Newly Identified Antibacterial Compounds Are Topoisomerase Poisons in African Trypanosomes

Human African trypanosomiasis, also known as African sleeping sickness, is a protozoal infection caused by Trypanosoma brucei that is fatal without treatment (3). Parasites are transmitted to humans via the tsetse fly, which is found exclusively in sub-Saharan Africa and thus limits the geographic distribution of the disease. Currently, there are an estimated 50,000 to 70,000 cases (25). Despite the lethality of sleeping sickness, the drugs currently available for its treatment are toxic and they require parenteral administration (10). In addition to these obstacles, resistance has been a growing concern, and availability and cost are major problems for the population affected. In light of these issues, new drugs for African trypanosomiasis are in demand. The recently completed T. brucei genome sequence has made possible the identification of potential drug targets which are essential to trypanosomes and have no human ortholog (2). Trypanosomes have a single mitochondrion with an unusual mitochondrial DNA, known as kinetoplast DNA (kDNA). The kinetoplast is a massive and topologically complex structure, comprised of thousands of interlocked ∼1-kb DNA minicircles and a few dozen ∼23-kb maxicircles (structure and replication of kDNA are reviewed in reference 16). This intricate network is in striking contrast with the monomeric circular DNA molecule found in several copies in each human mitochondrion. Replication of kDNA requires the release of individual covalently closed minicircles from the network into a pool of “free” minicircles, DNA synthesis via theta structure intermediates, and reattachment to the network of progeny minicircles, which retain nicks or gaps until all circles have been replicated. Every step of kDNA replication depends on the function of topoisomerases. These enzymes catalyze reactions that alter the topological state of DNA by cleaving the phosphodiester backbone through the formation of a covalent phosphotyrosine link, transferring segments of DNA through the break, and then ligating the DNA together (topoisomerases are reviewed in references 7, 9, and 28). They are classified into two types, based on the number of DNA strands cleaved: type I enzymes cleave one strand while type II enzymes cleave both strands in reactions requiring ATP. Type I enzymes are further classified into A and B subfamilies based on structural and mechanistic differences. Topoisomerase IA enzymes catalyze the relaxation of negatively supercoiled DNA through an “enzyme bridging mechanism.” After cleavage of a single strand, topoisomerase IA remains covalently bound to the 5′ end as it processively unwinds the DNA. If the enzyme acts across from a preexisting nick, it can pass a double helix through the resulting double-stranded break. The type II enzymes also utilize a 5′-end linkage during catalysis, in contrast to IB topoisomerases, which create a covalent link at the 3′ end of the cleavage site. T. brucei has at least six catalytically active topoisomerases, and two of these appear to function exclusively in the mitochondrion: topoisomerase IAmt and topoisomerase IImt (15, 21). These mitochondrial enzymes are especially attractive drug targets since they are essential according to RNA interference (RNAi) and drug studies (21), and their depletion leads to akinetoplasty (the loss of kDNA), which in turn results in death (13). Compounds that target topoisomerases can be classified as poisons or nonpoisoning inhibitors (17). Poisons bind to the Michaelis complex comprised of DNA and enzyme, forming a ternary DNA-protein-inhibitor “cleavable complex.” In situ, the presence of a poison prevents strand ligation, and thus, the topoisomerase remains in an inert complex that is fixed on the DNA helix. Covalent adducts of topoisomerase and DNA can be forged by rapid denaturation of the topoisomerase with detergents such as sodium dodecyl sulfate (SDS). Nonspecific inhibitors also decrease topoisomerase activity but do not promote the formation of cleavable complexes. They may include agents that deform the DNA substrate, bind proteins with high affinity, or compete for binding at active sites, such as DNA and ATP analogs. In the cell, stationary cleavable complexes prove lethal when replication machinery collides with them, producing breaks in the DNA (11, 17). Therapeutically useful topoisomerase-targeting agents are all poisons: fluoroquinolones and etoposide are topoisomerase II poisons for antibacterial and antitumor therapy, respectively, while camptothecin is an antitumor agent that poisons topoisomerase IB. For T. brucei, poisons against topoisomerase IAmt would be ideal since this enzyme has no human ortholog and is found exclusively in the mitochondrion with kDNA (21). Recently, a luciferase reporter system that detects the accumulation of covalent DNA complexes with recombinant Yersinia pestis topoisomerase IA in Escherichia coli was developed (8). The system was used in a high-throughput assay to screen for compounds that poisoned Y. pestis topoisomerase IA. Three compounds were found to promote intracellular cleavable complex formation by the recombinant topoisomerase IA and to inhibit relaxation of DNA by the isolated enzyme. These compounds also inhibited the growth of bacterial cells. Lead compounds 1 and 2 identified in the Y. pestis model (Fig. ​(Fig.1),1), hereafter referred to as 1895 and 0020, respectively, were tested against T. brucei to assess cytotoxic activity and ability to poison topoisomerase IAmt. These compounds were found to be trypanocidal at micromolar concentrations, they appear to intercalate into DNA, and, unexpectedly, they promote linearization of free minicircles by poisoning intracellular topoisomerase IImt. FIG. 1. Molecular structure of 1895 (A), which is N,N-dimethyl-2-(phenanthro[3,4-d][1,3]dioxol-5-yl)ethanamine, and 0020 (B), which is 2-(piperidin-1-ylmethyl)indeno[1,2,3-de]phthalazin-3-(2H)-one. Of interest, 1895 contains a methylenedioxy motif seen also in ...