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A Multifunctional NMR and Fluorescence‐based Assay Probe for Dehydrogenases, based on Privileged Scaffold: Application to an Anti‐Infective Drug Target
Author(s) -
Ge Xia,
Sem Daniel S.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a74-b
Subject(s) - chemistry , fluorescence , combinatorial chemistry , drug discovery , scaffold , rhodanine , molecular probe , biochemistry , stereochemistry , medicine , physics , quantum mechanics , biomedical engineering , dna
A catechol‐rhodanine (CR) privileged scaffold has recently been reported for dehydrogenases. This scaffold was used as a template in a focused combinatorial library, designed using the NMR SOLVE methodology, to identify potent (50‐200 nM inhibitors) for multiple dehydrogenases. We report herein that this CR‐scaffold is fluorescent, making it useful as an assay reagent for dehydrogenases and as a potential chemical proteomic probe. We have also developed versions of this probe that facilitate identification of “second‐site” fragments. Initial application of this multifunctional chemical proteomic probe is to dihydrodipicolinate reductases (DHPR), an anti‐infective drug target. First, 13 CH 3 labeled CR derivatives were prepared, where the label is located in the substrate site. This CR‐S‐ 13 CH 3 probe was used to screen for fragments that bind close to the 13 CH 3 label by using 13 C‐filtered NOEs. Second, since the methyl group is attached to a convenient thiol nucleophile, downstream fragment assembly/synthesis is simplified, using the demethylated version of this probe (CR‐SH). Third, the CR‐SH probe can be used in thiol‐based “Tethering” to second‐site ligands, as a complement to NMR‐based fragment assembly. Fourth, we have shown that the CR probe is fluorescent, so can be used in a complementary and universal fluorescence assay of dehydrogenases, which is: (a) better suited to initial HTS primary screens, and (b) potentially useful for in vivo molecular imaging and chemical proteomic assays.