohydrate-binding proteins) to detect HexNAc-O-Tyr modifications. These lectins are commonly used in glycoproteomic work flows to detect and enrich glycopeptides and -proteins. Additionally, HexNAc-O-Tyr-specific rabbit antibodies were raised and immunologically analyzed by enzyme-linked immunosorbent assays, western blot and microarray binding studies.In the second part of the presented thesis, synthetic mucin glycopeptide microarray libraries were prepared and employed to explore carbohydrate-protein interactions of galectins, bacterial lectins and tumor specific antibodies. Mucin glycoproteins are part of the mucus barrier that protects the host against invading pathogens. However, bacteria and viruses have co-evolved with the human host and have developed strategies to promote virulence, for example by adhering to glycans on the host cell-surface. To combat bacterial infections, their virulence and pathogenicity must be understood on a molecular level. In this work, mucin glycopeptides were enzymatically modified with different fucose motifs and used to determine the fine binding specificities of fucose-recognizing lectins LecB from Pseudomonas aeruginosa and the Clostridium difficile toxin A. Furthermore, a synthesis strategy was developed to generate simplified mucin core glycopeptides that could be used as scaffolds to enzymatically generate LacdiNAc modified glycopeptides. They could be used in microarray binding studies to evaluate the glycan binding preferences of various proteins, including the Helicobacter pylori lectin LabA and human galectins, which play roles in cancer development and progression. Aberrant glycosylation of mucin glycoproteins has been associated with various types of cancer. Tumor specific carbohydrate antigens on mucins represent attractive antigenic targets for the development of effective anti-cancer vaccines. In this work, antibodies induced by tumor-associated MUC1 glycopeptide-bacteriophage Qβ vaccine conjugates were immunologically analyzed using MUC1 glycopeptide microarray libraries.
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| 7. |
- Dingeldein, Artur
(författare)
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Bax and oxidized phospholipids - a deadly complex : Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are renowned for their vital role as cellular powerhouses because they provide ATP via cellular respiration. Additionally, these organelles also play an important role in other physiological processes, such as apoptosis. Apoptosis, or regulated cell death, is an important mechanism that regulates, for example, tissue homeostasis and embryonic development. Malfunctioning apoptosis can cause severe diseases such as various types of cancer and neurodegenerative diseases. The significance of mitochondria for apoptosis is that mitochondria host a variety of apoptogenic factors, such as cytochrome c. The release of these factors after the formation of an apoptotic pore can be regarded as a point of no return in the onset of apoptosis as these factors trigger the activation of caspases and consequently nuclear fragmentation.The mitochondrial outer membrane (MOM) is essential for deciding the cell’s fate, since the MOM provides an interaction surface for the pro- and anti-apoptotic proteins of the Bcl-2 protein family. Further, oxidized phospholipids (OxPls) within the MOM that are generated under oxidative stress conditions (a potent pro-apoptotic stimulus) can directly affect the equilibrium between pro- and anti-apoptotic proteins at the MOM surface, hence influencing the formation of apoptotic pores.To characterize the impact of different OxPls on membrane dynamics and organization, several MOM-mimicking systems were studied by solid-state magic angle spinning nuclear magnetic resonance spectroscopy (MAS NMR). These main experiments were accompanied by fluorescence spectroscopy and differential scanning calorimetry (DSC) studies to investigate the impact of OxPls and their interactions with the pro-apoptotic Bax protein at both the macroscopic and molecular levels. By combining several orthogonal methods, we were able to obtain a detailed description of the changes in MOM-mimicking vesicles induced by several types of OxPls. Moreover, we demonstrated how these changes impact the interaction between liposomes and the pro-apoptotic Bax protein.By using DSC, we were able to determine not only the macroscopic effect of two OxPls – PazePC and PoxnoPC – but also a concentration threshold. Both OxPls disrupted the membrane order such that the melting behavior of the MOM-mimicking vesicles became less cooperative. A decrease in cooperativity was detectable for OxPl concentrations of up to 5 mol%, after which the cooperativity remained constant. The addition of Bax resulted in an observable ordering effect, as some of the membrane disorder was negated by Bax and the melting process became more cooperative again. The ordering effect of Bax was subsequently confirmed by 31P MAS NMR experiments and cross polarization (CP) buildup curves. Analysis of the buildup curves revealed that at the molecular level, Bax enabled more efficient CP transfer, which indicated rigidification of the membranes after Bax insertion. Furthermore, the 31P NMR experiments provided the first molecular evidence of the importance of cardiolipin as a membrane contact site for Bax.Despite having similar disordering effects when studied with DSC, PoxnoPC and PazePC exhibited opposing effects on the pore formation ability of Bax. In studies with fluorescent dye-based leakage assays, Bax was able to form long-lived, stable pores in PazePC-containing giant unilamellar vesicles (GUVs). However, the observed leakage mechanism in PoxnoPC-containing GUVs could no longer be explained by an all-or-none leakage mechanism due to the brevity of the formed pores, leading to partially leaked vesicles, indicating a graded leakage mechanism instead.To investigate the possible reasons for the different leakage activities and to obtain mechanistic insights, we conducted 13C MAS NMR experiments. These experiments enabled us to pinpoint specific carbon sites in the different MOM-mimicking systems and to study their dynamic profile as a function of temperature. Though the OxPl-containing multilamellar vesicles (MLVs), compared with non-oxidized systems, also showed drastic dynamic changes, the molecular differences between PazePC- and PoxnoPC-containing vesicles were not significant enough to constitute a structural reason for the opposing leakage activities.In addition to investigating membrane dynamics, we were able to establish a novel strategy for producing cytotoxic Bax protein. This novel expression and purification strategy increased the obtained yields by an order of magnitude. By deploying a double fusion approach, we were able to inhibit both termini of the protein from exhibiting their disruptive, yield-diminishing interactions with the host cell membranes.In conclusion, over the course of this thesis we were able to develop fast, yet powerful tools to investigate the dynamic changes of MOM-mimicking vesicles under the influence of OxPls and pro-apoptotic Bax. In the future, these tools could be used to characterize the underlying protein-lipid interactions that are responsible for the opposing leakage activities. Due to the development of a novel Bax production strategy, future research could shift to protein-focused studies with the primary goal of determining the structure of the apoptotic Bax pore.
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| 8. |
- Engen, Karin, 1988-
(författare)
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Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP) : Identification, Synthesis and Evaluation
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Insulin-regulated aminopeptidase (IRAP) has emerged as a potential new therapeutic target for treatment of cognitive disorders. Inhibition of the enzymatic activity facilitates cognition in rodents. Potent and selective peptide and pseudopeptide based inhibitors have been developed, but most of them suffer from poor pharmacokinetics and blood-brain-barrier penetration. Hence, development of less-complex inhibitors with good pharmacokinetic properties are of great importance.The aim of this thesis was to identify and optimize new small-molecule based IRAP inhibitors for use as research tools to investigate the cognitive effects of IRAP inhibition. Adaptation of an existing enzymatic assay into a screening compatible procedure allowed the evaluation of 10,500 compounds as IRAP inhibitors. The screening campaign resulted in 23 compounds displaying more than 60% inhibition. Two of these compounds, a spiro-oxindole dihydroquinazolinone and an imidazo[1,5-α]pyridine, were further investigated in terms of structure-activity relationship, physicochemical properties, metabolic stability, and mechanism of inhibition.Spiro-oxindole dihydroquinazolinone based IRAP inhibitors were synthesized via fast and simple microwave-promoted reactions, either in batch or in a continuous flow approach. The most potent compounds displayed sub-µM affinity, and interestingly an uncompetitive mode of inhibition with the synthetic substrate used in the assay. Molecular modeling confirmed the possibility of simultaneous binding of the compounds and the substrate. Furthermore, the molecular modeling suggested that the S-enantiomer accounts for the inhibitory effect observed with this compound series. The compounds also proved inactive on the closely related enzyme aminopeptidase N. Unfortunately, the spiro-oxindole based inhibitors suffered from poor solubility and metabolic stability.Imidazo[1,5-α]pyridine based IRAP inhibitors were synthesized via a five step procedure, providing inhibitors in the low-µM range. The stereospecificity of a methyl group proved important for inhibition. The compound series displayed no inhibitory activity on aminopeptidase N. Intriguing, these compounds exhibit a noncompetitive inhibition mechanism with the model substrate. As observed for the spiro-compounds, the imidazopyridines suffered from both poor solubility and metabolic stability. In summary, the work presented in this thesis provide synthetic procedures, initial structure-activity relationship, and pharmacological evaluation of two distinct inhibitors classes. The compounds are among the first non-peptidic IRAP inhibitors presented, serving as interesting starting points in the development of research tools for use in models of cognition.
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| 9. |
- Johansson, Susanne, 1977-
(författare)
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Design and Synthesis of Sialic Acid Conjugates as Inhibitors of EKC-causing Adenoviruses
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.
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| 10. |
- Lindgren, Anders, 1985-
(författare)
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Synthesis of Small Molecules Targeting ADP-Ribosyltransferases and Total Synthesis of Resveratrol Based Natural Products
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diphtheria Toxin-like ADP-RibosyltransferasesThe Human ADP-ribosyl transferases (ARTDs) are a group of poorly studied enzymes which are believed to be involved in e.g. DNA repair, protein degradation, transcription regulation and cell death. Medicinal chemistry programmes aimed at developing selective inhibitors of these ARTDs were initiated. A suitable starting compound for one of these enzymes, ARTD3, was found by screening a library of NAD-mimics using a thermal shift assay. A virtual screening protocol was instead developed in order to find novel inhibitors of ARTD7, 8, and 10. The hit compounds were then further developed into selective inhibitors of the corresponding ARTDs by systematically varying different structural features using a combination of synthetic organic chemistry, computational chemistry and structural biology. Compounds were initially characterized using differential scanning fluorimetry which was later replaced with an enzymatic assay to obtain IC50 values. Biotinylated analogs were also synthesized in an attempt to develop an AlphaScreen assay. A selective ARTD3 inhibitor was ultimately identified and found to delay DNA repair in cells after γ-irradiation. These compounds are potentially valuable tools for elucidating the biological role of the poorly characterized ARTD-family of proteins.Total Synthesis of Resveratrol Based Natural ProductsThe polyphenolic natural product (-)-hopeaphenol was found to inhibit the type III secretion system present in certain gram-negative bacteria. (-)-Hopeaphenol is a tetramer of resveratrol and in order to investigate whether the entire structure was essential for inhibition two resveratrol dimers, ε-viniferin and ampelopsin B, were synthesized using a flexible and divergent synthetic route. Highlights of the synthetic strategy include the use of cyclopropylmethyl protecting groups, allowing an acid mediated three-step-one-pot deprotection-epimerization-cyclization of an advanced intermediate to form ampelopsin B. All previously reported syntheses of these two natural products include a dimerization of resveratrol which severly limits the possibilities to synthesize structural analogs. This new strategy enables the synthesis of a wide variety of analogs to ε-viniferin and ampelopsin B.
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