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- Abdurakhmanov, Eldar, 1977-, et al.
(författare)
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Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase
- 2017
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.
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| 5. |
- Nosrati, Masoumeh, et al.
(författare)
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Insights from engineering the Affibody-Fc interaction with a computational-experimental method
- 2017
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 30:9, s. 593-601
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.
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| 6. |
- Solbak, Sara M.Ø., et al.
(författare)
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Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA - effects on NS5B catalysis and allosteric inhibition
- 2017
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Ingår i: Virology Journal. - : Springer Science and Business Media LLC. - 1743-422X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy.Methods: Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition.Results: No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity (K D = 30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences.Conclusions: The complex interplay revealed between NS5B, AnxA2, RNA and filibuvir, suggests that AnxA2 may have an important role for the progression and treatment of HCV infections and the development of HCC, which should be considered also when designing new allosteric inhibitors.
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| 7. |
- 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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