| 1. |
- Adolfsson, Dan E., 1989-
(författare)
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Synthesis of Ring-fused Peptidomimetics : Interacting with Amyloid Fibrils
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson's and Alzheimer's disease are the two most common neurological disorders in humans. Both conditions involve progressive death of neurons in the central nervous system, decline in bodily functions and eventually (and invariably), death. So far, no cure exists and the available treatments can only ease symptoms. Despite substantial investments in research, the biomolecular processes are still far from fully understood. However, both diseases are associated with formation of fibrillar protein aggregates called amyloid deposits. Whereas Alzheimer’s disease involves aggregation of the Tau and Amyloid β proteins, α-Synuclein fibrilization plays a key role in Parkinson's disease. Although they are chemically distinct, the deposits consist of protein fibres with similar morphology and fold. Small molecules, such as the thiazoline fused 2-pyridones herein presented, can interfere with the formation of amyloid fibres, or bind to them. Besides having potential for diagnostication and treatment, such small molecules constitute valuable tool compounds in future research, to unravel the mechanisms of amyloid formation and pathology. The first step towards successful treatment, diagnostication and prevention of Alzheimer's and Parkinson's disease is understanding the causes and underlying mechanisms better. This thesis narrates the synthesis and development of novel chemical structures: multi ring fused peptidomimetics with the ability to bind mature amyloid fibrils, consisting of α-Synuclein or Amyloid β. The first project (articles I, III and VI) describes method development for the extension of bicyclic thiazolino 2-pyrdiones by fusion with aromatic nitrogen heterocycles, which enables the desired amyloid binding properties. Derivatisations of the newly generated central scaffold, and variation of the multiple attached substituents, were subsequently performed in efforts to improve binding strength and solubility, and gain selectivity towards certain fibrils. One of the most promising amyloid fibril binders was evaluated in a human cell line and in mice, and found to be protective against accelerator induced neurotoxicity. One pyrimidine fused compound moreover indicated potent inhibition of Amyloid b aggregation. The second project (articles II, IV and V) focuses on development of methods to modify the thiazoline ring. Ring opening induced by electrophiles generates N-alkenyl 2-pyridones but decreases amyloid binding potency. Introduction of a cyclobutane moiety fused with the thiazoline ring is better tolerated, and adds a terminal alkene moiety that can be exploited in future chemical modifications. Expansion of the five membered thiazoline ring to a six membered dihydrothiazine ring, equipped with a nitrophenyl substituent, provides compounds with enhanced fibril binding capacity, which further inhibits Amyloid β fibril formation in vitro. Taken together, the synthetic methodologies allow construction and late stage modification of complex fused heterocycles, with several points of variation. Thus, the developed methods may be of future value in our laboratories and elsewhere.
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| 2. |
- Alalam, Hanna, et al.
(författare)
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A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease
- 2021
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Ingår i: MSYSTEMS. - 2379-5077. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of the SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the Escherichia coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor by a robust growth increase, discernible down to 1 mM. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARSCoV-2 main protease or even for other proteases.
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| 3. |
- Battisti, Umberto Maria, et al.
(författare)
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Ellagic Acid and Its Metabolites as Potent and Selective Allosteric Inhibitors of Liver Pyruvate Kinase
- 2023
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Ingår i: Nutrients. - : MDPI AG. - 2072-6643. ; 15:3
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Tidskriftsartikel (refereegranskat)abstract
- Liver pyruvate kinase (PKL) has recently emerged as a new target for non-alcoholic fatty liver disease (NAFLD), and inhibitors of this enzyme could represent a new therapeutic option. However, this breakthrough is complicated by selectivity issues since pyruvate kinase exists in four different isoforms. In this work, we report that ellagic acid (EA) and its derivatives, present in numerous fruits and vegetables, can inhibit PKL potently and selectively. Several polyphenolic analogues of EA were synthesized and tested to identify the chemical features responsible for the desired activity. Molecular modelling studies suggested that this inhibition is related to the stabilization of the PKL inactive state. This unique inhibition mechanism could potentially herald the development of new therapeutics for NAFLD.
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| 4. |
- Battisti, Umberto Maria, et al.
(författare)
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Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase
- 2023
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Ingår i: Pharmaceuticals. - : MDPI AG. - 1424-8247. ; 16:5
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Tidskriftsartikel (refereegranskat)abstract
- The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.
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| 5. |
- Battisti, U. M., et al.
(författare)
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Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase
- 2023
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50=0.29 μM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.
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| 6. |
- Benitez-Martin, Carlos, 1994, et al.
(författare)
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Fluorescent Molecular Photoswitches for the Generation of All-Optical Encryption Keys
- 2023
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Ingår i: Chemphotochem. - 2367-0932.
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we report a tri-component photochromic molecular cocktail that can be used to encrypt and decrypt information. The time-dependent fluorescent response of this cocktail is highly non-linear with respect to the set of inputs used (concentrations of the three photochromic components, excitation- and emission wavelengths), a property required for the generation of so-called encryption keys. The all-optical system can generate more than 80 million unique fluorescence responses by applying different input combinations and is operated using a conventional fluorimeter.
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| 7. |
- Bood, Mattias, et al.
(författare)
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Interbase-FRET binding assay for pre-microRNAs
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Forster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA-target binding studies.
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| 8. |
- Bourgard, Catarina, 1985, et al.
(författare)
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Development of Dicationic Bisguanidine-Arylfuran Derivatives as Potent Agents against Gram-Negative Bacteria.
- 2022
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 11:8
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Tidskriftsartikel (refereegranskat)abstract
- Antibiotic resistance among bacteria is a growing global challenge. A major reason for this is the limited progress in developing new classes of antibiotics active against Gram-negative bacteria. Here, we investigate the antibacterial activity of a dicationic bisguanidine-arylfuran, originally developed as an antitrypanosomal agent, and new derivatives thereof. The compounds showed good activity (EC50 2-20 µM) against antibiotic-resistant isolates of the Gram-negative members of the ESKAPE group (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli with different antibiotic susceptibility patterns, including ESBL isolates. Cytotoxicity was moderate, and several of the new derivatives were less cytotoxic than the lead molecule, offering better selectivity indices (40-80 for several ESKAPE isolates). The molecular mechanism for the antibacterial activity of these molecules is unknown, but sensitivity profiling against human ESKAPE isolates and E. coli collections with known susceptibility patterns against established antibiotics indicates that it is distinct from lactam and quinolone antibiotics.
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| 9. |
- Fleming, Cassandra, 1987, et al.
(författare)
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All-photonic kinase inhibitors: light-controlled release-and-report inhibition
- 2024
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Ingår i: Chemical Science. - 2041-6539 .- 2041-6520. ; 15:18, s. 6897-6905
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Tidskriftsartikel (refereegranskat)abstract
- Light-responsive molecular tools targeting kinases affords one the opportunity to study the underlying cellular function of selected kinases. In efforts to externally control lymphocyte-specific protein tyrosine kinase (LCK) activity, the development of release-and-report LCK inhibitors is described, in which (i) the release of the active kinase inhibitor can be controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the active kinase inhibitor. This introduces an unprecedented all-photonic method for users to both control and monitor real-time inhibitory activity. A functional cellular assay demonstrated light-mediated LCK inhibition in natural killer cells. The use of coumarin-derived caging groups resulted in rapid cellular uptake and non-specific intracellular localisation, while a BODIPY-derived caging group predominately localised in the cellular membrane. This concept of release-and-report inhibitors has the potential to be extended to other biorelevant targets where both spatiotemporal control in a cellular setting and a reporting mechanism would be beneficial.
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| 10. |
- Fleming, Cassandra L., et al.
(författare)
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All-photonic kinase inhibitors: light-controlled release-and-report inhibition
- 2024
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Ingår i: CHEMICAL SCIENCE. - 2041-6520 .- 2041-6539. ; 15:18, s. 6897-6905
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Tidskriftsartikel (refereegranskat)abstract
- Light-responsive molecular tools targeting kinases affords one the opportunity to study the underlying cellular function of selected kinases. In efforts to externally control lymphocyte-specific protein tyrosine kinase (LCK) activity, the development of release-and-report LCK inhibitors is described, in which (i) the release of the active kinase inhibitor can be controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the active kinase inhibitor. This introduces an unprecedented all-photonic method for users to both control and monitor real-time inhibitory activity. A functional cellular assay demonstrated light-mediated LCK inhibition in natural killer cells. The use of coumarin-derived caging groups resulted in rapid cellular uptake and non-specific intracellular localisation, while a BODIPY-derived caging group predominately localised in the cellular membrane. This concept of release-and-report inhibitors has the potential to be extended to other biorelevant targets where both spatiotemporal control in a cellular setting and a reporting mechanism would be beneficial. An all-photonic method is described, in which (i) the release of an active kinase inhibitor is controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the inhibitor to its corresponding target.
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