| 1. |
- Cederfelt, Daniela
(författare)
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Structural studies of drug targets and a drug metabolizing enzyme
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis describes how structural information about a protein can be acquired, and how it can be used to answer scientific questions about proteins’ function, their dynamic behaviour and their interactions with other proteins or ligands.The catalytic function of the pyrimidine-degrading, drug metabolizing enzyme β-ureidopropionase (βUP) is dependent on the shift between oligomeric states. Substitution of amino acids H173 and H307 in the dimer-dimer interface and E207Q in the active site revealed that these are crucial for βUP activation. Inhibition studies of substrate-and product analogues allowed for a hypothesis that the ability to interact with F205 might distinguish activators from inhibitors. The first structure of the activated higher oligomer state of human βUP was determined using cryogenic electron microscopy, and confirmed that the closed entrance loop conformations and dimer-dimer interfaces are conserved between HsβUP and DmβUP. Interactions between the epigenetic drug target SET and MYND domain containing protein 3 (SMYD3) and possible inhibitors were investigated. A crystal structure confirmed the covalent bond of a rationally designed, targeted inhibitor to C186 in the active site of SMYD3. A new allosteric binding site was discovered using a biosensor screen with a blocked active site. Crystal structures revealed the location of the new binding site, and the binding mode of the (S)-and (R) enantiomers of the allosteric inhibitor. Lastly, a fragment based drug discovery approach was taken, co-crystallizing and soaking SMYD3 with hits from a fragment screen. This resulted in four crystal structures with weak electron density of fragments at several locations in the enzyme. The dynamic acetylcholine binding protein (AChBP) is a homologue of a Cys-loop type ligand gated ion channel. Hits from various biosensor screens, of which some indicated conformational changes, were co-crystallized with AChBP. Seven crystal structures of AChBP in complex with hit compounds from the biophysical screens were determined. Small conformational changes in the Cys-loop were detected in several of the crystal structures, coinciding with the results from the biosensor screens.In these studies, we explore new strategies for the investigation of the function and regulation of proteins relevant in drug discovery and optimization.
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| 2. |
- Cederfelt, Daniela, et al.
(författare)
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The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces
- 2023
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.
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| 3. |
- Cornelius Chukwu, Eugenia, et al.
(författare)
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Engineered Aldolases Catalyzing Stereoselective Aldol Reactions Between Aryl-Substituted Ketones and Aldehydes
- 2023
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Ingår i: Catalysis Science & Technology. - : Royal Society of Chemistry. - 2044-4753 .- 2044-4761.
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Tidskriftsartikel (refereegranskat)abstract
- An A129G/R134V/S166G triple mutant of fructose 6-phosphate aldolase (FSA) from Escherichia coli was further engineered with the goal to generate new enzyme variants capable of catalyzing aldol reactions between aryl substituted ketones and aldehydes. Residues L107 and L163 were subjected to saturation mutagenesis and the resulting library of FSA variants was screened for catalytic activity with 2-hydroxyacetophenone and phenylacetaldehyde as substrates. A selection of aldolase variants was identified that catalyze the synthesis of 2,3-dihydroxy-1,4-diphenylbutanone. The most active enzyme variants contained an L163C substitution. An L107C/L163C variant was further tested for activity with substituted phenylacetaldehydes, and was shown to afford the production of the corresponding diphenyl substituted butanones with good diastereoselectivities (anti : syn dr of 10 to 30) and reasonable to good enantioselectivities of syn enantiomers (er of 5 to 25).
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| 4. |
- FitzGerald, Edward A., et al.
(författare)
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Discovery of fragments inducing conformational effects in dynamic proteins using a second-harmonic generation biosensor
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 11:13, s. 7527-7537
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Tidskriftsartikel (refereegranskat)abstract
- Biophysical screening of compound libraries for the identification of ligands that interact with a protein is efficient, but does typically not reveal if (or how) ligands may interfere with its functional properties. For this a biochemical/functional assay is required. But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput. Here we have explored a high-throughput second-harmonic generation (SHG) biosensor to detect fragments that induce conformational changes upon binding to a protein in real time and identify dynamic regions. Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs). They were conjugated with second harmonic-active labels via amine or maleimide coupling. To validate the assay, it was confirmed that the conformational changes induced in AChBP by nicotinic acetyl choline receptor (nAChR) agonists and antagonists were qualitatively different. A 1056 fragment library was subsequently screened against all variants and conformational modulators of AChBP were successfully identified, with hit rates from 9–22%, depending on the AChBP variant. A subset of four hits was selected for orthogonal validation and structural analysis. A time-resolved grating-coupled interferometry-based biosensor assay confirmed the interaction to be a reversible 1-step 1 : 1 interaction, and provided estimates of affinities and interaction kinetic rate constants (KD = 0.28–63 μM, ka = 0.1–6 μM−1 s−1, kd = 1 s−1). X-ray crystallography of two of the fragments confirmed their binding at a previously described conformationally dynamic site, corresponding to the regulatory site of LGICs. These results reveal that SHG has the sensitivity to identify fragments that induce conformational changes in a protein. A selection of fragment hits with a response profile different to known LGIC regulators was characterized and confirmed to bind to dynamic regions of the protein.
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| 5. |
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| 6. |
- FitzGerald, Edward A., et al.
(författare)
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Multiplexed experimental strategies for fragment library screening using SPR biosensors
- 2020
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Surface plasmon resonance biosensor technology (SPR) is ideally suited for fragment-based lead discovery. However, generally suitable experimental procedures or detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are lacking. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. By adopting a multiplexed strategy, the range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded. We here illustrate innovative strategies using five challenging targets and variants thereof. Two libraries of 90 and 1056 fragments were screened using two different flow-based SPR biosensor systems, allowing different experimental approaches. Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted.Competing Interest StatementAnna Moberg, Maria T. Lindgren and Claes Holmgren work for Cytiva, which produce Biacore systems.
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| 7. |
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| 8. |
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| 9. |
- Parenti, Marco Daniele, et al.
(författare)
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Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3
- 2022
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier. - 0223-5234 .- 1768-3254. ; 243
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Tidskriftsartikel (refereegranskat)abstract
- Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-amino-piperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/ histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 mu M) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.
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| 10. |
- Talibov, Vladimir O, 1991-, et al.
(författare)
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Discovery of an allosteric ligand binding site in SMYD3 lysine methyltransferase
- 2021
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Ingår i: ChemBioChem (Print). - : Wiley. - 1439-4227 .- 1439-7633. ; 22:9, s. 1597-1608
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Tidskriftsartikel (refereegranskat)abstract
- SMYD3 is a multifunctional epigenetic enzyme with lysine methyl transferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened using a biosensor-based competition assay. Diperodon was identified as an allosteric ligand. The ( R )-and ( S )-enantiomers of the racemic drug were isolated and their affinities determined ( K D > = 42 and 84 ÎŒM). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although HSP90-SMYD3 binding was confirmed ( K D = 13 ÎŒM). The allosteric site appears to be druggable and suitable for exploration of non-catalytic SMYD3 functions and therapeutics with new mechanisms of action.
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