Based on the discoveries during the initial funding period, a former core has been converted to a Project for the renewal application. It has become increasingly important to the program to determine how the dicationic molecules exert their effects on the targeted pathogens. It has also become clear that while there are many similarities in the mode of action of compounds from both Tidwell and Boykin, their differences (ie. in toxicity and selectivity for different parasites) in their action is what makes them so amenable to development as therapeutic agents. The inclusion of a new target organism (Cryptococcus neoformans) that can be grown easily in vitro and can be genetically manipulated permits a level of analysis that still can only be contemplated for Pneumocystis carinii and Cryptosporidium parvum. Many of the ongoing studies will continue as new compounds are synthesized by Tidwell and Boykin; so that the database containing the activity of the compounds on target enzymes and their effects on DNA will be continuously updated. The compounds under development are all DNA minor groove binding agents. But, it is clear that topoisomerases are affected as well. For this reason, we will focus our studies more on the mechanism of inhibition of the enzymes that are selectively inhibited by the dicationic compounds, topoisomerases. The major objective of this project during the previous funding period was to identify the DNA-dependent enzyme(s) that is the most likely target(s) of the dicationic compounds. This goal has been achieved for most of the compound series with the identification of topoisomerases as the most likely target.. The question that needs to be addressed now is how this enzyme is affected. This will be achieved through a combination of in vitro and in vivo analysis of topoisomerase action in the presence of the compound. As new types of molecules are produced by Tidwell and Boykin, however, they will continue to be tested for more generalized effects. An important objective will be to determine structure-activity relationships between the existing compounds and C. neoformans topoisomerases in vitro. This will be accomplished in collaboration with Perfect. Once this is accomplished, drug resistant mutants isolated by Perfect will be used to identify regions on the genes important to drug action. Concurrently, the effect of the dicationic drugs on the topoisomerase dependent DNA cleavage activity ex vivo and in vitro will be determined. This analysis will performed for P. carinii, C. parvum, and mammalian cells. In summary, we have found that there is selectivity for inhibition of the parasite topoisomerases over those from mammalian cells, and consequently the major objective of the proposed studies is to determine the molecular basis of this selectivity.
| Patrick, D A; Hall, J E; Bender, B C et al. (1999) Synthesis and anti-Pneumocystis carinii pneumonia activity of novel dicationic dibenzothiophenes and orally active prodrugs. Eur J Med Chem 34:575-83 |
| Hall, J E; Kerrigan, J E; Ramachandran, K et al. (1998) Anti-Pneumocystis activities of aromatic diamidoxime prodrugs. Antimicrob Agents Chemother 42:666-74 |
| Tuttle, R H; Hall, J E; Tidwell, R R (1997) High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes. J Chromatogr B Biomed Sci Appl 688:319-24 |
| Tidwell, R R; Jones, S K; Naiman, N A et al. (1993) Activity of cationically substituted bis-benzimidazoles against experimental Pneumocystis carinii pneumonia. Antimicrob Agents Chemother 37:1713-6 |
