| 161. |
- Xu, Xingxing, et al.
(författare)
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Structural Changes of Mercaptohexanol Self-assembled Monolayers on Gold and their Influence on Impedimetric Aptamer Sensors
- 2019
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 91:22, s. 14697-14704
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Tidskriftsartikel (refereegranskat)abstract
- Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM) functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization. As a result, no significant difference between the charge transfer resistance (RCT) changes induced by the aptamer-target DNA hybridization and that caused by the drift could be identified. A conditioning of the electrode in the measurement solution for more than 12 hours was required to reach a stable RCT baseline prior to the aptamer-probe DNA hybridization. The monitored drift in RCT and CDL during the conditioning suggests that the MCH SAM on the gold surface reorganized to a thinner but more closely packed layer. We also observed that the hot binding buffer used in the following aptamer-probe DNA hybridization process could induce additional MCH and aptamer reorganization thus further drift in RCT. As a result, the RCT change caused by the aptamer-probe DNA hybridization was less than that caused by the hot binding buffer (blank control experiment). Therefore, it is suggested that the use of high temperature in the EIS measurement should be carefully evaluated or avoided. This work provides practical guidelines for the EIS measurements. Moreover, since SAM functionalized gold electrodes are widely used in biosensors, e.g., DNA sensors, an improved understanding of the origin of the observed drift is very important for the development of well-functioning and reproducible biosensors.
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| 162. |
- Yang, Jie, et al.
(författare)
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Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1
- 2020
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Ingår i: ACS Omega. - : AMER CHEMICAL SOC. - 2470-1343. ; 5:8, s. 3979-3995
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Tidskriftsartikel (refereegranskat)abstract
- Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent K-i = 2.3 mu M) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure-activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.
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| 163. |
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| 164. |
- Ålin, Per, et al.
(författare)
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Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing
- 1985
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 146:2, s. 313-320
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Tidskriftsartikel (refereegranskat)abstract
- Seven major isoenzymes of glutahione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W. B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539–2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.
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| 165. |
- Öhman Fuchs, Peder, 1984-, et al.
(författare)
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Fibrin fragment E potentiates TGF-beta-induced myofibroblast activation and recruitment
- 2020
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 72
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Tidskriftsartikel (refereegranskat)abstract
- Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-beta-induced myofibroblast formation. FnE forms a stable complex with alpha(v)beta(3) integrin, and the integrin beta(3) subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-beta-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis.
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| 166. |
- Örtqvist, Pernilla, et al.
(författare)
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Discovery of Achiral Inhibitors of the Hepatitis C Virus NS3 Protease based on 2(1H)-pyrazinones
- 2010
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 18:17, s. 6512-6525
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Tidskriftsartikel (refereegranskat)abstract
- Herein, the design, synthesis and inhibitory potency of a series of novel hepatitis C virus (HCV) NS3 protease inhibitors are presented. These inhibitors are based on a 2(1H)-pyrazinone P3 scaffold in combination with either a P2 phenylglycine or a glycine, and they were evaluated on the wild type as well as on two resistant variants of the enzyme, A156T and D168V. Molecular modelling suggested that the aromatic side-chain of the P2 phenylglycine occupies the same space as the substituent in position 6 on the pyrazinone core. The versatile synthetic route applied for the pyrazinone synthesis made a switch between the two positions easily feasible, resulting in phenyl- or benzyl substituted pyrazinones and leaving glycine as the P2 residue. Of several P1-P1′ residues evaluated, an aromatic P1-P1′ scaffold was found superior in combination with the new P3-P2 building block. As a result, an entirely new type of achiral and rigidified inhibitors was discovered, with the best of the novel inhibitors having fourfold improved potency compared to the corresponding tripeptide lead. We consider these achiral inhibitors highly suitable as starting points for further optimization.
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| 167. |
- Örtqvist, Pernilla, et al.
(författare)
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Phenylglycine as a Novel P2 Scaffold in Hepatitis C Virus NS3 Protease Inhibitors
- 2007
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 15:3, s. 1448-1474
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Tidskriftsartikel (refereegranskat)abstract
- Molecular modeling and inhibitory potencies of tetrapeptide protease inhibitors of HCV NS3 proposed phenylglycine as a new promising P2 residue. The results suggest that phenylglycine might be capable of interacting with the NS3 (protease-helicase/NTPase) in ways not possible for the common P2 proline-based inhibitors. Thus, a series of tripeptides, both linear and macrocyclic, based on p-hydroxy-phenylglycine in the P2 position were prepared and their inhibitory effect determined. When the p-hydroxy group was replaced by methoxy, isoquinolin-, or quinolinyloxy functions, inhibitors with improved potencies were obtained. The P2 phenylglycine-based inhibitors were further optimized by C-terminal extension to acyl sulfonamides and by P1–P3 cyclization, which gave products with inhibition constants in the nanomolar range (75 nM).
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| 168. |
- Örtqvist, Pernilla, et al.
(författare)
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Structure-activity relationships of HCV NS3 protease inhibitors evaluated on the drug-resistant variants A156T and D168V
- 2010
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Ingår i: Antiviral Therapy. - 1359-6535 .- 2040-2058. ; 15:6, s. 841-852
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: HCV infections are a serious threat to public health. An important drug target is the NS3 protease, for which several inhibitors are in clinical trials. Because of the high mutation rate of the virus, resistance against any HCV-specific drug is likely to become a substantial problem. Structure-activity data for the major resistant variants are therefore needed to guide future designs of protease inhibitors. METHODS: The inhibitory potency of tripeptide NS3 protease inhibitors, with either a P2 proline or phenylglycine, in combination with different P3 and P1-P1' groups, was assessed in enzyme activity assays using the full-length NS3 protein with known resistance-conferring substitutions A156T or D168V. The results obtained from these variants were compared with the inhibition of the wild-type enzyme. Molecular modelling was used to rationalize the biochemical results. RESULTS: Inhibitors combining the P2 proline and P1 (1R,2S)-1-amino-2-vinylcyclopropyl-carboxylic acid (vinylACCA) lost much of their potency on the resistant variants. Exchange of the P2 proline for phenylglycine yielded inhibitors that were equipotent on the wild-type and on the A156T and D168V variants. The same result was obtained from the combination of either the P2 residue with a norvaline or an aromatic scaffold in the P1 position. CONCLUSIONS: The combination of a substituted P2 proline and P1 vinylACCA appears to be the main problem behind the observed resistance. Molecular modelling suggests an enforced change in binding conformation for the P2 proline-based inhibitors, whereas the phenylglycine-based inhibitors retained their wild-type binding conformation in the substituted forms of the enzyme.
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| 169. |
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