Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces oxidative stress: proposed antimalarial mechanism of clotrimazole

Abstract

The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H₂O₂, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are k_i= 2.85 μm, k_inact = 0.9 min^-1, and t½ = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum.