Table 1.

Summary of parameters describing the inhibitory effect of hydroxamic and non-hydroxamic HDAC6 inhibitors.

Inhibitor	IC50, nM (s) a	Kiapp, nM b	Ki, nM c	konapp, M−1 min−1 b	koff, min−1 b
ITF2357	8.0 ± 0.4 (1.0 ± 0.06)	ND 9.2 e	-	ND	ND
ITF3756	3.40 ± 0.07 (1.04 ± 0.03)	ND 3.4 e	1.9 ± 0.1	ND	ND
1	6.6 ± 0.34 (0.96 ± 0.04)	8.90 ± 0.23	ND	5.6 ± 0.03 × 105	5.0 ± 0.13 × 10−3 (6.5 ± 0.2 × 10−3)d
2	10.8 ± 0.60 (0.81 ± 0.04)	13.0 ± 1.4	5.6 ± 0.6	6.5 ± 0.2 × 105	8.5 ± 0.9 × 10−3 (11 ± 0.2 × 10−3)d

^a The data from experiments shown in Figure 1 were used to calculate the IC₅₀ by calculating percentage residual activity from the steady-state velocities (v_s) and the initial velocity measured in the absence of inhibitors, and fitting to Equation (1). The slope factor (s) is in parenthesis. ^b The apparent K_i, k_on and k_off were calculated by fitting the rate constants for conversion into the inhibited complex at varying inhibitor concentrations (k_obs) to Equations (4) and (5). ^c The K_i was calculated from Equation (6) that describes competitive inhibition using the steady-state velocity calculated from progress curves at varying substrate concentrations in the presence of varying inhibitor concentrations (Figure 2). ^d The value was determined from jump-dilution assays. For ITF2357 and ITF3756, the onset of inhibition (k_obs) and inhibitor release from the enzyme were too fast to measure precisely. Thus, the values could not be determined (ND). ^e For ITF2357 a competitive mechanism of inhibition was determined with Yonetani –Theorell plots (Figure 3) knowing that ITF3756 competes with the substrate for binding to HDAC6 (Figure 2). The apparent inhibition constants of both ITF2357 and ITF3756 were determined at 2 µM Fluor de Lys Green, a concentration corresponding to the K_m value.