Bacterial β sliding clamp (β-clamp) is an emerging drug target currently lacking small-molecule inhibitors with good in vivo activity. Thus, there is a need for fast and simple screening methods for identifying inhibitor candidates. Here we demonstrate the use of nuclear magnetic resonance spectroscopy (NMR) for evaluating compound binding to the E. coli β-clamp. To identify suitable molecular probes, a series of tetrahydrocarbazoles were synthesized, some of which contain fluorine. Key challenges in the synthesis were formation of regioisomers during the Fischer indole reaction and reducing racemization at the stereogenic center. The tetrahydrocarbazoles were assayed against the E. coli β-clamp by saturation-transfer difference (STD) NMR, waterLOGSY and T1ρ. Analysis by isothermal titration calorimetry gave KD-values of 1.7-14 μM for three fluorinated probe candidates, and NMR chemical shift perturbation experiments confirmed these molecules to directly interact with the β-clamp binding pocket. Binding of the fluorinated molecules to β-clamp was easily observed with 19F-observed T2-based binding experiments, and proof of concept for a fluorine-based binding assay for E. coli β-clamp binders is provided.

Department of Biotechnology and Food Science Norwegian University of Science and Technology NO 7491 Trondheim Norway

Department of Chemistry Norwegian University of Science and Technology Høgskoleringen 5 NO 7491 Trondheim Norway

Department of Material Science Norwegian University of Science and Technology NO 7491 Trondheim Norway

Department of Structure Analysis FZU Institute of Physics of the Czech Academy of Sciences Na Slovance 1999 2 182 00 Prague 8 Czechia

REPIN and NMR Laboratory Department of Biology University of Copenhagen Ole Maaløes Vej 5 2200 Copenhagen N Denmark

Structural Biology and NMR Laboratory Department of Biology University of Copenhagen Ole Maaløes Vej 5 2200 Copenhagen N Denmark

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