| 1. |
- Anscombe, Elizabeth, et al.
(författare)
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Identification and Characterization of an Irreversible Inhibitor of CDK2
- 2015
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 22:9, s. 1159-1164
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Tidskriftsartikel (refereegranskat)abstract
- Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclo-hexylmethoxy)-9H-purin-2-yl) amino) benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl) phenyl)-9H- purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.
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| 2. |
- Brandt, Peter, et al.
(författare)
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Deconstruction of Non-Nucleoside Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Type 1 for Exploration of the Optimization Landscape of Fragments
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804.
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Tidskriftsartikel (refereegranskat)abstract
- This study has taken a closer look at the theoretical basis for protein-fragment interactions. The approach involved the deconstruction of 3 non-nucleoside inhibitors of HIV-1 reverse transcriptase and investigation of the interaction between 21 substructures and the enzyme. It focused on the concept of ligand efficiency and showed that ligand independent free energy fees (ΔG(ind)) are crucial for the understanding of the binding affinities of fragments. A value of 7.0 kcal mol(-1) for the ΔG(ind) term is shown to be a lower limit for the NNRTI binding pocket of HIV-1 RT. The addition of the ΔG(ind) term to the dissociation free energy in the calculation of a corrected ligand efficiency, in combination with the lack of an efficient ligand binding hot spot in the NNIBP, fully explains the existence of nonbinding NNRTI substructures. By applying the concept to a larger set of ligands, we could define a binding site profile that indicates the absence of an efficient fragment binding hot spot but an efficient binding of full-sized NNRTIs. The analysis explains some of the challenges in identifying fragments against flexible targets involving conformational changes and how fragments may be prioritized.
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| 3. |
- de Kloe, Gerdien E, et al.
(författare)
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Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands
- 2010
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 53:19, s. 7192-7201
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Tidskriftsartikel (refereegranskat)abstract
- The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.
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| 4. |
- Elinder, Malin, et al.
(författare)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Tidskriftsartikel (refereegranskat)abstract
- A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.
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| 5. |
- Elinder, Malin, 1977-, et al.
(författare)
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Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
- 2009
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Ingår i: Journal of Biomolecular Screening. - : SAGE. - 1087-0571 .- 1552-454X. ; 14:4, s. 395-403
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Tidskriftsartikel (refereegranskat)abstract
- A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance-based biosensor. the aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. the authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. to efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dissociation for the complete panel of enzyme variants. hits were confirmed by visually inspecting the complete sensorgrams. two structurally unrelated compounds fulfilled the hit criteria, but only 1 compound was found to (a) compete with a known NNRTI for binding to the NNRTI site, (b) inhibit HIV-1 RT activity, and (c) inhibit HIV-1 replication in cell culture, for all 4 enzyme variants. this novel screening methodology offers high-resolution real-time kinetic data for multiple targets in parallel. it is expected to have broad applicability for the discovery of compounds with defined kinetic profiles, crucial for optimal therapeutic effects.
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| 6. |
- Geitmann, Matthis, et al.
(författare)
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Additional level of information about complex interaction between non-nucleoside inhibitor and HIV-1 reverse transcriptase using biosensor-based thermodynamic analysis
- 2007
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Ingår i: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 15:23, s. 7344-7354
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Tidskriftsartikel (refereegranskat)abstract
- The thermodynamics of the interaction between mutant HIV-1 reverse transcriptase (K103N and Y181C) and a nonnucleoside reverse transcriptase inhibitor (NNRTI), the phenylethylthiazolylurea compound MIV-150, was obtained by determining the temperature dependence of the kinetic rate constants. Large entropic changes in the forward and backward steps of the isomerization between a non-binding competent and a binding competent conformation of the enzyme, as well as in the binding steps, implied the involvement of major structural rearrangements upon interaction with the inhibitor. Despite of the entropic character of the overall interaction, the equilibrium for the binding of inhibitor was found to be predominantly enthalpy-driven. The high affinity and the low affinity interactions of the heterogeneously interacting inhibitor showed different energetics in the analysis, revealing an expectedly higher enthalpic component for the high-affinity interaction. The thermodynamic profiles of the two enzyme variants displayed significant differences, which could not be derived from their kinetics at a single temperature.
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| 7. |
- Geitmann, Matthis, et al.
(författare)
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Biosensor-based kinetic characterization of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors
- 2006
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 49:8, s. 2367-74
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Tidskriftsartikel (refereegranskat)abstract
- Details of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors (NNRTIs) have been elucidated using a biosensor-based approach. This initial study was performed with HIV-1 reverse transcriptase mutant K103N, the phenethylthioazolylthiourea compound (PETT) MIV-150, and the three NNRTIs licensed for clinical use: nevirapine, delavirdine, and efavirenz. Mathematical evaluation of the experimental data with several interaction models revealed that the four inhibitors interacted with HIV-1 RT with varying degrees of complexity. The simplest adequate model accounted for two different conformations of the free enzyme, of which only one can bind the inhibitor, consistent with a previously hypothesized population-shift model including a preformation of the NNRTI binding site. In addition, a heterogeneous binding was observed for delavirdine, efavirenz, and MIV-150, indicating that two noncompetitive and kinetically distinct enzyme-inhibitor complexes could be formed. Furthermore, for these compounds, there were indications for ligand-induced conformational changes.
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| 8. |
- Geitmann, Matthis, 1972-
(författare)
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Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes : Applications for Antiviral Drug Development
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs.The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors.Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors.The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding.Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.
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| 9. |
- Geitmann, Matthis, et al.
(författare)
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Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:3, s. 699-708
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Tidskriftsartikel (refereegranskat)abstract
- A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).
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| 10. |
- Geitmann, Matthis, et al.
(författare)
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Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:37, s. 8143-8154
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein, and acetylcholine, nicotine, epibatidine and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model and a structural interpretation of the interaction was obtained by molecular modelling involving induced-fit and loop flexibility simulations. The data suggests that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.
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| 11. |
- Geitmann, Matthis, et al.
(författare)
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Interaction kinetic characterization of HIV-1 reverse transcriptase non-nucleoside inhibitor resistance
- 2006
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 49:8, s. 2375-87
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Tidskriftsartikel (refereegranskat)abstract
- To decipher the mechanism for non-nucleoside inhibitor resistance of HIV-1 reverse transcriptase, the kinetics of the interaction between wild type and drug-resistant variants of the enzyme and structurally diverse inhibitors were determined. Substitution of amino acid residues in the inhibitor binding site resulted in altered rate constants for the pre-equilibrium between two unliganded forms of the enzyme, and for the association and dissociation of the inhibitor-enzyme interaction. The Y181C, V108I, and P225H substitutions affected primarily the association and dissociation rate constants, while the K103N and the L100I substitutions also influenced the equilibrium between the two forms of the free enzyme. The K103N and the L100I substitutions were found to facilitate both the entry of the inhibitor into the binding pocket as well as its exit, in contrast to what has been reported elsewhere. Interaction kinetic-based resistance profiles showed that phenethylthiazolylthiourea compounds were relatively insensitive to the studied substitutions.
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| 12. |
- Geitmann, Matthis, et al.
(författare)
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Kinetic characterization of HCV NS3 inhibitors using an SPR biosensor
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied. The KD of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between koff or KD with Ki (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.
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| 13. |
- Geitmann, Matthis, et al.
(författare)
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Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technology
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Kinetic, mechanistic and chemodynamic aspects of the interaction between five non-nucleoside inhibitors and the HCV NS5B polymerase (genotype 2a) were assessed using SPR biosensor technology. The compounds were selected to represent different structural classes (benzothiadiazine, , α,γ-diketo acid, benzimidazole, thiophene carbocyclic acid and benzofuran), each known to interact with different binding sites. The viral polymerase interacted with the compounds with different kinetics and surprisingly also with different capacities. Cooperativity between the different allosteric inhibitor binding sites and the active site binding diketoacid was observed, but no cooperativity was seen between the allosteric sites. The interaction with diketoacid was stronger in phosphate buffer as compared to Tris buffer, indicating a phosphate ion-mediated interaction mechanism. The enzyme generally had reduced affinity for the inhibitors in the presence of RNA. Interaction parameters determined for human serum albumin revealed the propensity of the compounds to be distributed by HSA. This study provides important information for the design of optimized NS5B inhibitors and illustrates the complementarity of a biosensor-based analysis with inhibition studies, in particular for allosteric compounds with complex interaction mechanisms or when the target contains multiple ligand binding sites.
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| 14. |
- Geitmann, Matthis, et al.
(författare)
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Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors
- 2011
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 24:1, s. 60-70
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.
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| 15. |
- Geitmann, Matthis, et al.
(författare)
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Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology
- 2004
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Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 332:2, s. 203-214
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.
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| 16. |
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| 17. |
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| 18. |
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| 19. |
- Norgren, Anna S., et al.
(författare)
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Biomolecular Recognition of Glycosylated β3-Peptides by GalNAc Specific Lectins
- 2007
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 20:2, s. 132-138
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Tidskriftsartikel (refereegranskat)abstract
- The molecular recognition of a novel kind of hybrid conjugates, composed of artificial biomimetic β-peptide oligomers with an O-linked natural N-acetyl-galactosamine (the Tn-antigen) residue, by four different GalNAc specific lectins was investigated using surface plasmon biosensor technology. The influence of the peptide and the glycosyl moiety on the recognition was studied using two glycosylated β3-heptapeptides, a glycosylated α-heptapeptide, two β-amino acid containing dipeptides, and monomeric αGalNAc-O-Thr. Although all four lectins displayed a decreased affinity for the carbohydrate residue when attached to a peptide, as compared to the monomeric Tn-antigen, the peptide part was found to have distinct effects on the binding kinetics - indicating that varying degrees of protein-peptide interactions occurred in the recognition process. Likewise, the lectins did not discriminate between β3-peptides and the α-peptide, but the β- linkage of the galactose had a detrimental effect for at least two of the lectins.
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| 20. |
- Retra, Kim, et al.
(författare)
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Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands
- 2010
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 407:1, s. 58-64
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Tidskriftsartikel (refereegranskat)abstract
- Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z' factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.
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| 21. |
- Simister, Philip, et al.
(författare)
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Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase
- 2009
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 83:22, s. 11926-11939
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Tidskriftsartikel (refereegranskat)abstract
- The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.
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| 22. |
- Sookkongwaree, K, et al.
(författare)
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Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora
- 2006
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Ingår i: Pharmazie. - 0031-7144. ; 61:8, s. 717-721
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Tidskriftsartikel (refereegranskat)abstract
- In order to identify novel lead compounds with antiviral effect, methanol and aqueous extracts of eight medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). In general, the methanol extracts inhibited the enzymes more effectively than the aqueous extracts. HIV-1 protease was strongly inhibited by the methanol extract of Alpinia galanga. This extract also inhibited HCV and HCMV proteases, but to a lower degree. HCV protease was most efficiently inhibited by the extracts from Zingiber officinale, with little difference between the aqueous and the methanol extracts. Many of the methanol extracts inhibited HCMV protease, but the aqueous extracts showed weak inhibition. In a first endeavor to identify the active constituents, eight flavones were isolated from the black rhizomes of Kaempferia parviflora. The most effective inhibitors, 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease with IC50 values of 19 microM. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease with IC50 values of 190 and 250 microM, respectively.
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| 23. |
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| 24. |
- Wang, Xiaolu, et al.
(författare)
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A protein interaction node at the neurotransmitter release site : domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1
- 2009
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Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 29:40, s. 12584-12596
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Tidskriftsartikel (refereegranskat)abstract
- Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.
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| 25. |
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| 26. |
- Yang, Jie, et al.
(författare)
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Macrocyclic peptides as inhibitors of human LSD1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Lysine demethylase 1 (LSD1) regulates the degree of methylation of Lys4 of histone 3 in the nucleosome core particle. As LSD1 is overexpressed in certain cancers, inhibitors have potential for use as drugs. Guided by the structures of two peptidic ligands bound to LSD1 we prepared truncated, mono-substituted and macrocyclic peptides to find leads for development of specific and revserible inhibitors. Surface plasmon resonance biosensor analysis revealed that some stapled, macrocyclic peptides had up to 10-fold higher affinity for LSD1 than the corresponding linear native peptide. Furthermore, peptides cyclized by lactamization were low mM inhibitors of LSD1, with the most effective one being >25-fold more potent than the linear native reference.
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| 27. |
- 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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