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À propos de : High-Throughput Screening for Enzyme InhibitorsUsing Frontal Affinity Chromatography with LiquidChromatography and Mass Spectrometry        

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  • High-Throughput Screening for Enzyme InhibitorsUsing Frontal Affinity Chromatography with LiquidChromatography and Mass Spectrometry
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  • This work presents new frontal affinity chromatography(FAC) methodologies for high-throughput screening ofcompound libraries, designed to increase screening ratesand improve sensitivity and ruggedness in performance.A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented inthe identification of potential enzyme inhibitors from twolibraries of trisaccharides. Effluent from the FAC columnwas fractionated, sequentially processed via LC/MS, andreferenced to a similar analysis through a control FACcolumn lacking the enzyme. The resulting multidimensional data sets were compared across correspondingsample and control fractions to identify binders, in asemiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first libraryof 81 compounds, exhibiting an estimated Kd value of0.3 μM. Other binders yielded Kd values ranging from0.35 to 3.35 μM. To demonstrate the improvement inperformance of this FAC−LC/MS approach over theconventional online FAC/MS approach, 15 compoundsfrom this library were blended with a second library of1000 synthetic trisaccharides and screened againstGnT-V. All ligands in the 15-compound set were identifiedin this larger screen, and no ligands of greater affinitythan compound 1 were found. Our results show thatFAC−LC/MS is a reliable method for screening largecompound libraries directly and useful for large-scaleligand discovery initiatives.
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