Summary /

As 8-hydroyquinoline are known metal chelators, the above data suggest that these inhibitors bind directly to the active site iron, which raises the possibility that these inhibitors could have the necessary redox potential to reduce the active ferric iron to the resting ferrous state, as has been reported previously. To investigate the redox potential of this chemotype, DPPH, a free radical scavenger, was incubated with the probe compound; no reduction of DPPH was observed. It should be noted that stoichiometric reduction of DPPH was achieved by the known reductive LO inhibitor, NDGA, suggesting that these 8-HQ inhibitors are not reductive in nature.

In addition to characterizing the inhibition constants, the mode of binding was investigated with steady state kinetics using the probe compound by monitoring the formation of 12-HPETE as a function of substrate and inhibitor concentration in the presence of 0.01% Triton X-100. Re-plots of KM/kcat and 1/kcat versus inhibitor concentration (Figure 2) yielded linear plots, with Kic equaling 0.8 +/- 0.1μM, which is defined as the equilibrium dissociation constant. The similar affinity of inhibitor binding to both the enzyme and the enzyme substrate complex (Kic is approximately Kiu) is a rare example of true non-competitive inhibition, which is indicative of a distal site from the catalytic site, whose inhibitor affinity is not affected by substrate binding. It should be noted that attempts to determine the reversibility of inhibition were made, but the enzyme dies too fast to allow for attempted wash-out experiments. No timedependent inhibition was displayed when ML127was incubated with 12-hLO, unlike the time-dependent inhibition seen for the bidentate catechol inhibitors against sLO-1.

Figure 1. UV-Vis Assay Dose Response for ML127

Figure 2. Steady-state kinetics data for the determination of Kic for 12-hLO with inhibitor. KM/Vmax (slope, KM units are μM) versus [Inhibitor] (μM) is the secondary replot of the inhibition data, which yielded a Kic of 0.8 +/- 0.1μM.