Affinity-based chemical proteomic probe for dehydrogenases: Fluorescence and visible binding assays in gels

Abstract

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 (50–200 nM) 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.