| 1. |
- Andréasson, Måns, 1994-
(författare)
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Redefining the essential molecular aspects that drive interactions between small molecules and G-quadruplex DNA
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- G-Quadruplex (G4) structures are secondary nucleic acid structures located in guanine-rich regions of DNA and RNA sequences, involved in gene regulation and cellular maintenance. Efforts to target G4s in a therapeutic setting are scarce, mainly due to vague details about the binding interactions between the ligands and the G4 structure combined with the lack of emphasis on drug-like properties early in the ligand development process. Furthermore, the ability to target specific G4 structures with small drug-like molecules remains a big challenge to overcome in the field. In this thesis, extensive organic synthesis developments coupled with computational-aided design and orthogonal in vitro assays has been used in tandem to reveal in-depth knowledge about ligand-to-G4 interactions. First, a macrocyclic approach was applied to design and discover novel G4 ligands which showed that macrocycles offer a solid foundation for ligand design. Next, computational tools to optimise the macrocyclic molecular conformation were used based on the macrocycles' abilities to stack on the G4 surface. In addition, macrocyclic, and non-macrocyclic ligands that bound G4 with high potency were shown to correlate with electron-deficient electrostatic potential (ESP) maps. The frequent inclusion of cationic residues in G4 ligands and their enhancement on ligand-to-G4 binding was, thereof, ascribed to their impact on the electrostatic character of the ligands' arene-arene interactions with the G4 surface, and not through direct electrostatic ionic interactions. In addition, the dispersion energetic component in the arene-arene interactions between the G4 ligand and the G4 was discovered to be paramount for ligand-to-G4 binding. The implementation of these descriptors in practice resulted in the discovery of potent G4 binders with adequate pharmacokinetic (PK) properties, accentuating the significance of understanding the molecular interactions between ligands and G4s in rational ligand design. Finally, a G4 ligand conjugated to an oligonucleotide was demonstrated as a modular approach to achieve selective binding of a ligand to a specific G4 structure.
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| 2. |
- Andersson, Ida E., 1982-
(författare)
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Modified Glycopeptides Targeting Rheumatoid Arthritis : Exploring molecular interactions in class II MHC/glycopeptide/T-cell receptor complexes
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that leads to degradation of cartilage and bone mainly in peripheral joints. In collagen-induced arthritis (CIA), a mouse model for RA, activation of autoimmune CD4+ T cells depends on a molecular recognition system where T-cell receptors (TCRs) recognize a complex between the class II MHC Aq protein and CII259-273, a glycopeptide epitope from type II collagen (CII). Interestingly, vaccination with the Aq/CII259-273 complex can relieve symptoms and cause disease regression in mice. This thesis describes the use of modified glycopeptides to explore interactions important for binding to the Aq protein and recognition by autoimmune T-cell hybridomas obtained from mice with CIA. The CII259-273 glycopeptide was modified by replacement of backbone amides with different amide bond isosteres, as well as substitution of two residues that anchor the glycopeptide in prominent pockets in the Aq binding site. A three-dimensional structure of the Aq/glycopeptide complex was modeled to provide a structural basis for interpretation of the modified glycopeptide’s immunological activities. Overall, it was found that the amide bond isosteres affected Aq binding more than could be explained by the static model of the Aq/glycopeptide complex. Molecular dynamics (MD) simulations, however, revealed that the introduced amide bond isosteres substantially altered the hydrogen-bonding network formed between the N-terminal 259-265 backbone sequence of CII259-273 and Aq. These results indicated that the N-terminal hydrogen-bonding interactions follow a cooperative model, where the strength and presence of individual hydrogen bonds depended on the neighboring interactions. The two important anchor residues Ile260 and Phe263 were investigated using a designed library of CII259-273 based glycopeptides with substitutions by different (non-)natural amino acids at positions 260 and 263. Evaluation of binding to the Aq protein showed that there was scope for improvement in position 263 while Ile was preferred in position 260. The obtained SAR understanding provided a valuable basis for future development of modified glycopeptides with improved Aq binding. Furthermore, the modified glycopeptides elicited varying T-cell responses that generally could be correlated to their ability to bind to Aq. However, in several cases, there was a lack of correlation between Aq binding and T-cell recognition, which indicated that the interactions with the TCRs were determined by other factors, such as presentation of altered epitopes and changes in the kinetics of the TCR’s interaction with the Aq/glycopeptide complex. Several of the modified glycopeptides were also found to bind well to the human RA-associated DR4 protein and elicit strong responses with T-cell hybridomas obtained from transgenic mice expressing DR4 and the human CD4 co-receptor. This encourages future investigations of modified glycopeptides that can be used to further probe the MHC/glycopeptide/TCR recognition system and that also constitute potential therapeutic vaccines for treatment of RA. As a step towards this goal, three modified glycopeptides presented in this thesis have been identified as candidates for vaccination studies using the CIA mouse model.
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| 3. |
- Begnini, Fabio
(författare)
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Discovery of Novel Macrocyclic Ligands for Difficult Targets : Applications to Natural Product Derived Keap1 Inhibitors
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of small-molecule ligands for biological targets that possess large, featureless or highly polar binding sites is a challenging task. This thesis is focused on a novel lead generation strategy to identify macrocyclic ligands for difficult-to-drug targets, as well as on the relationship between cell permeability and the conformations of macrocycles.A database of natural products was investigated to compile a set of macrocyclic cores to be used for in silico screening on difficult-to-drug targets. Docking of this set on Keap1, a target considered challenging due to its large and polar binding site, identified the core of the natural product cyclothialidine as a starting point for lead generation. Synthesis and evaluation of a small number of analogues provided a novel macrocyclic Keap1 inhibitor with potency in the low micromolar range that displayed cellular activity. Investigation of the structure-activity relationship of the lead inhibitor identified two positions amenable for optimization. In silico libraries were constructed at both positions using structure-based design to improve the affinity for Keap1. Subsequent synthesis of approximately 100 compounds led to an optimized lead series with potency in the low nanomolar range, providing a 100-fold improvement from the starting point. Additionally, the difference in passive cell permeability for a pair of diastereoisomeric macrocycles was rationalized on the basis of differences in their solution-phase conformations, that were determined by NMR spectroscopy. Moreover, for two sets of moderately flexible isomeric macrocycles, it was shown that the molecular descriptors predicted from conformational sampling correlated with cell permeability. This method may find use for prioritization of macrocycles prior to embarking on demanding synthetic routes.
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| 4. |
- Blomberg, David, 1971-
(författare)
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Synthesis of β-turn and pyridine based peptidomimetics
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite the unfavorable pharmacokinetic properties associated with peptides, they are still of great interest in drug development due to a multitude of interesting biological functions. The development of peptidomimetics strives to maintain or improve the biological activity of a peptide concurrently with removing the unwanted properties. This thesis describes two synthetic approaches to peptidomimetics with particular emphasis on secondary structure mimetics. First the design, synthesis and evaluation of two beta-turn mimetics incorporated in the endorphin Leu-enkephalin is presented. The beta-turn mimetics were stabilized by replacement of the intramolecular hydrogen bond with an ethylene bridge, and the amide bond between Tyr and Gly was replaced with an ether linkage. Linear analogues of the two mimetics were also synthesized. The peptidomimetics and their linear analogues were evaluated in a competitive binding assay at two opiate receptors, my and delta. One of the cyclized beta-turn mimetics was found to be a delta receptor antagonist with an IC50 value of 160 nM. Second a synthetic strategy to a beta-strand mimetic using 2-fluoro-4-iodopyridine as scaffold is described. The synthesis involved a Grignard exchange reaction on the pyridine scaffold using an amino acid derivative as electrophile followed by an SNAr reaction using an amine as nucleophile. The synthesis of a tripeptidomimetic of Leu-Gly-Gly and attempts to introduce chiral building blocks at the C-terminal, as well as studies towards elongated mimetics are presented. Two additional studies deal with the synthesis of two classes of potential thrombin inhibitors based on the pyridine scaffold. The first class contain pyridine as central fragment (P2 residue) substituted with a para-amidinobenzylamine group as P1 residue and various benzoyl groups as P3 residues. Three potential thrombin inhibitors were synthesized and found to be microM inhibitors in an enzymatic assay. In the second class, the pyridine ring serves as P3 residue. This class also lacks a strongly basic group in the P1 position. A small library of eight compounds were synthesized and evaluated in the enzymatic assay. Unfortunately, these compounds lacked inhibitory activity.
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| 5. |
- Hedenström, Mattias, 1976-
(författare)
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NMR as a tool in drug research : Structure elucidation of peptidomimetics and pilicide-chaperone complexes
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the last decades NMR spectroscopy has become an invaluable tool both in academic research and in the pharmaceutical industry. This thesis describes applications of NMR spectroscopy in biomedicinal research for structure elucidation of biologically active peptides and peptidomimetics as well as in studies of ligand-protein interactions. The first part of this thesis describes the theory and methodology of structure calculations of peptides using experimental restraints derived from NMR spectroscopy. This methodology has been applied to novel mimetics of the peptide hormones desmopressin and Leu-enkephalin. The results of these studies highlight the complicating issue of conformational exchange often encountered in structural determination of peptides and how careful analysis of experimental data as well as optimization of experimental conditions can enable structure determinations in such instances. Although the mimetics of both desmopressin and Leu-enkephalin were found to adopt the wanted conformations, they exhibited no or very poor biological activity. These results demonstrate the difficulties in designing peptidomimetics without detailed structural information of the receptors. A stereoselective synthetic route towards XxxΨ[CH2O]Ala pseudodipeptides is also presented. Such pseudodipeptides can be used as isosteric amide bond replacements in peptides in order to increase their resistance towards proteolytic degradation. The second part of this thesis describes the study of the interaction between compounds that inhibit pilius assembly, pilicides, and periplasmic chaperones from uropathogenic Escherichia coli. Periplasmic chaperones are key components in assembly of pili, i.e. hair-like protein complexes located on the surface of Escherichia coli that cause urinary tract infections. Detailed knowledge about this interaction is important in understanding how pilicides can inhibit pilus assembly by binding to chaperones. Relaxation-edited NMR experiments were used to confirm the affinity of the pilicides for the chaperones and chemical shift mapping was used to study the pilicide-chaperone interaction surface. These studies show that at least two interaction sites are present on the chaperone surface and consequently that two different mechanisms resulting in inhibition of pilus assembly may exist.
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| 6. |
- 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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| 7. |
- Kihlberg, Johan, 1970-
(författare)
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Magnetic Resonance Imaging of Myocardial Deformation and Scarring in Coronary Artery Disease.
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Although improved treatments have reduced the rates of acute complications from myocardial infarction, sequelae such as heart failure and sudden death threaten the future wellbeing of those patients. Secondary prevention after myocardial infarction is related to cardiovascular risk factors and the effect of the infarct on left ventricular function. Cardiovascular magnetic resonance imaging (CMR) is necessary to determine the size of the infarct scar and can with great precision determine left ventricular volumes, left ventricular ejection fraction, and deformation (strain and torsion). The purpose of this thesis was to improve on CMR methods to facilitate image acquisition and post processing in patients with high risk of coronary artery disease (CAD).In Paper 1, a three-dimensional phase-sensitive inversion-recovery (3D PSIR) sequence was modified to measure T1 during a single breath hold. The measured T1 values were used to extrapolate a map of T1 relaxation, which avoided the time-consuming manual determination of the inversion time. The data collection consisted of phantom experiments, Monte Carlo simulations of the effect of various heart rates, and clinical investigation of 18 patients with myocardial infarction. Scar images created with the modified sequence were compared to those created with the standard sequence. The 3D PSIR sequence was able to measure T1 relaxation with a high accuracy up to 800 ms, which is in the suitable range for scar imaging. Simulated arrhythmias showed that the method was robust and able to tolerate some variation in heart rate. The modified sequence provides measurements of inversion time that can be used to facilitate standard scar imaging or to reconstruct synthetic scar images. Images of infarct scar obtained with the 3D PSIR sequence bore striking similarity to images obtained with the standard sequence.In Paper 2, 125 patients with high risk of CAD were investigated using the displacement encoding with stimulated echoes (DENSE) sequence. Image segments with infarct scar area >50% (transmurality) could be identified with a sensitivity of 95% and a specificity of 80% based on circumferential strain calculated from the DENSE measurements. The DENSE sequence was also applied in other directions, but its sensitivity and specificity to detect scar was lower than when used for circumferential strain.In Paper 3, 90 patients with high risk of CAD were examined by DENSE, tagging with harmonic phase (HARP) imaging and cine imaging with feature tracking (FT), to detect cardiac abnormalities as manifested in end-systolic circumferential strain. Circumferential strain calculated with DENSE had higher sensitivity and specificity than the competing methods to detect infarction with transmurality >50%. Global circumferential strain measured by DENSE correlated better with global parameters such as left ventricular ejection fraction, myocardial wall mass, left ventricular end-diastolic and end-systolic volume; than strain measured by FT or HARP.In Paper 4, myocardial torsion was investigated using DENSE, HARP, and FT in 48 patients with high risk of CAD. Torsion measured by each of the three methods was correlated with other global measures such as left ventricular ejection fraction, left ventricular mass, and left ventricular end-diastolic and end-systolic volumes. The torsion measurements obtained with DENSE had a stronger relationship with left ventricular ejection fraction, left ventricular mass, and volumes than those obtained with HARP or FT.DENSE was superior to the other methods for strain and torsion measurement and can be used to describe myocardial deformation quantitatively and objectively.
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| 8. |
- Lindfors, Lina
(författare)
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Molecular Imaging of Diabetic Kidney Tissue and Binding Studies of Proinsulin C-peptide
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diabetic kidney disease is a serious complication of diabetes with a complex and incompletely understood pathology. In this work, the molecular changes in diabetic rat kidneys at a very early disease stage were studied using nanospray desorption electrospray ionisation mass spectrometry imaging. Our results demonstrate how disease-relevant metabolites and lipids can be conveniently analysed on intact kidney tissue sections. A number of significantly increased metabolites were identified in the diabetic kidney, revealing disturbances in energy metabolism detectable before histological changes.Proinsulin C-peptide is produced in the pancreas along with insulin and has shown beneficial effects in diabetes, but its mode of action is not yet known. 125I radiolabelled C-peptide was used to study its tissue distribution in healthy and diabetic rats after intravenous injection. The majority of C-peptide accumulated in renal tissues, with lower levels in the diabetic animals, showing that there are significant changes in kidney – C-peptide interactions in early stage diabetes.The interactions of C-peptide with the orphan receptor GPR146, which has been proposed as its receptor, were also investigated using Chinese hamster ovary cells overexpressing human GPR146. Neither dynamic mass redistribution nor β-arrestin recruitment assays showed any significant response to human or murine C-peptides in the GPR146 overexpressing cells compared to controls. Fluorescence confocal microscopy revealed no surface binding or cellular uptake of C-peptides by GPR146 overexpressing cells compared to controls. These combined results refute the suggestion that GPR146 is the C-peptide receptor.To further probe the function of C-peptide, 15N-labelled residues were incorporated into the peptide in preparation for nanoscale secondary ion mass spectrometry imaging of cells and intact kidney tissue sections. A number of crosslinking C-peptides were also designed and synthesised for experiments aimed at identifying its binding target. These studies have not yet been completed. Finally, to investigate the structure-activity relationship of C-peptide, a library of modified pentapeptide analogues was created for medium-throughput testing in a cell assay.
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| 9. |
- Larsson, Andreas, 1972-
(författare)
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Antiadhesive agents targeting uropathogenic Escherichia coli : Multivariate studies of protein-protein and protein-carbohydrate interactions
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes studies directed towards development of novel antiadhesive agents, with particular emphasis on compounds that prevent attachment of bacteria to a host-cell. Three different proteins involved in the assembly or function of adhesive pili in uropathogenic Escherichia coli have been targeted either by rational structure based design or statistical molecular methods. A library of substituted galabiose (Galα1-4Gal) derivatives was screened for binding to the E. coli adhesin PapG in an assay based on surface plasmon resonance, and for inhibition of Streptococcus suis adhesins PN and PO in a hemagglutination assay. The results were used to generate QSAR models which had good predictive powers and provided further insight in the structural requirements needed for high affinity binding. 2-pyridones and amino acid derivatives were modelled into the binding site of chaperones involved in pilus assembly in E. coli and a heuristic method, VALIDATE, was used for affinity prediction. The affinity of the compounds for the chaperones PapD and FimC were assessed in assays based on surface plasmon resonance and relaxation-edited NMR spectroscopy. Their ability to disrupt chaperone/subunit complexes was investigated in vitro through a FPLC assay and their capacity to inhibit pilus formation in vivo was determined via hemagglutination and confirmed with atomic force microscopy. Statistical molecular design was used to design a diverse peptide library targeting pili subunits, and an ELISA was developed to investigate the ability of the peptides to inhibit chaperone/subunit complexation. The resulting QSAR model provided extensive information regarding binding of the peptides to the subunits. Because the peptides were suggested to bind in an extended β-strand formation, β-strand mimetics consisting of oligomeric enaminones were designed. Finally, new methods to synthesize enaminone building blocks were developed using microwave assisted chemistry. The projects described have generated compounds that besides their value as leads for developing novel antibacterial agents, also constitute new chemical tools to study the mechanisms underlying bacterial virulence.
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| 10. |
- Luttens, Andreas, et al.
(författare)
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Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:7, s. 2905-2920
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Tidskriftsartikel (refereegranskat)abstract
- Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.
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