Antimalarial Quinoline Drugs Inhibit \u03b2-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases

Tomás Herraiz,Vicente J Arán,Hugo Guillén,Diana González-Peña

doi:10.1038/s41598-019-51604-z

Abstract

Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (β-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (β-carbolines or nitroindazoles) were studied as inhibitors of β-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and β-carbolines had no effect. Compounds that inhibited β-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.

Highlights

The synthesis of hemozoin constitutes a unique system of Plasmodium to detoxify heme; its inhibition is a useful target for antimalarial drugs action[2,18,31,32]
Hemin incubated at 37 °C and pH 4.8 in presence of tween 20 crystallized and precipitated as a dark powder that was isolated and had IR spectra exhibiting bands at 1210, 1663 and 1712 cm−1 (Supplementary Figure 1) corresponding to β-hematin or hemozoin, the pigment of Plasmodium18,24,46. β-Hematin has UV-VIS spectrum with lower relative absorbance at 415 nm and higher at 630 nm than free hemin, so that the concentration and relative contribution of free hemin can be assessed from A415 nm-A630 nm[47,48]
Heme crystallization system in the digestive vacuole of Plasmodium has been a useful target for antimalarial drugs[16,49,50]

Summary

Introduction

The synthesis of hemozoin constitutes a unique system of Plasmodium to detoxify heme; its inhibition is a useful target for antimalarial drugs action[2,18,31,32]. Quinoline drugs (e.g. chloroquine) (Fig. 1) have been successfully used for years to treat malaria These drugs are less effective as parasites are becoming resistant. Quinoline drugs exert their antimalarial action by interfering with heme detoxification[33,34,35]. This work highlights a link between free hemin build-up by quinoline drugs with peroxidative effects (oxidative effects) and inhibition of cysteine proteases (inhibition of proteolysis). These events may underlay the basics for toxicity of antimalarial quinoline drugs in blood feeding organisms including Plasmodium and could be useful for the development of new antimalarial agents

Methods

Results

Conclusion