| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | All ITC experiments for the ester series compounds were carried out at 25 C. on a MicroCal Auto iTC200 (GE Healthcare) instrument. The buffer used in the titrations of the compounds belonging to the ester series was 50 mM phosphate buffer, pH 6.5 and the concentration of DMSO was 2% v/v for all the compounds, unless otherwise stated in Table 51. Final compound solutions were heated to 65 C. and/or sonicated prior to the experiment. Each experiment consisted of an initial injection of 0.4 μL followed by nineteen 2 μL injections and in most of cases for these compounds, the continue injections protocol was used leaving the cell intact and performing a second series of titrations using the above protocol to achieve saturation. Control experiments were performed, where each compound was titrated into buffer and when small amount of heat was detected due to heat of dilution, it was subtracted when processing the data using a linear fit method. In all cases the first injection was omitted from the data processing. All data were analysed using the MicroCal PEAQ-ITC Analysis software. Because some compounds have affinity that lie in the low-mid micromolar range and the c value (Wiseman constant) is very small, a fixed stoichiometry to 1 was applied during the non-linear regression of the raw data for fitting the data (Tellinghuisen, J., Isothermal titration calorimetry at very low c. Analytical biochemistry, 373 (2), 395-7, 2008). Control runs on literature compounds were run as a control at the end of each experiment to verify that CypA remained active during the duration of the experiment. |
| Affinity data for this assay | |
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