TARGETS AND MECHANISMS OF ACTION FOR PARASITICAL AGENTS

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Our collaborative group as a whole has taken a multifaceted approach to the development of new chemotherapeutic strategies for AIDS- toxoplasmosis. In addition to working towards the improvement of existing drugs and the validation of parasite-specific targets, we have also examined novel classes of parasiticidal agents active against T. gondii. Focusing on these latter compounds, Project I seeks to discover the relevant targets for existing drugs of proven efficacy, taking advantage of molecular tools (many developed under the auspices of this grant) which now render Toxoplasma uniquely accessible to genetic manipulation. Macrolide and lincosamide antibiotics are highly effective against T. gondii, but in vitro studies reveal unusual pharmacokinetics: parasites die only after entry into the next parasitophorous vacuole, hours or even days after treatment. Cross-resistance profiles of mutant parasites support a protein-inhibitory mechanism, but molecular sequence analysis and biochemical studies on intact parasites and isolated ribosomes indicates that neither cytoplasmic nor mitochondrial ribosomes is likely to be involved. We have identified alternative ribosomal targets, however, including genes associated with the novel plastid-like extrachromosomal genome. In addition to examining appropriate ribosomal genes in mutant parasites, we propose to identify the macrolide-resistance locus by genetic means. Returning to the plastid-like genome, we will determine the subcellular location of this parasite-specific structure and investigate associated targets with therapeutic potential. Among the many compounds screened for parasiticidal activity, the dinitroaniline herbicides are a particularly intriguing group, inhibiting T. gondii replication via disruption of the parasite's intranuclear mitotic spindle. Their disruption of plant microtubules appears to be an indirect effect, and the precise molecular target (and basis for resistance) remains unclear. We have isolated herbicide resistant T. gondii mutants for genetic characterization, and will test suitable analogs of the original lead compounds for in vivo activity against Toxoplasma. In other experiments, cyclosporins were also found to be potent parasiticidal drugs, presumably through interaction with parasite cyclophilins. Two species of cyclophilin cDNAs have been cloned from T. gondii, and we will investigate the biological basis of their function using a combination of biochemical, immunological, and molecular genetic techniques. Both the dinitroanilines and cyclophilins exhibit features which yield new insights into the cell biology of the protein secretory pathway, which may be expected to provide additional strategies for intervention. Further pharmacological screening (and genetic analysis of promising compounds) will focus on herbicides and coccidiostats known from studies on Eimeria and Plasmodium species.