A new class of antiparasitic drugs

Transmitted by insect vectors, human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis cause millions of infections, which are often fatal unless treated with toxic drugs. Although there are promising new therapeutics for HAT (1), development of reliable, safe, and affordable drugs for Chagas disease and leishmaniasis remains challenging. On page 1349 of this issue, Rao et al. (2) introduce cyanotriazoles (CTs), a new class of compounds with potent and broad trypanocidal activity. These covalent inhibitors of topoisomerase II, an enzyme involved in nuclear DNA replication, show high efficacy in clearing trypanosomes and leishmania parasites from infected animals, efficient tissue distribution, and low toxicity in the mammalian host. Acting on a different target than those of existing interventions, these drug candidates bring the possibility of eradication and effective management of many neglected tropical diseases.

Trypanosomiasis, Chagas disease, and leishmaniasis are all caused by protozoan hemoflagellates belonging to the class Kinetoplastea. They are typified by the “kinetoplast,” a condensed body of mitochondrial DNA, and include agents of many tropical diseases. Historically called “sleeping sickness,” West and East HAT are caused by Trypanosoma brucei gambiense and T. brucei rhodesiense subspecies, respectively. During a tsetse fly blood meal, parasites enter the circulatory system, where they differentiate into proliferative form and divide rapidly. Ultimately, the trypanosomes populate adipose and other tissues (3) and penetrate the blood-brain barrier, leading to neurological damage and death.

Leishmania donovani and Leishmania infantum are introduced into hosts through sand fly bite, which triggers systemic life-threatening visceral leishmaniasis. Both parasites infect and proliferate in macrophages, with L. infantum also persisting extracellularly. A combinatorial drug regimen is often required to clear both intracellular and extracellular forms. Also spread by sandflies, other Leishmania species give rise to mucocutaneous leishmaniasis that inflicts disfigurements or skin lesions. An infection by Trypanosoma cruzi could be transmitted through the bite of the triatomine bug, congenitally, or through contaminated blood. After the initial acute phase with nonspecific symptoms, the often undiagnosed Chagas disease may remain asymptomatic for years while progressing to heart failure or digestive tract damage (4).

Rao et al. used an automated assay to evaluate inhibitory effects of approximately 2 million compounds on proliferation of a nonpathogenic T. brucei strain, Lister 427, in axenic culture. This phenotypic screen identified a heterocyclic CT molecule as a potent growth inhibitor that displayed similar effects on pathogenic T. brucei gambiense and T. brucei rhodesiense. Remarkably, the lead compound also demonstrated effectiveness in killing T. cruzi and L. donovani parasites in vitro and low cytotoxicity for human and mouse cultured cells and in mouse models of infection. During lead optimization, chemical modifications improved efficacy, and pharmacological properties manifested from a robust clearance of T. brucei and T. cruzi systemic infections in mice. A single dose of a variant molecule named CT3 demonstrated fast-sterilizing activity by curing early and late stages of HAT in mouse models more effectively than the current standard-of-care drugs.

High selectivity and comprehensive activity of CT compounds against multiple kinetoplastid protozoans hinted at a conserved mechanism of action. Rao et al. gained initial insights from observations of nuclear DNA damage and cell cycle arrest after CT3 treatment, whereas mitochondrial DNA remained unaffected. With the understanding that CTs damage nuclear but not organellar DNA, the authors obtained a series of drug-resistant T. brucei isolates, most of which carried mutations in the topoisomerase II (Top2) gene that is essential for parasite viability (5). Further analyses of these drug-resistant strains established CTs as a new class of covalent topoisomerase II inhibitors.

An adenosine triphosphate (ATP)–dependent molecular machine, topoisomerase II modulates supercoiling and resolves topological conflicts that arise during DNA replication. By transiently breaking double-stranded DNA (dsDNA) and forming a covalent adduct with a nucleic acid, the enzyme translocates the double helix through the gap and repairs breaks. Because DNA breaks may destabilize the genome, ligation is kinetically favored, and the topoisomerase II–DNA cleavage complexes are short-lived and readily reversible. It follows that a compound that can stabilize the topoisomerase II–DNA complex, a so-called topoisomerase II “poison,” would trigger accumulation of dsDNA breaks and eventual cell death (6). In vitro activity assays demonstrated that CTs induce accumulation of linearized DNA in a reaction catalyzed by the recombinant T. cruzi topoisomerase II, indicating that CTs indeed stabilize the topoisomerase II–trypanosomal DNA cleavage complex but spare the homologous human topoisomerase II enzyme. These findings reveal a new class of covalent topoisomerase II inhibitors but raise the question of selectivity. Topoisomerase II poisons are commonly used as antibiotics (7) and in cancer chemotherapeutics (8) and tend to exhibit general inhibitory activity against this family of essential cellular enzymes.

To understand the specificity of CTs to kinetoplastids, Rao et al. used cryo–electron microscopy (cryo-EM) to determine a high-resolution structure of the T. cruzi topoisomerase II bound to DNA and their lead compound, CT1. The structure revealed intercalation of the aromatic trifluoromethylphenyl moiety shared by all CT compounds between dsDNA bases and spatial occupancy of the active site, such as those observed with human and bacterial topoisomerase II poisons. Conversely, formation of a covalent bond between cyanoazole heterocycle and a cysteine residue distinctly conserved in topoisomerase II among trypanosomatids provides a structural basis for the observed selectivity of CT compounds toward trypanosomal enzymes.

Therapeutic potency of CTs—which exceeds that of currently available drugs—and their broad trypanocidal activity, low cytotoxicity, and oral bioavailability approaching 100% in a mouse model provide strong encouragement for future clinical trials. As a new class of trypanocides, these molecules have great promise to aid the World Health Organization’s efforts in eliminating West African trypanosomiasis by 2030 and to improve treatment of Chagas disease and leishmaniasis. The potential for using CTs to treat African animal trypanosomiasis, arguably the most economically important livestock disease of the continent (9), also seems apparent. Rightfully called “neglected,” these diseases are ultimately yielding to collective efforts of international organizations and synergistic research prowess.

These pathogens have likely played an important role in early hominid evolution, and kinetoplastids have been studied as a model of early branching eukaryotes. This work has yielded discoveries of antigenic variation, trans-splicing, polycistronic transcription, RNA editing and guide RNAs, and other phenomena of general biological importance (10). Although the threat of parasites to human life and economy is reduced by advances in pharmacological interventions, these organisms remain an endless source of unconventional biology. J

Shown is a colored scanning electron micrograph of Leishmania donovani promastigotes. Transmitted to humans by the bite of phlebotomine sand flies, these parasites cause visceral leishmaniasis.