Work

Synthesis and evaluation of fluorinated tetrahydrocarbazoles as probes in NMR based binding assay of the E. coli β sliding clamp

Year: 2025

Type: article

Abstract: • Synthesis of tetrahydrocarbazoles by Fisher-Indole reaction. • Fluoro-containing tetrahydracarbazoles identified as E. coli β-clamp binders. • Binding to the canonical binding pocket of β-clamp is shown by NMR. • K D values in the range of 1.7–14 μM (ITC). • Proof of concept for an 19 F NMR based assay for small-molecular binding with β-clamp. 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 T 1ρ . Analysis by isothermal titration calorimetry gave K D -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 19 F-observed T 2 -based binding experiments, and proof of concept for a fluorine-based binding assay for E. coli β-clamp binders is provided.

Source: Bioorganic & Medicinal Chemistry

DOI: 10.1016/j.bmc.2025.118139