Figure 6. Competitive binding of 2-f-LIGRLO-dtpa with peptidomimetic PAR₂ agonists and controls.

16HBE14o- cells plated onto 96-well plates were simultaneously exposed to 300 nM 2-f-LIGRLO-dtpa and various concentrations of known agonists 2-f-LIGRLO, 2-at-LIGRL, 6-an-LIGRL or a control peptidomimetic, 3-ia-LIGRL and assessed for competitive binding. Binding is expressed as a percentage of the maximal net Eu fluorescence measured from 2-f-LIGRLO-dtpa (300 nM) binding (% Max). The two most potent agonists, 2-f-LIGRLO and 2-at-LIGRL displayed sigmoidal curves consistent with high competition for PAR₂ binding. The slightly less potent full agonist, 6-an-LIGRL, displayed a right-shifted sigmoidal binding curve, indicative of reduced ability to compete against 2-f-LIGRLO-dtpa for receptor binding. The control peptidomimetic, which does not initiate PAR₂ signaling, displayed limited binding, and was best fit with a linear function. Apparent IC₅₀ values for agonists showing competitive binding for PAR₂ are as follows: 2-f-LIGRLO: 3.29 µM; 2-at-LIGRL, 2.06 µM and; 6-an-LIGRL, 18.54 µM. Data of all agonists are from n ≥ 3 samples at each concentration of all agonists in each multi-well experiment, and from at least two independent multi-well experiments. Control peptidomimetic (3-ia-LIGRL) data is from one multi-well experiment.