INHIBITION OF MYELOPEROXIDASE BY BENZOIC-ACID HYDRAZIDES

Myeloperoxidase is the most abundant protein in neutrophils and cataly ses the conversion of H2O2 and chloride into HOCl. To help clarify the role of this enzyme in bacterial killing and inflammation, a specific and potent inhibitor needs to be identified. We have studied a series of benzoic acid hydrazides and found that in general they inhibit the peroxidation activity of myeloperoxidase with an IC50 value of less t han 10 mu M. The IC50 values of derivatives with substituents containi ng oxygen or nitrogen were related to their Hammett substituent consta nts. This indicates that myeloperoxidase oxidizes the hydrazide group of these compounds, and the degree to which they inhibit the enzyme is dependent on the ease of their oxidation. Unsubstituted benzoic acid hydrazide and its 4-chloro derivative were poor inhibitors of peroxida tion. Thus it is likely that hydrogen-bonding of the enzyme to substit uents containing oxygen or nitrogen increases the binding affinity of the hydrazides and enhances their oxidation by myeloperoxidase. 4-Amin obenzoic acid hydrazide (ABAH) was the most potent inhibitor of peroxi dation. It irreversibly inhibited HOCl production by the purified enzy me, having an IC50 value of 0.3 mu M. With neutrophils stimulated with opsonized zymosan or phorbol myristate acetate, ABAH inhibited HOCl p roduction by up to 90 % and the IC50 values were 16 mu M and 2.2 mu M respectively. In the presence of superoxide dismutase, these values de creased to 6.4 mu M and 0.6 mu M respectively. ABAH had no effect on s uperoxide radical (O-2(-.)) production and degranulation by neutrophil s, nor did it inhibit catalase or glutathione peroxidase. Thus ABAH is an effective and selective inhibitor that should be useful for determ ining the contribution of myeloperoxidase to oxidant-mediated reaction s of neutrophils.