Table 7.

Enzyme binding kinetics of BAY 85-8501 (29) and the endogenous α-proteinase inhibitor (αPI).

Compd	kon [106 m−1 s−1][a]	koff [10−3 s−1][b]	Residence time [h]
29	12.6	1.0	∼0.3
αPI	14.5[c]	0.0002[d]	∼1900[e]

^[300] The on-rates at which elastase inhibitors bind to the target were determined by applying a functional biochemical assay using a substrate with a modified fluorescent label, MeOSuc-AAPV-umbelliferyl; this allows very sensitive detection of substrate hydrolysis on the millisecond timescale, in the presence or absence of elastase inhibitor. Using nonlinear regression of the reaction progress curves, the observed rate constant of the onset of inhibition (k_obs) was obtained and plotted against the inhibitor concentration. The slope of the linear regression revealed the estimated k_on value (Supporting Information).

^[b] Calculated from k_on and K_i according to the equation k_off=k_on×K_i.

^[c] Data from Sinden et al.[33]

^[d] Calculated with K_i∼1×10⁻¹⁴ m, taken from Beatty et al.[32]

^[e] Calculated according to target residence time, 1/k_off.