External ID: IucA Pilot Assay Tocris Library

Protocol: A solution containing 55.6 mM HEPES pH 7.5, 0.11% Tween 20, 16.7 mM MgCl2, 55.6 mM hydroxylamine, 55.6 microM ATP, 55.6 microM citrate, and 0.28 U/mL IPP was dispensed (45 microL) into clear polystyrene microplates (Corning, Inc.) using a BioTek MicroFlo dispenser.

Next, 40 nL of test compounds (10 mM in DMSO, 8 microM final concentration) were transferred from deep-well blocks to the reaction solution using a stainless-steel pin tool operated by a robotic workstation (JANUS, PerkinElmer, Waltham, MA). The IucA-catalyzed reaction was initiated by adding 5 microL of 3 microM IucA in 25 mM HEPES, 75 mM NaCl, and 0.1 mM TCEP at pH 7.5.

The reactions were allowed to proceed for 30 min at room temperature before being quenched by dispensing (microFill, BioTek) 13 microL of MG developing solution, containing 1.0 mg/mL MG oxalate, 1.5% (w/v) ammonium molybdate, 0.15% (v/v) Tween 20, and 4.7 N sulfuric acid. After allowing the assay color to develop/stabilize for 30 min, the absorbance at 620 nm was measured (EnVision 2103 Multilabel Microplate Reader, PerkinElmer).

Average positive controls from 24 wells with no test compound and average negative controls from 8 wells with no enzyme were calculated. Dynamic range was calculated by difference between Avg Pos Control and Avg Neg Control. Percent inhibition was calculated by the ratio of (Avg. Pos. Ctrl - Sample OD) to Dynamic Range.

Comment: Protein Target is
IucA

EMB09144
574 aa
G057_19877
Klebsiella pneumoniae hvKP1

Active compounds were defined by <80% activity at 8 microM screening concentration.