African trypanosomes are extracellular blood parasites that cause sleeping sickness in humans and Nagana in cattle. The drugs used to treat these diseases are far from ideal and are associated with a variety of problems, including acute toxicity, short duration of action, and the emergence of resistant parasites. Therefore, the search for novel antitrypanocidal compounds is an important task. Our HHMI-funded research focuses on developing RNA-based therapeutic tools against trypanosome infections. Within this context, we follow three concepts: (1) we design parasite-specific RNA aptamers, which, according to our results, can be converted into "drug-like" molecules; (2) we use aptamers as screening tools for identifying novel small-molecule drug leads; and (3) we investigate parasite-specific RNAs and RNA-driven reaction pathways as potential drug targets.
African trypanosomes are transmitted by tsetse flies. They multiply within the peripheral blood and tissue fluids of an infected host and evade the immune system by antigenic variation. This phenomenon has its molecular basis in the surface presentation of structurally polymorphic N-terminal domains of an abundant glycoprotein known as variant surface glycoprotein (VSG). VSG molecules have a molecular mass of approximately 60 kDa. They homodimerize and are anchored by glycosylphosphatidylinositol within the plasma membrane. The VSG surface induces a strong T cell–independent IgM response as well as a T cell–dependent B cell response that elicits VSG-specific IgG. The parasites, however, evade the host immune system by temporarily expressing different VSG variants. The trypanosome genome encodes a repertoire of about 1,000 different vsg genes, but only one VSG variant is expressed at any given time. The switching frequency from one type to the next has been estimated to range from 10−2 to 10−7 per cell generation, and switching seems to be a stochastic event. Taken together, the VSG surface acts as an exclusion barrier for larger molecules, such as antibodies, while its variable characteristics render the infected host unable to clear the infection.
Despite these features, the trypanosome surface is not an impenetrable casing. Molecules of lower molecular mass, such as the protease trypsin (23 kDa), have been shown to cleave VSG molecules at a site deep within the protein layer. This suggests that molecular cavities exist within the VSG mantle, which, in turn, can be used as binding sites for small molecules capable of penetrating the protein layer. To identify such molecules, we rely on combinatorial nucleic acid libraries of very high complexity in combination with an in vitro evolution method known as SELEX (systematic evolution of ligands by exponential enrichment). The SELEX protocol has been widely used to isolate high-affinity binders, so-called aptamers, to a variety of biological targets. Over the past several years, we have shown that SELEX technology can be used to identify RNA aptamers that bind with high specificity and high affinity to live African trypanosomes. We demonstrated that RNA aptamers can be used as lead structures for the development of novel trypanocidal strategies. However, the RNA molecules have to be converted into pharmacologically active compounds. Within this context, two key issues must be addressed: (1) ribonucleolytic stability, and (2) the systemic half-life of the RNAs within an infected organism. Chemical modification strategies can be used to address both topics. For the serum stability, we use 2′-amino or 2′-fluoro ribose substitutions to generate ribonuclease-resistant aptamer preparations with serum half-lives in the range of hours to several days. Renal clearance can be side-stepped by increasing the molecular mass of the RNAs, which we achieve by coupling the aptamers to high-molecular-mass carrier compounds such as polyethylene glycol (PEG). Both modifications result in aptamer preparations that can be analyzed in animal systems.
We have selected a specific RNA aptamer known as 2-16 RNA to specifically bind to the flagellar pocket of the African trypanosomes. The flagellar pocket represents the main endocytosis–exocytosis site of the parasite, and, as a consequence, the RNA becomes internalized by receptor-mediated endosomal uptake and is routed to the lysosome. We demonstrated that these characteristics of the aptamer can be used to target toxins to the lysosomal compartment of the parasite. We use small synthetic molecules such as the pH-responsive peptide GALA (glutamic acid-alanine-leucine-alanine repeat) as lysolytic compounds. GALA undergoes a transition from a random coil conformation at neutral pH to an amphiphatic helix when shifted to mild acidic conditions. The peptide is covalently attached to 3′-oxidized aptamer preparations by reductive amination.
Infections with African trypanosomes are characterized by the parasite crossing the blood-brain barrier (BBB), and thereby causing severe neurological symptoms. Thus, based on the clinical need for trypanocidal drugs capable of crossing the BBB, we have started to develop an in vitro BBB model. The system will be used to directly measure BBB permeability of trypanocidal compounds and also to identify trypanosome genes that contribute to the BBB passage by using an RNA interference-based forward genetic approach. The in vitro system uses brain microvascular endothelial cells (PBMEC) that form a polarized cell monolayer on top of a permeable polyester/polycarbonate membrane support. The brain microvascular endothelial cells show the expected cell type–specific expression of the endothelial cell marker von Willebrand factor (Factor VIII) in addition to the marker polypeptide P-glycoprotein. P-glycoprotein is a member of the multiple-drug resistance (Mdr) efflux transporter family; its expression thus allows for the removal of contaminating pericyte cells by the addition of puromycin. This results in more than 99 percent homogeneity of the endothelial cell monolayer. Immunocytochemical analysis of the tight junction–associated protein zonula occludens 1 (ZO-1) verified the formation of tight junctions at day three after seeding. The physical tightness can be verified by a high transendothelial electric resistance (TEER) and a very low permeability of marker molecules for paracellular diffusion such as 14C-sucrose.
Parasite surface-specific RNA aptamers can also be used to screen small-molecule libraries for compounds with aptamer-displacing activity in competition binding experiments. We used two of our selected aptamers in a first trial experiment. The screening was done with a library of small organic compounds, including a panel of anionic dyes; a set of substituted carbohydrate derivatives; and a series of synthetic aminoglycoside mimetics. The aptamer displacement reaction was performed with live parasites at a concentration of 100 µM for the organic compounds. This represents a concentration about 1,000-fold above the Kd of the aptamer/parasite interaction. Five compounds were identified with aptamer displacing activity. We performed cytotoxicity assays to rule out that the displacement reaction was due to the cytotoxic effects of the individual compounds. The experiments verified a toxic effect for only one compound (LD50 of 5 µM) while the other molecules were nontoxic to bloodstream-stage trypanosomes even at concentrations above 100 µM. Thus, we successfully identified four low-molecular-mass compounds with aptamer displacement activities in the low micromolar range. The molecules can now be used as novel lead compounds for designing alternative therapeutic intervention strategies.
Last updated August 2008
