Table 2. Dissociation Constants (K_d) Measured by Titration Series of [¹⁵N,¹H]-HSQC with Increasing Ligand Concentrations and 200 μM PIN1^a.

compound	Kd T154 (mM)	Kd A124 (mM)	polarization ratio	T1ρ decay ratio	STD
1	1.5	3.6	0.42	0.67	+
13	1.7	4.5	0.38	0.45	+
10	8.4*	11.8*	0.38	0.90	–
11	5.4*	8.8*	0.53	0.92	–
3	12*	8.6*	0.75	0.98	–

^aThe K_ds were obtained from fitting with eq 6 in the CcpNMR software 3.0.⁴⁰ Fitting curves are provided in Figure S7. The values annotated with * could not be fitted to a maximum and therefore should be considered with caution. The proposed K_ds are most likely to be higher than these values. The polarization ratios were calculated according to eq 6 and showed a trend with low values for higher affinities, with the exception of compound 10, which shows a low polarization ratio despite a low affinity. The T_1ρ decay ratio and the saturation transfer difference (STD) were measured for samples containing 200 μM ligand (L) and 200 μM ligand with 20 μM protein (PL). The T_1ρ decay ratio was calculated with the formula (Γ_400ms/Γ_10ms)_PL/(Γ_400ms/Γ_10ms)_L. The observation of ligand signals in the STD NMR spectra detects a protein–ligand interaction; (+) the absence of a signal fails to detect protein–ligand interaction (−). Figure S8 provides examples of spectra for high (compound 1) and low (compound 11) T_1ρ decay ratios and the positive STD spectrum for compound 1.