African trypanosomiasis (sleeping sickness), caused by the protozoan parasite Trypanosoma brucei
, is a devastating disease transmitted by tsetse flies and is prevalent throughout sub-Saharan Africa. Lethal if untreated, this infection is responsible for substantial morbidity and mortality (40
). The impact of the disease is exacerbated by the paucity of effective drugs, their toxicity, and the emergence of drug resistance (14
). This is exemplified by the fact that melarsoprol, an organoarsenical that is the only drug effective against the central nervous system stage of both East and West African sleeping sickness, itself causes 5% mortality and has a disturbing failure rate (14
). There is a pressing need to develop new drugs as well as to identify molecular targets for new therapies.
DNA topoisomerases have proven to be effective drug targets in prokaryotic and eukaryotic systems (16
). These enzymes catalyze topological changes in DNA and have vital roles in many aspects of nucleic acid metabolism. Based on their mechanism of action, topoisomerases can be classified as type I enzymes, which break a single strand of the DNA helix during the catalytic cycle, and type II enzymes, which make double-stranded breaks. On the basis of primary sequence and reaction mechanism, type I topoisomerases are further subdivided into type IA and type IB (21
). Type IB topoisomerases are clinically relevant by virtue of being the target of anticancer camptothecins. The latter are exquisitely specific for topoisomerase IB (38
) and belong to a class of agents collectively known as topoisomerase “poisons.” The poisons are a distinct subgroup of topoisomerase inhibitors that bind to and stabilize a normally transient DNA-topoisomerase catalytic intermediate termed the cleavable complex. Persistence of such complexes leads to strand breaks in DNA and cell death, making topoisomerase poisons effective antiproliferative agents (22
Kinetoplastid topoisomerases have been a focus of much study with a view toward their utilization as drug targets (4
). The type IB topoisomerase of T. brucei
(and Leishmania donovani
, a related kinetoplastid pathogen) is an enzyme singularly distinct from its mammalian counterpart. While all other described type IB topoisomerases are single polypeptide moieties, the enzyme from kinetoplastids is heteromultimeric, composed of two distinct proteins encoded by two independent genes (7
). Knock-down studies have revealed that this enzyme is essential for trypanosomes (5
), validating its status as a potential drug target. Camptothecin kills trypanosomes by targeting topoisomerase IB (9
), and structure-activity studies with camptothecin analogs have illuminated the possibility of generating agents that can selectively target the trypanosomal enzyme (10
). Taken together, these aspects make topoisomerase IB poisons a promising source of lead compounds for antitrypanosomal drug development.
Indenoisoquinolines, a noncamptothecin class of synthetic compounds (Fig. ), were originally identified in a search for anticancer agents. In the 60-cell-line National Cancer Institute anticancer drug screen, indenoisoquinolines demonstrated a cell-line-specific cytotoxicity profile consistent with topoisomerase IB poisons, and they can trap topoisomerase IB-DNA covalent complexes (19
). Some of the indenoisoquinolines have the ability to bind and intercalate into DNA in the absence of the enzyme (30
), and others may even interact directly with the mammalian topoisomerase IB protein at high drug concentrations (29
). We have evaluated a battery of indenoisoquinolines (26
) against T. brucei
and we find that they have potent antitrypanosomal activities in vitro, inhibit nucleic acid synthesis in the parasite, act as topoisomerase poisons within the cell, and show preliminary evidence of efficacy in mice challenged with trypanosomes.
The indenoisoquinoline scaffold. Structure-activity analysis was performed with substitutions at the numbered positions.