| 1. |
- Anscombe, Elizabeth, et al.
(author)
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Identification and Characterization of an Irreversible Inhibitor of CDK2
- 2015
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In: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 22:9, s. 1159-1164
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Journal article (peer-reviewed)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.
(author)
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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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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804.
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Journal article (peer-reviewed)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.
(author)
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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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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 53:19, s. 7192-7201
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Journal article (peer-reviewed)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.
(author)
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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
- 2011
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In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 16:1, s. 15-25
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Journal article (peer-reviewed)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.
(author)
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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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In: Journal of Biomolecular Screening. - : SAGE. - 1087-0571 .- 1552-454X. ; 14:4, s. 395-403
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Journal article (peer-reviewed)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.
(author)
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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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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 15:23, s. 7344-7354
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Journal article (peer-reviewed)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.
(author)
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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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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 49:8, s. 2367-74
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Journal article (peer-reviewed)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-
(author)
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Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes : Applications for Antiviral Drug Development
- 2005
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Doctoral thesis (other academic/artistic)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.
(author)
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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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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:3, s. 699-708
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Journal article (peer-reviewed)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.
(author)
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Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein
- 2010
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:37, s. 8143-8154
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Journal article (peer-reviewed)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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