Affinity-based chemical proteomic probe for dehydrogenases: fluorescence and visible binding assays in gels.
A catechol rhodanine (CR)-based privileged scaffold, tailored to dehydrogenase enzymes, has recently been reported. This scaffold was used as a template in a focused combinatorial library, designed using the NMR SOLVE methodology, to prepare potent (50-200nM) biligand inhibitors for multiple dehydrogenases. It is reported here that this CR scaffold is also a fluorescent and visible probe for solution and in-gel (native) binding assays, making it a useful screening reagent for dehydrogenases, with applications as an affinity-based chemical proteomic probe. Initial application of this fluorescent CR probe was to dihydrodipicolinate reductase, an anti-infective drug target. The probe also shows in-gel binding affinity to two lactate dehydrogenase isozymes and to 1-deoxy-d-xylulose-5-phosphate reductoisomerase, making it a generally useful in-gel staining reagent for multiple dehydrogenases. Because binding is noncovalent, such a reagent could be used in a displacement assay performed in a native gel, monitoring decrease in fluorescent band intensity. But, because the probe has the added function of serving as a privileged scaffold for dehydrogenase-targeted combinatorial libraries, it could also be used in a direct binding assay to screen for the highest-affinity biligand inhibitors that contain the fluorescent CR. Finally, the CR probe could be used as a stain in proteomic studies, to profile mixtures of proteins run on a native gel, to identify proteins that are likely to be dehydrogenases.
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Chemical Proteomics Facility at Marquette, Chemistry Department, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.
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Daniel S
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D005782:Gels; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D020543:Proteome; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D014325:Tromethamine
D005782:Gels; D020543:Proteome; D014325:Tromethamine; D010088:Oxidoreductases