Exploring the active site of human factor Xa protein by NMR screening of small molecule probes

Fielding, Lee A.; Fletcher, Dan; Rutherford, Samantha H.; Kaur, Jaspreet; Mestres, Jordi

doi:10.1039/b310265c

Cited by 17 publications

(10 citation statements)

References 22 publications

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“…1 H NMR (500 MHz, DMSO) δ 7.84 (s, 1H), 7.45 (m, 3H), 7.37 (m, 4H), 5.56 (s, 2H), 3.09 (t, J = 7.5, 2H), 2.99 (t, J = 7.5, 2H). 13 3-(2-(1-Hydroxy-1H-benzo[d][1,2,3]triazol-5-yl)ethyl)-1,2,4-oxadiazole-5(4H)-thione (14).…”

Section: -(2-(1-hydroxy-1h-benzo[d][123]triazol-5-yl)ethyl)-124-mentioning

confidence: 99%

Rational Design of Small-Molecule Inhibitors for β-Catenin/T-Cell Factor Protein–Protein Interactions by Bioisostere Replacement

Huang

Zhang

et al. 2013

ACS Chem. Biol.

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A new hot spot-based design strategy using bioisostere replacement is reported to rationally design nonpeptidic small-molecule inhibitors for protein-protein interactions. This method is applied to design new potent inhibitors for β-catenin/T-cell factor (Tcf) interactions. Three hot spot regions of Tcf for binding to β-catenin were quantitatively evaluated; the key binding elements around K435 and K508 of β-catenin were derived; a bioisostere library was used to generate new fragments that can match the proposed critical binding elements. The most potent inhibitor, with a molecular weight of 230, has a Kd of 0.531 μM for binding to β-catenin and a Ki of 3.14 μM to completely disrupt β-catenin/Tcf interactions. The binding mode of the designed inhibitors was validated by the site-directed mutagenesis and structure-activity relationship (SAR) studies. This study provides a new approach to design new small-molecule inhibitors that bind to β-catenin and effectively disrupt β-catenin/Tcf interactions specific for canonical Wnt signaling.

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Section: -(2-(1-hydroxy-1h-benzo[d][123]triazol-5-yl)ethyl)-124-mentioning

confidence: 99%

Rational Design of Small-Molecule Inhibitors for β-Catenin/T-Cell Factor Protein–Protein Interactions by Bioisostere Replacement

Huang

Zhang

et al. 2013

ACS Chem. Biol.

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“…Further discussion of the graphical fitting of linewidth data can be found in a report on the binding of sialyloligosaccharides to wheat germ agglutin, itself a popular subject of NMR protein-ligand studies [58]. The hits from an exploratory screening exercise to find new inhibitors of human factor Xa were followed by construction of 1 H NMR linewidth isotherms to establish quantitative binding affinities [59].…”

Section: Ligand Observed Relaxation Ratesmentioning

confidence: 99%

NMR Methods for the Determination of Protein- Ligand Dissociation Constants

Fielding¹

2003

CTMC

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207

104

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This article is a review with 83 references of the application of NMR to the measurement of the dissociation constants of protein-ligand complexes. After briefly discussing some general concepts of molecular stability, the text turns to consider which NMR parameters are reporters of complex formation. The available data treatments required to translate observed NMR effects into quantitative measurements of the stability of the complex in the form of the dissociation constant (KD) are introduced. Linearisation methods and curve fitting methods are explained in detail and are illustrated with examples drawn from recent reports of protein-small molecule interactions. Throughout the text examples of the commonly observed NMR parameters , 1 / T1 and 1 / T2 are drawn from biological studies of 1H, 31P, 19F 15N (and other nuclei). The advantages of NMR diffusion experiments as a measure of KD are considered. Some less frequently used NMR approaches, some new ideas and some non-general methods are grouped together in a miscellaneous section. The major sources of errors in the determination of KD are identified. This allows recommendations for optimal experimental set up. Options for dealing with strong binding are reviewed. Finally, the implications of abstracting KD data from high throughput screening experiments are considered and several different approaches to generate this data are discussed.

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“…The fragment library used herein was specifically designed to include fragments from previous NMR 27 and X-ray screenings, moieties known to bind S1 and S4 pockets, and in-house designed fragments likely to exhibit affinity. In detail, the final focused library contained 42 compounds, divided into four overlapping sets (Figure 2).…”

Section: ■ Introductionmentioning

confidence: 99%

Insights from Fragment Hit Binding Assays by Molecular Simulations

Ferruz

Harvey

Mestres

et al. 2015

J. Chem. Inf. Model.

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Novel bioactive molecules can be rationally designed by growing and linking small fragments. Because fragments are fast and promiscuous, it is common to have contradictory hit results between different experimental screening techniques. Here, we simultaneously determine fragment binding poses, affinities, and kinetics on a focused library of 42 fragments against the serine protease factor Xa using multimillisecond molecular dynamics simulations. We predict experimental poses of 12 over 15 S1 crystal structures, and affinities are recovered for 4 out of 6. A kinetic map of protein cavities is computed in terms of on- and off-rates as well as insights into secondary ligand poses. The results suggest that the approach can be useful to recapitulate discordant fragment screening data.

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Exploring the active site of human factor Xa protein by NMR screening of small molecule probes

Cited by 17 publications

References 22 publications

Rational Design of Small-Molecule Inhibitors for β-Catenin/T-Cell Factor Protein–Protein Interactions by Bioisostere Replacement

Rational Design of Small-Molecule Inhibitors for β-Catenin/T-Cell Factor Protein–Protein Interactions by Bioisostere Replacement

NMR Methods for the Determination of Protein- Ligand Dissociation Constants

Insights from Fragment Hit Binding Assays by Molecular Simulations

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