Activity of Aromathecins against African Trypanosomes

Abstract

African sleeping sickness is responsible for thousands of deaths annually, and new therapeutics are needed. This study evaluated aromathecins, experimental inhibitors of mammalian topoisomerase IB, against Trypanosoma brucei African trypanosomes. The compounds had selectively toxic antiparasitic potency, in situ poisoning activity against the phylogenetically unique topoisomerase in these parasites, and a representative compound intercalated into DNA with micromolar affinity. DNA intercalation and topoisomerase poisoning may contribute to the antitrypanosomal activity of aromathecins.

Keywords: Trypanosoma brucei; aromathecin; sleeping sickness; structure-activity; topoisomerase.

FIG 1

Type IB topoisomerase inhibitors. Compound 1, natural product camptothecin; compound 2, indenoisoquinoline scaffold; and compound 3, aromathecin scaffold with relevant positions numbered.

FIG 2

Unwinding of pHOT1 by m-AMSA or compound 11. Negatively supercoiled pHOT1 plasmid DNA substrate was incubated in topoisomerase I reaction buffer (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin [BSA], 0.1 mM spermidine, 5% glycerol) either with or without human topoisomerase I prior to the addition of solvent or drug. Reactions were quenched with SDS, digested with protease, and extracted with chloroform, and DNA was separated by gel electrophoresis (1% agarose, 36 mM Tris, 1 mM EDTA, 30 mM NaH₂PO₄ [pH 7.7], with 0.2 μg/ml chloroquine; 18 h, 1 V/cm), stained with ethidium bromide, and visualized by fluorescence. Lane 1, relaxed pHOT1 plasmid DNA standard; lane 2, negatively supercoiled pHOT1 substrate; lane 3, substrate plus topoisomerase; lanes 4 and 5, substrate plus topoisomerase and then 2% or 1% dimethyl sulfoxide (DMSO), respectively; lane 6, substrate plus 100 μM m-AMSA; lane 7, substrate plus topoisomerase, and then 100 μM m-AMSA; lanes 8 and 9, same as lane 7 but with 10 or 1 μM m-AMSA, respectively; lane 10, pHOT1 substrate plus 50 μM compound 11; lanes 11 to 16, substrate plus topoisomerase, and then 50, 25, 10, 5, 1, or 0.5 μM compound 11, respectively. W, well; R, relaxed pHOT1 plasmid; ISOMERS, partially supercoiled topoisomers; SC, negatively supercoiled substrate pHOT1.